Modulation of a voltage-gated Na+ channel by sevoflurane involves multiple sites and distinct mechanisms

Halogenated inhaled general anesthetic agents modulate voltage-gated ion channels, but the underlying molecular mechanisms are not understood. Many general anesthetic agents regulate voltage-gated Na⁺ (Na_V) channels, including the commonly used drug sevoflurane. Here, we investigated the putative binding sites and molecular mechanisms of sevoflurane action on the bacterial Na_V channel NaChBac by using a combination of molecular dynamics simulation, electrophysiology, and kinetic analysis. Structural modeling revealed multiple sevoflurane interaction sites possibly associated with NaChBac modulation. Electrophysiologically, sevoflurane favors activation and inactivation at low concentrations (0.2 mM), and additionally accelerates current decay at high concentrations (2 mM). Explaining these observations, kinetic modeling suggests concurrent destabilization of closed states and low-affinity open channel block. We propose that the multiple effects of sevoflurane on NaChBac result from simultaneous interactions at multiple sites with distinct affinities. This multiple-site, multiple-mode hypothesis offers a framework to study the structural basis of general anesthetic action.General anesthetic agents have been in use for more than 160 y. However, we still understand relatively little about their mechanisms of action, which greatly limits our ability to design safer and more effective general anesthetic agents. Ion channels of the central nervous system are known to be key targets of general anesthetic agents, as their modulation can account for the endpoints and side effects of general anesthesia (1–4). Many families of ion channels are modulated by general anesthetic agents, including ligand-gated, voltage-gated, and nongated ion channels (2, 5–7). Mammalian voltage-gated Na⁺ (Na_V) channels, which mediate the upstroke of the action potential, are regulated by numerous inhaled general anesthetic agents (8–14), which generally cause inhibition. Previous work showed that inhaled general anesthetic agents, including sevoflurane, isoflurane, desflurane, and halothane, mediate inhibition by increasing the rate of Na⁺ channel inactivation, hyperpolarizing steady-state inactivation, and slowing recovery from inactivation (11, 15–18). Inhibition of presynaptic Na_V channels in the spinal cord is proposed to lead to inhibition of neurotransmitter release, facilitating immobilization—one of the endpoints of general anesthesia (14, 19, 20). Despite the importance of Na_V channels as general anesthetic targets, little is known about interaction sites or the mechanisms of action.What is known about anesthetic sites in Na_V channels comes primarily from the local anesthetic field. Local anesthetic agent binding to Na_V channels is well characterized. These amphiphilic drugs enter the channel pore from the intracellular side, causing open-channel block (21). Investigating molecular mechanisms of mammalian Na_V channel modulation by general anesthetic agents has been complicated by the lack of high-resolution structures of these channels as a result of their large size and pseudotetrameric organization. However, the recent discovery of the smaller, tetrameric bacterial Na⁺ channel family has provided an invaluable tool to characterize the structural features of Na_V channels and investigate their interactions with general anesthetic agents at the molecular level (22, 23). Several bacterial Na⁺ channels have been crystallized (24–27). These channels have a classical domain structure in which helices S1–S4 form the voltage sensor domain (VSD), S5 and S6 form the pore, and the S4–S5 linker connects the voltage sensor to the pore domain. One notable structural feature is the presence of “fenestrations” or hydrophobic tunnels through the pore domain (24).Although crystal structures are not yet reported, the bacterial Na⁺ channel NaChBac has been extensively characterized by electrophysiology (22, 28–36). Additionally NaChBac exhibits conserved slow open channel block in response to local and general anesthetic agents (15, 37). These anesthetic agents reduce peak current and accelerate current decay, making it conceivable that local and general anesthetic agents could share a site of action in NaChBac. The local anesthetic binding site identified in the central cavity of the mammalian Na_V1.2 channel, which mediates open channel block, is partially conserved in NaChBac (37, 38). A recent molecular dynamics (MD) modeling study found that isoflurane, which inhibits NaChBac (15), interacts with multiple regions of this channel, including the pore, the selectivity filter, and the S4–S5 linker/S6 interface (39). Although the importance of these interactions on the modulation of mammalian Na_V channels remains to be determined, the available data indicate that NaChBac is currently one of the best starting points to investigate the mechanisms of action of sevoflurane.Here, we investigated NaChBac to gain structural insight into the mechanisms of inhaled anesthetic modulation of Na_V channels. The focus of this work is sevoflurane because this anesthetic is commonly used in clinical settings and is a known inhibitor of several mammalian Na_V channels (Na_V 1.4, 1.7, and 1.8) (11, 13). A three-pronged approach incorporating MD simulation, whole-cell patch-clamp electrophysiology, and kinetic modeling suggests that sevoflurane acts on multiple sites to alter gating and permeation. Whereas the effect on gating results from modulating activation and inactivation gating at low concentrations (0.2 mM), the permeation effect is apparent at high concentrations (2 mM) and results from open channel block (2 mM). Although the net inhibitory effect of these multisite interactions is consistent with anesthetic-induced reduction of neuronal firing, general anesthesia does not simply result from a global reduction in firing. General anesthesia depends on complex mechanisms throughout the brain, which include increases and decreases in firing (3). Thus, precisely how Na⁺ channel activation by sevoflurane fits into the global effects of anesthesia remains to be seen. The present work helps elucidate the molecular mechanism of sevoflurane action on Na_V channels.     

Nonfunctional NaV1.1 familial hemiplegic migraine mutant transformed into gain of function by partial rescue of folding defects

Familial hemiplegic migraine (FHM) is a rare subtype of migraine with aura. Mutations causing FHM type 3 have been identified in SCN1A, the gene encoding the Na_v1.1 Na⁺ channel, which is also a major target of epileptogenic mutations and is particularly important for the excitability of GABAergic neurons. However, functional studies of Na_V1.1 FHM mutations have generated controversial results. In particular, it has been shown that the Na_V1.1-L1649Q mutant is nonfunctional when expressed in a human cell line because of impaired plasma membrane expression, similarly to Na_V1.1 mutants that cause severe epilepsy, but we have observed gain-of-function effects for other Na_V1.1 FHM mutants. Here we show that Na_V1.1-L1649Q is nonfunctional because of folding defects that are rescuable by incubation at lower temperatures or coexpression of interacting proteins, and that a partial rescue is sufficient for inducing an overall gain of function because of the modifications in gating properties. Strikingly, when expressed in neurons, the mutant was partially rescued and was a constitutive gain of function. A computational model showed that 35% rescue can be sufficient for inducing gain of function. Interestingly, previously described folding-defective epileptogenic Na_V1.1 mutants show loss of function also when rescued. Our results are consistent with gain of function as the functional effect of Na_V1.1 FHM mutations and hyperexcitability of GABAergic neurons as the pathomechanism of FHM type 3.Epilepsy and migraine are common neurologic disorders that may have pathophysiological links (1–3). Mutations have been identified for some rare types of epilepsy and migraine (1, 4–6), opening a window for investigating their pathogenic mechanisms, which may provide useful information also about more common forms. The Na⁺ channel α subunit Na_V1.1, encoded by the SCN1A gene, is the target of hundreds of epileptogenic mutations (7–9), and of mutations causing familial hemiplegic migraine type 3 (FHM-3), a rare subtype of migraine with aura characterized by hemiplegia during the attacks, which can also be caused by mutations of Ca_V2.1 Ca²⁺ channels and the α2 subunit of the Na⁺/K⁺ ATPase (FHM types 1 and 2) (1, 6). The results of most studies suggest that epileptogenic Na_V1.1 mutations cause variable degrees of loss of function of Na_V1.1, leading to reduced Na⁺ current and excitability in GABAergic neurons, and resulting in decreased inhibition in neuronal networks (10–14). The most severe phenotypes (e.g., Dravet syndrome, an extremely severe epileptic encephalopathy) are in general caused by mutations that induce complete Na_V1.1 loss of function, leading to haploinsufficiency (15). Thus, it has been hypothesized that a more severe loss of function would cause more severe epilepsy (8). Functional studies of Na_V1.1 FHM mutations have generated more confusing results (1). For instance, we have reported gain-of-function effects for the mutant Q1489K causing pure FHM (16), and modulable gain-/loss-of-function effects for the mutant T1174S associated with FHM or mild epilepsy in different branches of the family (17). Overall, our results are consistent with a gain of function of Na_V1.1 as the cause of FHM, which might induce cortical spreading depression (CSD), a probable pathomechanism of migraine, because of hyperexcitability of GABAergic interneurons (16). However, a study has reported loss of function for FHM hNa_V1.1 mutants expressed in the human cell line tsA-201—in particular, complete loss of function for the L1649Q mutant because of lack of cell surface expression (18). L1649Q has been identified in a four-generation family with eight members presenting with FHM, without epilepsy or other neurologic symptoms (19); this is a puzzling result more consistent with a phenotype of severe epilepsy (7, 8). We have found that Na_V1.1 epileptogenic mutations can induce loss of function by causing folding defects (20), which can be partially rescued by incubation of the transfected cells at lower temperatures (≤30 °C) or by molecular interactions (21, 22), as recently confirmed also for other epileptogenic Na_V1.1 mutants (23, 24). We report here that L1649Q is a folding-defective mutant that, when partially rescued, is characterized by an overall gain of function, consistent with our hypothesis of FHM type 3 pathomechanism (16).     

Voltage sensor interaction site for selective small molecule inhibitors of voltage-gated sodium channels

Voltage-gated sodium (Na_v) channels play a fundamental role in the generation and propagation of electrical impulses in excitable cells. Here we describe two unique structurally related nanomolar potent small molecule Na_v channel inhibitors that exhibit up to 1,000-fold selectivity for human Na_v1.3/Na_v1.1 (ICA-121431, IC₅₀, 19 nM) or Na_v1.7 (PF-04856264, IC₅₀, 28 nM) vs. other TTX-sensitive or resistant (i.e., Na_v1.5) sodium channels. Using both chimeras and single point mutations, we demonstrate that this unique class of sodium channel inhibitor interacts with the S1–S4 voltage sensor segment of homologous Domain 4. Amino acid residues in the “extracellular” facing regions of the S2 and S3 transmembrane segments of Na_v1.3 and Na_v1.7 seem to be major determinants of Na_v subtype selectivity and to confer differences in species sensitivity to these inhibitors. The unique interaction region on the Domain 4 voltage sensor segment is distinct from the structural domains forming the channel pore, as well as previously characterized interaction sites for other small molecule inhibitors, including local anesthetics and TTX. However, this interaction region does include at least one amino acid residue [E1559 (Na_v1.3)/D1586 (Na_v1.7)] that is important for Site 3 α-scorpion and anemone polypeptide toxin modulators of Na_v channel inactivation. The present study provides a potential framework for identifying subtype selective small molecule sodium channel inhibitors targeting interaction sites away from the pore region.Voltage-gated sodium (Na_v) channels play an important role in the generation and propagation of electrical signals in excitable cells (1–3). Eukaryotic Na_v channels are heteromeric membrane proteins composed of a pore-forming α-subunit and auxiliary β-subunits (3, 4). The mammalian genome encodes nine distinct α (Na_v1.1–1.9) and four β subunits (3). The α-subunit comprises four homologous domains (D1–D4), each of which contains six transmembrane segments (S1–S6) (3–5). The S5 and S6 segments form the central pore separated by the SS1 and SS2 segments, which form the ion selectivity filter at its extracellular end. The S1 to S4 segments form the voltage sensor (3–5).Both naturally occurring and synthetic pharmacological modulators of sodium channel have been identified (6–11), and for many, their site of interaction has been defined. For example, the marine toxin TTX inhibits several Na_v subtypes by interacting with amino acid residues within the SS1–SS2 segments that define the outer pore of the channel (12, 13). In contrast, the polypeptide α- and β-scorpion venom toxins, which enhance sodium channel activation or delay inactivation, and spider venom toxin sodium channel inhibitors like Protx II interact with specific residues on the S1–S4 voltage sensor regions within homologous Domain 2 (i.e., β-scorpion toxins, Protx II) or Domain 4 (i.e., α-scorpion toxins, anemone toxins) of the channel (8, 14–18). Many synthetic small molecule inhibitors of Na_v channels, including local anesthetic, antiepileptic, and antiarrhythmic agents, are believed to interact with amino acid residues within the S6 segment in Domain 4, which forms part of the pore lining and is structurally highly conserved across subclasses of mammalian Na_v channels (11, 19–22). This structural homology probably accounts for many clinically used local anesthetics and related antiepileptic and antiarrhythmic inhibitors, exhibiting little or no selectivity across the nine subtypes of mammalian Na_v channels (23). In the clinic this absence of subtype selectivity can result in toxicities associated with unwanted interactions with off-target Na_v channels (e.g., cardiac toxicity due to inhibition of cardiac Na_v1.5 channels) (24, 25). Therefore, because of their importance in normal physiology and pathophysiology, identification of selective pharmacological modulators of Na_v channels is of considerable interest to the scientific and medical communities (9, 23, 26–29). For example, in addition to the therapeutic utility of sodium channel inhibitors described above, there has been recent interest in potentially targeting inhibition of specific Na_v channel subtypes (i.e., Na_v1.7, Na_v1.8, and Na_v1.3) for the treatment of pain (9, 30–32).The present study describes the characterization of a class of subtype selective sodium channel inhibitor that interacts with a unique site on the voltage sensor region of homologous Domain 4. This inhibitory interaction site differs from previously reported inhibitor binding sites for TTX and local anesthetic-like modulators (11, 12).     

Structural basis for gating charge movement in the voltage sensor of a sodium channel

Voltage-dependent gating of ion channels is essential for electrical signaling in excitable cells, but the structural basis for voltage sensor function is unknown. We constructed high-resolution structural models of resting, intermediate, and activated states of the voltage-sensing domain of the bacterial sodium channel NaChBac using the Rosetta modeling method, crystal structures of related channels, and experimental data showing state-dependent interactions between the gating charge-carrying arginines in the S4 segment and negatively charged residues in neighboring transmembrane segments. The resulting structural models illustrate a network of ionic and hydrogen-bonding interactions that are made sequentially by the gating charges as they move out under the influence of the electric field. The S4 segment slides 6–8 Å outward through a narrow groove formed by the S1, S2, and S3 segments, rotates ∼30°, and tilts sideways at a pivot point formed by a highly conserved hydrophobic region near the middle of the voltage sensor. The S4 segment has a 3₁₀-helical conformation in the narrow inner gating pore, which allows linear movement of the gating charges across the inner one-half of the membrane. Conformational changes of the intracellular one-half of S4 during activation are rigidly coupled to lateral movement of the S4–S5 linker, which could induce movement of the S5 and S6 segments and open the intracellular gate of the pore. We confirmed the validity of these structural models by comparing with a high-resolution structure of a NaChBac homolog and showing predicted molecular interactions of hydrophobic residues in the S4 segment in disulfide-locking studies.Voltage-gated sodium (Na_V) channels are responsible for initiation and propagation of action potentials in nerve, muscle, and endocrine cells (1, 2). They are members of the structurally homologous superfamily of voltage-gated ion channel proteins that also includes voltage-gated potassium (K_V), voltage-gated calcium (Ca_V), and cyclic nucleotide-gated (CNG) channels (3). Mammalian Na_V and Ca_V channels consist of four homologous domains (I through IV), each containing six transmembrane segments (S1 through S6) and a membrane-reentrant pore loop between the S5 and S6 segments (1, 3). Segments S1–S4 of the channel form the voltage-sensing domain (VSD), and segments S5 and S6 and the membrane-reentrant pore loop form the pore. The bacterial Na_V channel NaChBac and its relatives consist of tetramers of four identical subunits, which closely resemble one domain of vertebrate Na_V and Ca_V channels, but provide much simpler structures for studying the mechanism of voltage sensing (4, 5). The hallmark feature of the voltage-gated ion channels is the steep voltage dependence of activation, which derives from the voltage-driven outward movement of gating charges in response to the membrane depolarization (6, 7). The S4 transmembrane segment in the VSD has four to seven arginine residues spaced at 3-aa intervals, which serve as gating charges in the voltage-sensing mechanism (8–15). The intracellular S4–S5 linker that connects the VSD to the pore plays a key role in coupling voltage-dependent conformational changes in the VSD to opening and closing of the pore (16). The gating charges are pulled in by the internally negative transmembrane electric field and released to move out on depolarization. Their outward movement must be catalyzed by the voltage sensor to reduce the large thermodynamic barrier to movement of charged amino acid residues across the membrane. The molecular mechanism by which the gating charges are stabilized in the hydrophobic transmembrane environment and the catalytic mechanism through which they are transported across the membrane in response to changes in membrane potential are the subjects of intense research efforts.Progress has been made in determining high-resolution structures of voltage sensors of K_V and Na_V channels in activated states (17–20). However, high-resolution structures of resting and intermediate states of voltage sensors are unknown. The majority of evidence supports a sliding helix model of the voltage-dependent gating in which the gating charge-carrying arginines in S4 are proposed to sequentially form ion pairs with negatively charged residues in S1–S3 segments during activation of the channel (9–11, 21). However, the structural basis for stabilization of the gating charges in the membrane and catalysis of their movement through the hydrophobic membrane environment remain uncertain. Here, we have integrated bioinformatics analysis of Na_V and K_V channel families using the HHPred homology detection server (22–24), high-resolution structural modeling using the Rosetta Membrane (25–27) and Rosetta Symmetry methods (28), the X-ray structures of the K_v1.2-K_v2.1 chimeric channel and NavAb with activated VSDs (19, 20) and the MlotiK1 CNG channel in the resting state (29), and experimental data showing sequential state-dependent interactions between gating charges in S4 and negatively charged residues in S1–S3 (this work and refs. 30–33). Predictions of the resulting voltage-sensing model are confirmed in this work by disulfide-locking studies and mutant cycle analysis of the interactions of hydrophobic residues in the S4 segment. This model reveals structural details of the voltage-dependent conformational changes in the VSD that stabilize and catalyze gating charge movement and are coupled to opening and closing of the intracellular activation gate of the ion-conducting pore.     

Pathophysiological implication of CaV3.1 T-type Ca2+ channels in trigeminal neuropathic pain

A crucial pathophysiological issue concerning central neuropathic pain is the modification of sensory processing by abnormally increased low-frequency brain rhythms. Here we explore the molecular mechanisms responsible for such abnormal rhythmicity and its relation to neuropathic pain syndrome. Toward this aim, we investigated the behavioral and electrophysiological consequences of trigeminal neuropathic pain following infraorbital nerve ligations in Ca_V3.1 T-type Ca²⁺ channel knockout and wild-type mice. Ca_V3.1 knockout mice had decreased mechanical hypersensitivity and reduced low-frequency rhythms in the primary somatosensory cortex and related thalamic nuclei than wild-type mice. Lateral inhibition of gamma rhythm in primary somatosensory cortex layer 4, reflecting intact sensory contrast, was present in knockout mice but severely impaired in wild-type mice. Moreover, cross-frequency coupling between low-frequency and gamma rhythms, which may serve in sensory processing, was pronounced in wild-type mice but not in Ca_V3.1 knockout mice. Our results suggest that the presence of Ca_V3.1 channels is a key element in the pathophysiology of trigeminal neuropathic pain.Since 1911, when H. Head and G. M. Holmes first addressed the relevance of the thalamus as a central pattern generator for neuropathic pain (1), many clinical studies have indicated the coexistence of pathophysiological thalamocortical activity and the occurrence of neuropathic pain. Compared with healthy controls, patients with neuropathic pain show increased low-frequency thalamocortical oscillations in magnetoencephalogram (MEG) recordings. Such low-frequency oscillations are a typical thalamocortical dysrhythmia (TCD) syndrome (2). In agreement with such MEG findings, the excess power of low-frequency oscillation was marked in local-field potential (LFP) recordings from the thalamus (3–5) and electroencephalogram (EEG) recordings from the cortex (6, 7) of patients with neuropathic pain. In addition, the presence of thalamic burst firing, which is a well-known underlying mechanism for cortical low-frequency oscillations through the thalamocortical recurrent network (8, 9), has been confirmed in patients with neuropathic pain (10–13). Results from small lesions in the posterior part of the central lateral nucleus of the medial thalamus, which reduce tonic hyperpolarization of thalamic neurons in chronic neuropathic pain patients, have also provided insight into the role of low-frequency thalamic rhythmicity in neuropathic pain. Following such interventions, a marked decrease in low-frequency EEG power was observed as well as pain relief (7, 12), indicating that alteration of thalamocortical rhythms plays a crucial role in the development and/or persistence of neuropathic pain.Trigeminal neuropathic pain (TNP) is characterized by unilateral chronic facial pain limited to one or more divisions of the trigeminal nerve. There is increasing evidence that TNP is associated with anatomical and biochemical changes in the thalamus (14–16). Moreover, patients with TNP display significant reductions in thalamic volume and neural viability (15), indicating that altered thalamic anatomy, physiology, and biochemistry may result in disturbed thalamocortical oscillatory properties.Abnormal thalamic activity has been investigated in patients with neuropathic pain (3–7, 10–13, 17, 18), including TNP (14–16). Furthermore, the potential role of thalamic burst firing in abnormally increased low-frequency oscillations has been proposed as a pathophysiological mechanism (2, 12). Because T-type Ca²⁺ channels are known to underlie thalamic burst firing (8, 9), it is reasonable to propose that pathophysiological low-frequency rhythms, such as those seen in central neuropathic pain, may be mediated by these calcium channels. Nevertheless, this hypothesis has not been directly tested. To determine whether T-type Ca²⁺ channels play a role in the generation of neuropathic pain, thalamocortical oscillatory properties were examined in mice lacking Ca_V3.1 channels following induction of TNP through partial ligation of the inferior orbital nerve (IoN). This channel represents the major T-type Ca²⁺ channel isoform in thalamocortical projection neurons (19). Following IoN ligations, Ca_V3.1 knockout (KO) mice showed significantly attenuated mechanical hypersensitivity, compared with wild-type (WT) mice. Moreover, spectral analysis of thalamocortical rhythms from Ca_V3.1 KO mice showed decreased low-frequency rhythm propensity, compared with WT mice. In addition, response to gamma activation and the spatiotemporal patterns of primary somatosensory (S1) cortex activity were altered in WT but not in KO mice after IoN ligation. Moreover, the cross-frequency interactions between low-frequency and gamma rhythms were significantly increased in WT but not in Ca_V3.1 KO mice. These findings indicate that TNP is associated with altered thalamocortical rhythms, resulting in increased sensitivity to pain as well as pain generation in response to nonnoxious stimuli. In addition, these results indicate that Ca_V3.1 T-type Ca²⁺ channels are fundamentally associated with the alteration of thalamocortical rhythms seen in TNP.     

Impaired excitability of somatostatin- and parvalbumin-expressing cortical interneurons in a mouse model of Dravet syndrome

Haploinsufficiency of the voltage-gated sodium channel Na_V1.1 causes Dravet syndrome, an intractable developmental epilepsy syndrome with seizure onset in the first year of life. Specific heterozygous deletion of Na_V1.1 in forebrain GABAergic-inhibitory neurons is sufficient to cause all the manifestations of Dravet syndrome in mice, but the physiological roles of specific subtypes of GABAergic interneurons in the cerebral cortex in this disease are unknown. Voltage-clamp studies of dissociated interneurons from cerebral cortex did not detect a significant effect of the Dravet syndrome mutation on sodium currents in cell bodies. However, current-clamp recordings of intact interneurons in layer V of neocortical slices from mice with haploinsufficiency in the gene encoding the Na_V1.1 sodium channel, Scn1a, revealed substantial reduction of excitability in fast-spiking, parvalbumin-expressing interneurons and somatostatin-expressing interneurons. The threshold and rheobase for action potential generation were increased, the frequency of action potentials within trains was decreased, and action-potential firing within trains failed more frequently. Furthermore, the deficit in excitability of somatostatin-expressing interneurons caused significant reduction in frequency-dependent disynaptic inhibition between neighboring layer V pyramidal neurons mediated by somatostatin-expressing Martinotti cells, which would lead to substantial disinhibition of the output of cortical circuits. In contrast to these deficits in interneurons, pyramidal cells showed no differences in excitability. These results reveal that the two major subtypes of interneurons in layer V of the neocortex, parvalbumin-expressing and somatostatin-expressing, both have impaired excitability, resulting in disinhibition of the cortical network. These major functional deficits are likely to contribute synergistically to the pathophysiology of Dravet syndrome.Dravet syndrome (DS), also referred to as “severe myoclonic epilepsy in infancy,” is a rare genetic epileptic encephalopathy characterized by frequent intractable seizures, severe cognitive deficits, and premature death (1–3). DS is caused by loss-of-function mutations in SCN1A, the gene encoding type I voltage-gated sodium channel Na_V1.1, which usually arise de novo in the affected individuals (4–7). Like DS patients, mice with heterozygous loss-of-function mutations in Scn1a exhibit ataxia, sleep disorder, cognitive deficit, autistic-like behavior, and premature death (8–14). Like DS patients, DS mice first become susceptible to seizures caused by elevation of body temperature and subsequently experience spontaneous myoclonic and generalized tonic-clonic seizures (11). Global deletion of Na_V1.1 impairs Na⁺ currents and action potential (AP) firing in GABAergic-inhibitory interneurons (8–10), and specific deletion of Na_V1.1 in forebrain interneurons is sufficient to cause DS in mice (13, 15). These data suggest that the loss of interneuron excitability and resulting disinhibition of neural circuits cause DS, but the functional role of different subtypes of interneurons in the cerebral cortex in DS remains unknown.Neocortical GABAergic interneurons shape cortical output and display great diversity in morphology and function (16, 17). The expression of parvalbumin (PV) and somatostatin (SST) defines two large, nonoverlapping groups of interneurons (16, 18, 19). In layer V of the cerebral cortex, PV-expressing fast-spiking interneurons and SST-expressing Martinotti cells each account for ∼40% of interneurons, and these interneurons are the major inhibitory regulators of cortical network activity (17, 20). Layer V PV interneurons make synapses on the soma and proximal dendrites of pyramidal neurons (18, 19), where they mediate fast and powerful inhibition (21, 22). Selective heterozygous deletion of Scn1a in neocortical PV interneurons increases susceptibility to chemically induced seizures (23), spontaneous seizures, and premature death (24), indicating that this cell type may contribute to Scn1a deficits. However, selective deletion of Scn1a in neocortical PV interneurons failed to reproduce the effects of DS fully, suggesting the involvement of other subtypes of interneurons in this disease (23, 24). Layer V Martinotti cells have ascending axons that arborize in layer I and spread horizontally to neighboring cortical columns, making synapses on apical dendrites of pyramidal neurons (17, 25, 26). They generate frequency-dependent disynaptic inhibition (FDDI) that dampens excitability of neighboring layer V pyramidal cells (27–29), contributing to maintenance of the balance of excitation and inhibition in the neocortex. However, the functional roles of Martinotti cells and FDDI in DS are unknown.Because layer V forms the principal output pathway of the neocortex, reduction in inhibitory input to layer V pyramidal cells would have major functional consequences by increasing excitatory output from all cortical circuits. However, the effects of the DS mutation on interneurons and neural circuits that provide inhibitory input to layer V pyramidal cells have not been determined. Here we show that the intrinsic excitability of layer V fast-spiking PV interneurons and SST Martinotti cells and the FDDI mediated by Martinotti cells are reduced dramatically in DS mice, leading to an imbalance in the excitation/inhibition ratio. Our results suggest that loss of Na_V1.1 in these two major types of interneurons may contribute synergistically to increased cortical excitability, epileptogenesis, and cognitive deficits in DS.     

Mutant cycle analysis with modified saxitoxins reveals specific interactions critical to attaining high-affinity inhibition of hNaV1.7

Improper function of voltage-gated sodium channels (Na_Vs), obligatory membrane proteins for bioelectrical signaling, has been linked to a number of human pathologies. Small-molecule agents that target Na_Vs hold considerable promise for treatment of chronic disease. Absent a comprehensive understanding of channel structure, the challenge of designing selective agents to modulate the activity of Na_V subtypes is formidable. We have endeavored to gain insight into the 3D architecture of the outer vestibule of Na_V through a systematic structure–activity relationship (SAR) study involving the bis-guanidinium toxin saxitoxin (STX), modified saxitoxins, and protein mutagenesis. Mutant cycle analysis has led to the identification of an acetylated variant of STX with unprecedented, low-nanomolar affinity for human Na_V1.7 (hNa_V1.7), a channel subtype that has been implicated in pain perception. A revised toxin-receptor binding model is presented, which is consistent with the large body of SAR data that we have obtained. This new model is expected to facilitate subsequent efforts to design isoform-selective Na_V inhibitors.Modulation of action potentials in electrically excitable cells is controlled by tight regulation of ion channel expression and distribution. Voltage-gated sodium ion channels (Na_Vs) constitute one such family of essential membrane proteins, encoded in 10 unique genes (Na_V1.1–Na_V1.9, Na_x) and further processed through RNA splicing, editing, and posttranslational modification. Sodium channels are comprised of a large (∼260 kDa) pore-forming α-subunit coexpressed with ancillary β-subunits. Misregulation and/or mutation of Na_Vs have been ascribed to a number of human diseases including neuropathic pain, epilepsy, and cardiac arrhythmias. A desire to understand the role of individual Na_V subtypes in normal and aberrant signaling motivates the development of small-molecule probes for regulating the function of specific channel isoforms (1–4).Nature has provided a collection of small-molecule toxins, including (+)-saxitoxin (STX, 1) and (−)-tetrodotoxin (TTX), which bind to a subset of mammalian Na_V isoforms with nanomolar affinity (5–7). Guanidinium toxins inhibit Na⁺ influx through Na_Vs by occluding the outer pore above the ion selectivity filter (site 1). This proposed mechanism for toxin block follows from a large body of electrophysiological and site-directed mutagenesis studies (Fig. 1A and refs. 8–10). The detailed view of toxin binding, however, is unsupported by structural biology, as no high-resolution structure of a eukaryotic Na_V has been solved to date (11–16). Na_V homology models, constructed based on X-ray analyses of prokaryotic Na⁺ and K⁺ voltage-gated channels, do not sufficiently account for experimental structure–activity relationship (SAR) data (6, 17–20), and the molecular details underlying distinct differences in toxin potencies toward individual Na_V subtypes remain undefined (5, 6, 21–23). The lack of structural information motivates a comprehensive, systematic study of toxin–protein interactions.Open in a separate windowFig. 1.(A) Schematic drawing of 1 bound in the Na_V outer pore as suggested by previous electrophysiology and mutagenesis experiments. Each of the four domains (I, orange; II, red; III, gray; and IV, teal) is represented by a separate panel. (B) Schematic representation of double-mutant cycle analysis and mathematical definition of coupling energy (ΔΔE_Ω). X¹ = IC₅₀(WT⋅STX)/IC₅₀(MutNa_V⋅STX), X² = IC₅₀(WT⋅MeSTX)/IC₅₀(MutNa_V⋅MeSTX), Y¹ = IC₅₀(MutNa_V⋅STX)/IC₅₀(MutNa_V⋅MeSTX), and Y² = IC₅₀(WT⋅STX)/IC₅₀(WT⋅MeSTX).Double-mutant cycle analysis has proven an invaluable experimental method for assessing protein–protein, protein–peptide, and protein–small-molecule interactions in the absence of crystallographic data (Fig. 1B and Fig. S1 and refs. 9, 10, and 24–31). Herein, we describe mutant cycle analysis with Na_Vs using STX and synthetically modified forms thereof. Our results are suggestive of a toxin–Na_V binding pose distinct from previously published views. Our studies have resulted in the identification of a natural variant of STX that is potent against the STX-resistant human Na_V1.7 isoform (hNa_V1.7). Structural insights gained from these studies provide a foundation for engineering guanidinium toxins with Na_V isoform selectivity.Open in a separate windowFig. S1.Mutant cycle analysis definition and examples. (A) Schematic of a single mutant cycle with mathematical expressions for coupling energy ΔΔE_Ω. R is the ideal gas constant and T is temperature. Each IC₅₀ is the half maximal inhibition concentration determined by whole-cell voltage-clamp electrophysiology. When the separation between IC₅₀ values for the reference compound and the modified compound is different with a mutant than with the WT protein, a nonzero value for ΔΔE_Ω is obtained (B), but when the separation is the same (C), ΔΔE_Ω is equal to 0. In B, the difference in the relative affinity of 1 and 4 with Y401A is smaller than the difference with the WT channel, indicating a positive coupling (ΔΔE_Ω > 0). In C, the relative affinities of 1 and 8 against WT rNa_V1.4 and Y401A are similar, and ΔΔE_Ω ∼0 kcal/mol.     

Fine-tuning synaptic plasticity by modulation of CaV2.1 channels with Ca2+ sensor proteins

Modulation of P/Q-type Ca²⁺ currents through presynaptic voltage-gated calcium channels (Ca_V2.1) by binding of Ca²⁺/calmodulin contributes to short-term synaptic plasticity. Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2 (VILIP-2) are neurospecific calmodulin-like Ca²⁺ sensor proteins that differentially modulate Ca_V2.1 channels, but how they contribute to short-term synaptic plasticity is unknown. Here, we show that activity-dependent modulation of presynaptic Ca_V2.1 channels by CaBP1 and VILIP-2 has opposing effects on short-term synaptic plasticity in superior cervical ganglion neurons. Expression of CaBP1, which blocks Ca²⁺-dependent facilitation of P/Q-type Ca²⁺ current, markedly reduced facilitation of synaptic transmission. VILIP-2, which blocks Ca²⁺-dependent inactivation of P/Q-type Ca²⁺ current, reduced synaptic depression and increased facilitation under conditions of high release probability. These results demonstrate that activity-dependent regulation of presynaptic Ca_V2.1 channels by differentially expressed Ca²⁺ sensor proteins can fine-tune synaptic responses to trains of action potentials and thereby contribute to the diversity of short-term synaptic plasticity.Neurons fire repetitively in different frequencies and patterns, and activity-dependent alterations in synaptic strength result in diverse forms of short-term synaptic plasticity that are crucial for information processing in the nervous system (1–3). Short-term synaptic plasticity on the time scale of milliseconds to seconds leads to facilitation or depression of synaptic transmission through changes in neurotransmitter release. This form of plasticity is thought to result from residual Ca²⁺ that builds up in synapses during repetitive action potentials and binds to a Ca²⁺ sensor distinct from the one that evokes neurotransmitter release (1, 2, 4, 5). However, it remains unclear how changes in residual Ca²⁺ cause short-term synaptic plasticity and how neurotransmitter release is regulated to generate distinct patterns of short-term plasticity.In central neurons, voltage-gated calcium (Ca_V2.1) channels are localized in high density in presynaptic active zones where their P/Q-type Ca²⁺ current triggers neurotransmitter release (6–11). Because synaptic transmission is proportional to the third or fourth power of Ca²⁺ entry through presynaptic Ca_V2.1 channels, small changes in Ca²⁺ current have profound effects on synaptic transmission (2, 12). Studies at the calyx of Held synapse have provided important insights into the contribution of presynaptic Ca²⁺ current to short-term synaptic plasticity (13–17). Ca_V2.1 channels are required for synaptic facilitation, and Ca²⁺-dependent facilitation and inactivation of the P/Q-type Ca²⁺ currents are correlated temporally with synaptic facilitation and rapid synaptic depression (13–17).Molecular interactions between Ca²⁺/calmodulin (CaM) and Ca_V2.1 channels induce sequential Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents in nonneuronal cells (18–21). Facilitation and inactivation of P/Q-type currents are dependent on Ca²⁺/CaM binding to the IQ-like motif (IM) and CaM-binding domain (CBD) of the Ca_V2.1 channel, respectively (20, 21). This bidirectional regulation serves to enhance channel activity in response to short bursts of depolarizations and then to decrease activity in response to long bursts. In synapses of superior cervical ganglion (SCG) neurons expressing exogenous Ca_V2.1 channels, synaptic facilitation is induced by repetitive action potentials, and mutation of the IM and CBD motifs prevents synaptic facilitation and inhibits the rapid phase of synaptic depression (22). Thus, in this model synapse, regulation of presynaptic Ca_V2.1 channels by binding of Ca²⁺/CaM can contribute substantially to the induction of short-term synaptic plasticity by residual Ca²⁺.CaM is expressed ubiquitously, but short-term plasticity has great diversity among synapses, and the potential sources of this diversity are unknown. How could activity-dependent regulation of presynaptic Ca_V2.1 channels contribute to the diversity of short-term synaptic plasticity? CaM is the founding member of a large family of Ca²⁺ sensor (CaS) proteins that are differentially expressed in central neurons (23–25). Two CaS proteins, Ca²⁺-binding protein-1 (CaBP1) and Visinin-like protein-2(VILIP-2), modulate facilitation and inactivation of Ca_V2.1 channels in opposite directions through interaction with the bipartite regulatory site in the C-terminal domain (26, 27), and they have varied expression in different types of central neurons (23, 25, 28). CaBP1 strongly enhances inactivation and prevents facilitation of Ca_V2.1 channel currents, whereas VILIP-2 slows inactivation and enhances facilitation of Ca_V2.1 currents during trains of stimuli (26, 27). Molecular analyses show that the N-terminal myristoylation site and the properties of individual EF-hand motifs in CaBP1 and VILIP-2 determine their differential regulation of Ca_V2.1 channels (27, 29–31). However, the role of CaBP1 and VILIP-2 in the diversity of short-term synaptic plasticity is unknown, and the high density of Ca²⁺ channels and unique Ca²⁺ dynamics at the presynaptic active zone make extrapolation of results from studies in nonneuronal cells uncertain. We addressed this important question directly by expressing CaBP1 and VILIP-2 in presynaptic SCG neurons and analyzing their effects on synaptic plasticity. Our results show that CaM-related CaS proteins can serve as sensitive bidirectional switches that fine-tune the input–output relationships of synapses depending on their profile of activity and thereby maintain the balance of facilitation versus depression by the regulation of presynaptic Ca_V2.1 channels.     

Acceleration of conduction velocity linked to clustering of nodal components precedes myelination

High-density accumulation of voltage-gated sodium (Na_v) channels at nodes of Ranvier ensures rapid saltatory conduction along myelinated axons. To gain insight into mechanisms of node assembly in the CNS, we focused on early steps of nodal protein clustering. We show in hippocampal cultures that prenodes (i.e., clusters of Na_v channels colocalizing with the scaffold protein ankyrinG and nodal cell adhesion molecules) are detected before myelin deposition along axons. These clusters can be induced on purified neurons by addition of oligodendroglial-secreted factor(s), whereas ankyrinG silencing prevents their formation. The Na_v isoforms Na_v1.1, Na_v1.2, and Na_v1.6 are detected at prenodes, with Na_v1.6 progressively replacing Na_v1.2 over time in hippocampal neurons cultured with oligodendrocytes and astrocytes. However, the oligodendrocyte-secreted factor(s) can induce the clustering of Na_v1.1 and Na_v1.2 but not of Na_v1.6 on purified neurons. We observed that prenodes are restricted to GABAergic neurons, whereas clustering of nodal proteins only occurs concomitantly with myelin ensheathment on pyramidal neurons, implying separate mechanisms of assembly among different neuronal subpopulations. To address the functional significance of these early clusters, we used single-axon electrophysiological recordings in vitro and showed that prenode formation is sufficient to accelerate the speed of axonal conduction before myelination. Finally, we provide evidence that prenodal clusters are also detected in vivo before myelination, further strengthening their physiological relevance.Voltage-gated sodium (Na_v) channels are highly enriched at the axon initial segment (AIS) and the node of Ranvier, allowing generation and rapid propagation of action potentials by saltatory conduction in myelinated fibers. These axonal domains also contain cell adhesion molecules [e.g., neurofascin 186 (Nfasc186)] and the scaffolding proteins ankyrinG (AnkG) and βIV spectrin, which provide a potential link with the actin cytoskeleton (1). Flanking the nodes are the paranodes, where axoglial junctions between paranodal myelin loops and the axon are formed through interactions between axonal contactin-associated protein (Caspr)/contactin and glial Nfasc155 (2, 3). Although the mechanisms of nodal assembly are best characterized in the peripheral nervous system (4–9), less is known about the cellular and molecular mechanisms underlying node assembly in the CNS. ECM proteins, adhesion molecules, such as Nfasc186, and also, axoglial paranodal junctions have been shown to trigger CNS nodal clustering, although their respective roles remain uncertain (10–19). Moreover, axonal clustering of Na_v channels before myelin deposition and oligodendroglial contact has been shown to occur in retinal ganglion cell (RGC) cultures, where these clusters were induced by oligodendroglial-secreted factor(s) (20, 21).Here, we have investigated the cellular and molecular mechanisms underlying nodal protein assembly in hippocampal neuron cultures. We first showed that evenly spaced clusters of Na_v channels, colocalizing with Nfasc186 and AnkG, are detected along axons before myelination. Strikingly, this prenode assembly is restricted to GABAergic interneurons, suggesting the existence of different mechanisms of nodal assembly. The prenodal clustering can be induced on purified neurons by the addition of oligodendroglial-secreted factor(s) and also depends on intrinsic cues, such as AnkG. Furthermore, we also provide evidence that these clusters are detected in vivo before myelination on hippocampal tissue sections. Finally, to gain insight into their functional significance, we performed in vitro simultaneous somatic and axonal recordings and showed that the presence of prenodes increases the speed of action potential propagation along axons before myelination.     

Luminescence color switching of supramolecular assemblies of discrete molecular decanuclear gold(I) sulfido complexes

A series of discrete decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of various lengths on the aminodiphosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂, has been synthesized and characterized. These complexes have been shown to form supramolecular nanoaggregate assemblies upon solvent modulation. The photoluminescence (PL) colors of the nanoaggregates can be switched from green to yellow to red by varying the solvent systems from which they are formed. The PL color variation was investigated and correlated with the nanostructured morphological transformation from the spherical shape to the cube as observed by transmission electron microscopy and scanning electron microscopy. Such variations in PL colors have not been observed in their analogous complexes with short alkyl chains, suggesting that the long alkyl chains would play a key role in governing the supramolecular nanoaggregate assembly and the emission properties of the decanuclear gold(I) sulfido complexes. The long hydrophobic alkyl chains are believed to induce the formation of supramolecular nanoaggregate assemblies with different morphologies and packing densities under different solvent systems, leading to a change in the extent of Au(I)–Au(I) interactions, rigidity, and emission properties.Gold(I) complexes are one of the fascinating classes of complexes that reveal photophysical properties that are highly sensitive to the nuclearity of the metal centers and the metal–metal distances (1–59). In a certain sense, they bear an analogy or resemblance to the interesting classes of metal nanoparticles (NPs) (60–69) and quantum dots (QDs) (70–76) in that the properties of the nanostructured materials also show a strong dependence on their sizes and shapes. Interestingly, while the optical and spectroscopic properties of metal NPs and QDs show a strong dependence on the interparticle distances, those of polynuclear gold(I) complexes are known to mainly depend on the nuclearity and the internuclear separations of gold(I) centers within the individual molecular complexes or clusters, with influence of the intermolecular interactions between discrete polynuclear molecular complexes relatively less explored (34–38), and those of polynuclear gold(I) clusters not reported. Moreover, while studies on polynuclear gold(I) complexes or clusters are known (34–54), less is explored of their hierarchical assembly and nanostructures as well as the influence of intercluster aggregation on the optical properties (34–38). Among the gold(I) complexes, polynuclear gold(I) chalcogenido complexes represent an important and interesting class (44–51). While directed supramolecular assembly of discrete Au₁₂ (52), Au₁₆ (53), Au₁₈ (51), and Au₃₆ (54) metallomacrocycles as well as trinuclear gold(I) columnar stacks (34–38) have been reported, there have been no corresponding studies on the supramolecular hierarchical assembly of polynuclear gold(I) chalcogenido clusters.Based on our interests and experience in the study of gold(I) chalcogenido clusters (44–46, 51), it is believed that nanoaggegrates with interesting luminescence properties and morphology could be prepared by the judicious design of the gold(I) chalcogenido clusters. As demonstrated by our previous studies on the aggregation behavior of square-planar platinum(II) complexes (77–80) where an enhancement of the solubility of the metal complexes via introduction of solubilizing groups on the ligands and the fine control between solvophobicity and solvophilicity of the complexes would have a crucial influence on the factors governing supramolecular assembly and the formation of aggregates (80), introduction of long alkyl chains as solubilizing groups in the gold(I) sulfido clusters may serve as an effective way to enhance the solubility of the gold(I) clusters for the construction of supramolecular assemblies of novel luminescent nanoaggegrates.Herein, we report the preparation and tunable spectroscopic properties of a series of decanuclear gold(I) μ₃-sulfido complexes with alkyl chains of different lengths on the aminophosphine ligands, [Au₁₀{Ph₂PN(C_nH_2n+1)PPh₂}₄(μ₃-S)₄](ClO₄)₂ [n = 8 (1), 12 (2), 14 (3), 18 (4)] and their supramolecular assembly to form nanoaggregates. The emission colors of the nanoaggregates of 2−4 can be switched from green to yellow to red by varying the solvent systems from which they are formed. These results have been compared with their short alkyl chain-containing counterparts, 1 and a related [Au₁₀{Ph₂PN(C₃H₇)PPh₂}₄(μ₃-S)₄](ClO₄)₂ (45). The present work demonstrates that polynuclear gold(I) chalcogenides, with the introduction of appropriate functional groups, can serve as building blocks for the construction of novel hierarchical nanostructured materials with environment-responsive properties, and it represents a rare example in which nanoaggregates have been assembled with the use of discrete molecular metal clusters as building blocks.     

Calcium sensor regulation of the CaV2.1 Ca2+ channel contributes to short-term synaptic plasticity in hippocampal neurons

Short-term synaptic plasticity is induced by calcium (Ca²⁺) accumulating in presynaptic nerve terminals during repetitive action potentials. Regulation of voltage-gated Ca_V2.1 Ca²⁺ channels by Ca²⁺ sensor proteins induces facilitation of Ca²⁺ currents and synaptic facilitation in cultured neurons expressing exogenous Ca_V2.1 channels. However, it is unknown whether this mechanism contributes to facilitation in native synapses. We introduced the IM-AA mutation into the IQ-like motif (IM) of the Ca²⁺ sensor binding site. This mutation does not alter voltage dependence or kinetics of Ca_V2.1 currents, or frequency or amplitude of spontaneous miniature excitatory postsynaptic currents (mEPSCs); however, synaptic facilitation is completely blocked in excitatory glutamatergic synapses in hippocampal autaptic cultures. In acutely prepared hippocampal slices, frequency and amplitude of mEPSCs and amplitudes of evoked EPSCs are unaltered. In contrast, short-term synaptic facilitation in response to paired stimuli is reduced by ∼50%. In the presence of EGTA-AM to prevent global increases in free Ca²⁺, the IM-AA mutation completely blocks short-term synaptic facilitation, indicating that synaptic facilitation by brief, local increases in Ca²⁺ is dependent upon regulation of Ca_V2.1 channels by Ca²⁺ sensor proteins. In response to trains of action potentials, synaptic facilitation is reduced in IM-AA synapses in initial stimuli, consistent with results of paired-pulse experiments; however, synaptic depression is also delayed, resulting in sustained increases in amplitudes of later EPSCs during trains of 10 stimuli at 10–20 Hz. Evidently, regulation of Ca_V2.1 channels by CaS proteins is required for normal short-term plasticity and normal encoding of information in native hippocampal synapses.Modification of synaptic strength in central synapses is highly dependent upon presynaptic activity. The frequency and pattern of presynaptic action potentials regulates the postsynaptic response through diverse forms of short- and long-term plasticity that are specific to individual synapses and depend upon accumulation of intracellular Ca²⁺ (1–4). Presynaptic plasticity regulates neurotransmission by varying the amount of neurotransmitter released by each presynaptic action potential (1–5). P/Q-type Ca²⁺ currents conducted by voltage-gated Ca_V2.1 Ca²⁺ channels initiate neurotransmitter release at fast excitatory glutamatergic synapses in the brain (6–9) and regulate short-term presynaptic plasticity (3, 10). These channels exhibit Ca²⁺-dependent facilitation and inactivation that is mediated by the Ca²⁺ sensor (CaS) protein calmodulin (CaM) bound to a bipartite site in their C-terminal domain composed of an IQ-like motif (IM) and a CaM binding domain (CBD) (11–14). Ca²⁺-dependent facilitation and inactivation of P/Q-type Ca²⁺ currents correlate with facilitation and rapid depression of synaptic transmission at the Calyx of Held (15–18). Elimination of Ca_V2.1 channels by gene deletion prevents facilitation of synaptic transmission at the Calyx of Held (19, 20). Cultured sympathetic ganglion neurons with presynaptic expression of exogenous Ca_V2.1 channels harboring mutations in their CaS regulatory site have reduced facilitation and slowed depression of postsynaptic responses because of reduced Ca²⁺-dependent facilitation and Ca²⁺-dependent inactivation of Ca_V2.1 currents (21). The CaS proteins Ca²⁺-binding protein 1 (CaBP-1), visinin-like protein-2 (VILIP-2), and neuronal Ca²⁺ sensor-1 (NCS-1) induce different degrees of Ca²⁺-dependent facilitation and inactivation of channel activity (22–26). Expression of these different CaS proteins with Ca_V2.1 channels in cultured sympathetic ganglion neurons results in corresponding bidirectional changes in facilitation and depression of the postsynaptic response (25, 26). Therefore, binding of CaS proteins to Ca_V2.1 channels at specific synapses can change the balance of CaS-dependent facilitation and inactivation of Ca_V2.1 channels, and determine the outcome of synaptic plasticity (27). Currently, it is not known whether such molecular regulation of Ca_V2.1 by CaS proteins induces or modulates synaptic plasticity in native hippocampal synapses.To understand the functional role of regulation of Ca_V2.1 channels by CaS proteins in synaptic plasticity in vivo, we generated knock-in mice with paired alanine substitutions for the isoleucine and methionine residues in the IM motif (IM-AA) in their C-terminal domain. Here we investigated the effects of mutating this CaS regulatory site on hippocampal neurotransmission and synaptic plasticity. This mutation had no effect on basal Ca²⁺ channel function or on basal synaptic transmission. However, we found reduced short-term facilitation in response to paired stimuli in autaptic synapses in hippocampal cultures and in Schaffer collateral (SC)-CA1 synapses in acutely prepared hippocampal slices. Moreover, synaptic facilitation in mutant SC-CA1 synapses developed and decayed more slowly during trains of stimuli. These results identify a critical role for modulation of Ca_V2.1 channels by CaS proteins in short-term synaptic plasticity, which is likely to have important consequences for encoding and transmitting information in the hippocampus.     

In vitro selection of a sodium-specific DNAzyme and its application in intracellular sensing

Over the past two decades, enormous progress has been made in designing fluorescent sensors or probes for divalent metal ions. In contrast, the development of fluorescent sensors for monovalent metal ions, such as sodium (Na⁺), has remained underdeveloped, even though Na⁺ is one the most abundant metal ions in biological systems and plays a critical role in many biological processes. Here, we report the in vitro selection of the first (to our knowledge) Na⁺-specific, RNA-cleaving deoxyribozyme (DNAzyme) with a fast catalytic rate [observed rate constant (k_obs) ∼0.1 min⁻¹], and the transformation of this DNAzyme into a fluorescent sensor for Na⁺ by labeling the enzyme strand with a quencher at the 3′ end, and the DNA substrate strand with a fluorophore and a quencher at the 5′ and 3′ ends, respectively. The presence of Na⁺ catalyzed cleavage of the substrate strand at an internal ribonucleotide adenosine (rA) site, resulting in release of the fluorophore from its quenchers and thus a significant increase in fluorescence signal. The sensor displays a remarkable selectivity (>10,000-fold) for Na⁺ over competing metal ions and has a detection limit of 135 µM (3.1 ppm). Furthermore, we demonstrate that this DNAzyme-based sensor can readily enter cells with the aid of α-helical cationic polypeptides. Finally, by protecting the cleavage site of the Na⁺-specific DNAzyme with a photolabile o-nitrobenzyl group, we achieved controlled activation of the sensor after DNAzyme delivery into cells. Together, these results demonstrate that such a DNAzyme-based sensor provides a promising platform for detection and quantification of Na⁺ in living cells.Metal ions play crucial roles in a variety of biochemical processes. As a result, the concentrations of cellular metal ions have to be highly regulated in different parts of cells, as both deficiency and surplus of metal ions can disrupt normal functions (1–4). To better understand the functions of metal ions in biology, it is important to detect metal ions selectively in living cells; such an endeavor will not only result in better understanding of cellular processes but also novel ways to reprogram these processes to achieve novel functions for biotechnological applications.Among the metal ions in cells, sodium (Na⁺) serves particularly important functions, as changes in its concentrations influence the cellular processes of numerous living organisms and cells (5–8), such as epithelial and other excitable cells (9). As one of the most abundant metal ions in intracellular fluid (10), Na⁺ affects cellular processes by triggering the activation of many signal transduction pathways, as well as influencing the actions of hormones (11). Therefore, it is important to carefully monitor the concentrations of Na⁺ in cells. Toward this goal, instrumental analyses by atomic absorption spectroscopy (12), X-ray fluorescence microscopy (13), and ²³Na NMR (14) have been used to detect the concentration of intracellular Na⁺. However, it is difficult to use these methods to obtain real-time dynamics of Na⁺ distribution in living cells. Fluorescent sensors provide an excellent choice to overcome this difficulty, as they can provide sensitive detection with high spatial and temporal resolution. However, despite significant efforts in developing fluorescent metal ion sensors, such as those based on either genetically encoded probes or small molecular sensors, most fluorescent sensors reported so far can detect divalent metal ions such as Ca²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ (15–21). Among the limited number of Na⁺ sensors, such as sodium-binding benzofuran isophthalate (22), Sodium Green (23), CoroNa Green/Red (24, 25), and Asante NaTRIUM Green-1/2 (26), most of them are not selective for Na⁺ over K⁺ (22–25, 27, 28) or have a low binding affinity for Na⁺ (with a K_d higher than 100 mM) (25, 27–31). Furthermore, the presence of organic solvents is frequently required to achieve the desired sensitivity and selectivity for many of the Na⁺ probes (32–34), making it difficult to study Na⁺ under physiological conditions. Therefore, it is still a major challenge to design fluorescent sensors with strong affinity for Na⁺ and high selectivity over other monovalent and multivalent metal ions that work under physiological conditions.To meet this challenge, our group and others have taken advantage of an emerging class of metalloenzymes called DNAzymes (deoxyribozymes or catalytic DNA) and turned them into metal ion probes. DNAzymes were first discovered in 1994 through a combinatorial process called in vitro selection (35). Since then, many DNAzymes have been isolated via this selection process. Among them, RNA-cleaving DNAzymes are of particular interest for metal ion sensing, due to their fast reaction rate and because the cleavage, which is catalyzed by a metal ion cofactor, can easily be converted into a detectable signal (36–38). Unlike the rational design of either small-molecule or genetically encoded protein sensors, DNAzymes with desired sensitivity and specificity for a metal ion of interest can be selected from a large library of DNA molecules, containing up to 10¹⁵ different sequences (35, 39). A major advantage of DNAzymes as metal ion sensors is that metal-selective DNAzymes can be obtained without prior knowledge of necessary metal ion binding sites or specific metal–DNA interaction (40, 41). In addition, through the in vitro selection process, metal ion binding affinity and selectivity can be improved by tuning the stringency of selection pressure and introducing negative selection against competing metal ions (39, 40). Finally, DNA is easily synthesized with a variety of useful modifications and its biocompatibility makes DNAzyme-based sensors excellent tools for live-cell imaging of metal ions. As a result, several metal-specific DNAzymes have been isolated and converted into sensors for their respective metal ion cofactors, including Pb²⁺ (35, 42, 43), Cu²⁺ (44, 45), Zn²⁺ (46), UO₂²⁺ (47), and Hg²⁺ (48). They have recently been delivered into cells for monitoring UO₂²⁺ (41, 49), Pb²⁺ (50), Zn²⁺ (51), and histidine (52) in living cells.However, in contrast to the previously reported DNAzymes with divalent metal ion selectivity, no DNAzymes have been reported to have high selectivity toward a specific monovalent metal ion. Although DNAzymes that are independent of divalent metal ions have been obtained (53–55), including those using modified nucleosides with protein-like functionalities (i.e., guanidinium and imidazole) (56–58), no DNAzyme has been found to be selective for a specific monovalent metal ion over other monovalent metal ions. For example, the DNAzyme with the highest reported selectivity for Na⁺ still binds Na⁺ over K⁺ with only 1.3-fold selectivity (54). More importantly, those DNAzymes require very high concentrations of monovalent ions (molar ranges) to function and display very slow catalytic rates (e.g., 10⁻³ min⁻¹) (53–55). The poor selectivity, sensitivity, and slow catalytic rate render these DNAzymes unsuitable for cellular detection of Na⁺, due to interference from other monovalent ions such as K⁺ (which is present in concentrations about 10-fold higher than Na⁺), and the need to image the Na⁺ rapidly.In this study, we report the in vitro selection and characterization of an RNA-cleaving DNAzyme with exceptionally high selectivity (>10,000-fold) for Na⁺ over other competing metal ions, with a dynamic range covering the physiological Na⁺ concentration range (0.135–50 mM) and a fast catalytic rate (k_obs, ∼0.1 min⁻¹). This Na⁺-specific DNAzyme was transformed into a DNAzyme-based fluorescent sensor for imaging intracellular Na⁺ in living cells, by adopting an efficient DNAzyme delivery method using a cationic polypeptide, together with a photocaging strategy to allow controllable activation of the probe inside cells.     

SCN5A variant that blocks fibroblast growth factor homologous factor regulation causes human arrhythmia

Na_v channels are essential for metazoan membrane depolarization, and Na_v channel dysfunction is directly linked with epilepsy, ataxia, pain, arrhythmia, myotonia, and irritable bowel syndrome. Human Na_v channelopathies are primarily caused by variants that directly affect Na_v channel permeability or gating. However, a new class of human Na_v channelopathies has emerged based on channel variants that alter regulation by intracellular signaling or cytoskeletal proteins. Fibroblast growth factor homologous factors (FHFs) are a family of intracellular signaling proteins linked with Na_v channel regulation in neurons and myocytes. However, to date, there is surprisingly little evidence linking Na_v channel gene variants with FHFs and human disease. Here, we provide, to our knowledge, the first evidence that mutations in SCN5A (encodes primary cardiac Na_v channel Na_v1.5) that alter FHF binding result in human cardiovascular disease. We describe a five*generation kindred with a history of atrial and ventricular arrhythmias, cardiac arrest, and sudden cardiac death. Affected family members harbor a novel SCN5A variant resulting in p.H1849R. p.H1849R is localized in the central binding core on Na_v1.5 for FHFs. Consistent with these data, Na_v1.5 p.H1849R affected interaction with FHFs. Further, electrophysiological analysis identified Na_v1.5 p.H1849R as a gain-of-function for I_Na by altering steady-state inactivation and slowing the rate of Na_v1.5 inactivation. In line with these data and consistent with human cardiac phenotypes, myocytes expressing Na_v1.5 p.H1849R displayed prolonged action potential duration and arrhythmogenic afterdepolarizations. Together, these findings identify a previously unexplored mechanism for human Na_v channelopathy based on altered Na_v1.5 association with FHF proteins.Encoded by 10 different genes, Na_v channel α-subunits regulate excitable membrane depolarization and are therefore central to metazoan physiology (1). Na_v channel function is critical for neuronal firing and communication (1, 2), cardiac excitation–contraction coupling (3), and skeletal and intestinal function (4, 5). The impact of Na_v channel function for human biology has been elegantly defined by nearly two decades of studies directly linking Na_v channel gene variants with human disease. To date, the field of human Na_v channelopathies has exploded to include wide spectrums of neurological [epilepsy (1), pain (6), ataxia (7)] and cardiovascular diseases (8) as well as myotonia congenital (9) and even irritable bowel syndrome (5). Though the majority of these diseases are based on gene variants in Na_v channel transmembrane segments that affect the channel pore or channel gating (10), a new paradigm for Na_v channelopathies has emerged based on variants that alter association of Na_v channels with essential regulatory proteins. To date, human Na_v channel gene variants linked with neurological and cardiovascular disease have not only provided new insight on the pathophysiology of excitable cell disease, but also identified and/or validated key in vivo Na_v channel regulatory pathways [syntrophin (11), ankyrin-G (12, 13), Na_v β1 (14), calmodulin (15), protein kinase A (16), and CaMKIIδ (17, 18)]. However, in many cases, whereas animal and cellular findings may strongly support the role of regulatory proteins for human Na_v channel function, human variants that may serve to validate the association have remained elusive, likely due to redundancy of signaling pathways or extreme severity of the disease.Identified in the retina nearly two decades ago, fibroblast growth factor homologous factors (FHFs; FGF11–14) are a family of signaling proteins with key roles in ion channel regulation (19). Distinct from canonical FGFs that are secreted and bind to extracellular FGF receptors, FGF11–14 lack signal sequences and thus regulate intracellular targets. Currently, Na_v channels are the most characterized FHF target (20–22), and recent structural data mapped the FHF binding site to the Na_v channel C terminus (23, 24). Notably, FHFs display multiple roles in Na_v channel regulation, including expression, trafficking, and channel gating. However, each FHF appears to show unique regulatory roles for Na_v channel regulation that are cell type and Na_v channel isoform dependent. Though FHF signaling is complex, the roles of FHFs in vertebrate physiology are clearly illustrated by dysfunction of FHFs in human disease. To date, FHF loci have been linked with spinocerebellar ataxia 27, X-linked mental retardation, and cardiac arrhythmia (25–27). In animals, FHF deficiency results in severe neurological phenotypes associated with altered Na_v channel function (28). Despite the overwhelming biochemical, functional, and in vivo animal data linking Na_v channels and FHF proteins, and in contrast to many other Na_v channel regulatory pathways, there are surprisingly little data linking human Na_v channel variants with FHFs in any disease.Here we provide, to our knowledge, the first evidence that human Na_v channel gene variants that alter FHF binding result in potentially fatal human disease. We describe a five-generation kindred with a history of atrial and ventricular arrhythmias, cardiac arrest, and sudden cardiac death. Genetic testing revealed a SCN5A variant, resulting in p.H1849R, in affected family members. The identified SCN5A variant p.H1849R is novel and located at a site in the Na_v1.5 C-terminal domain identified to associate with FHFs. Notably, the human p.H1849R variant markedly altered interaction with FHFs, and functional analysis of the variant identified Na_v1.5 p.H1849R as a gain-of-function variant. Further, consistent with LQT3 phenotypes observed in the family, expression of this variant resulted in prolonged action potential duration and arrhythmogenic afterdepolarizations. Together, our findings define a previously unidentified mechanism for human Na_v channelopathies based on loss of Na_v1.5 association with FHF proteins and further confirm the critical link between these intracellular proteins and Na_v channels in excitable cells.     

Catalysis of Na+ permeation in the bacterial sodium channel NaVAb

Determination of a high-resolution 3D structure of voltage-gated sodium channel Na_VAb opens the way to elucidating the mechanism of ion conductance and selectivity. To examine permeation of Na⁺ through the selectivity filter of the channel, we performed large-scale molecular dynamics simulations of Na_VAb in an explicit, hydrated lipid bilayer at 0 mV in 150 mM NaCl, for a total simulation time of 21.6 μs. Although the cytoplasmic end of the pore is closed, reversible influx and efflux of Na⁺ through the selectivity filter occurred spontaneously during simulations, leading to equilibrium movement of Na⁺ between the extracellular medium and the central cavity of the channel. Analysis of Na⁺ dynamics reveals a knock-on mechanism of ion permeation characterized by alternating occupancy of the channel by 2 and 3 Na⁺ ions, with a computed rate of translocation of (6 ± 1) × 10⁶ ions⋅s⁻¹ that is consistent with expectations from electrophysiological studies. The binding of Na⁺ is intimately coupled to conformational isomerization of the four E177 side chains lining the extracellular end of the selectivity filter. The reciprocal coordination of variable numbers of Na⁺ ions and carboxylate groups leads to their condensation into ionic clusters of variable charge and spatial arrangement. Structural fluctuations of these ionic clusters result in a myriad of ion binding modes and foster a highly degenerate, liquid-like energy landscape propitious to Na⁺ diffusion. By stabilizing multiple ionic occupancy states while helping Na⁺ ions diffuse within the selectivity filter, the conformational flexibility of E177 side chains underpins the knock-on mechanism of Na⁺ permeation.The rapid passage of cations in and out of excitable cells through selective pathways underlies the generation and regulation of electrical signals in all living organisms (1–4). The metazoan cell membrane is exposed to a high-Na⁺, low-K⁺ concentration on the extracellular (EC) side, and to a low-Na⁺, high-K⁺ concentration on the intracellular (IC) side. Selective voltage-gated Na⁺ and K⁺ channels control the response of the cell to changes in the membrane potential. In particular, voltage-gated Na⁺ channels (Na_V) are responsible for the initiation and propagation of action potentials in cardiac and skeletal myocytes, neurons, and endocrine cells (1–4). Mutations in Na_V channel genes are responsible for a wide range of debilitating channelopathies, including congenital epilepsy, paramyotonia, erythromelalgia, familial hemiplegic migraine, paroxysmal extreme pain disorder, and periodic paralyses (5, 6), underlining the importance of deciphering the relationship between the structure and function of Na_V channels. Here, we use molecular simulations to study the binding and permeation of Na⁺ in bacterial sodium channel Na_VAb.Although several atomic structures of K⁺-selective channels have been solved over the past decade (7–12), the atomic structure of an Na⁺-selective channel from the bacterium Arcobacter butzleri, Na_VAb, was reported only recently (13). In the preopen state of Na_VAb (13), the pore is closed at the IC gate, but the selectivity filter (SF) appears to be in its open, functional state. The molecular structure of the SF of Na_VAb (TLESW) differs significantly from that of potassium channels such as KcsA (TVGYG), in that it is both wider and shorter. In KcsA, channel coordination of permeating cations consists almost entirely of direct interactions with backbone carbonyl oxygen atoms. In contrast, in Na_VAb, the SF is lined with amino acid side chains from S178 and E177 in addition to backbone carbonyl groups from T175 and L176 (7, 8, 10, 13). Due to the tetrameric domain arrangement of Na_VAb, the E177 site forms a ring of four glutamate side chains (EEEE) in the same sequence positions as the characteristic DEKA ring of eukaryotic sodium channels (14, 15). The presence of charged and titratable carboxylate groups in the SF of Na_v channels raises major questions about the catalytic mechanism for ionic permeation and the structural basis for ion selectivity.As a first step toward elucidating the structural basis of ionic permeation and selectivity, we examine the movement of Na⁺ ions in and out of the pore from equilibrium molecular dynamics (MD) simulations of Na_VAb in a hydrated lipid bilayer (Fig. S1). Forty-seven time trajectories totaling 21.6 μs were generated at 300 K in the presence of 150 mM NaCl to mimic the physiological environment of the periplasm. We analyzed Na⁺ diffusion at a potential of 0 mV, similar to the peak of macroscopic Na⁺ current during an action potential or a voltage clamp experiment in nerve or muscle cells. The analysis of hundreds of spontaneous events of Na⁺ diffusion through the SF provides detailed insight into a knock-on mechanism of Na⁺ permeation involving alternating ion-occupancy states and resulting in an estimated translocation rate of (6 ± 1) × 10⁶ ions⋅s⁻¹.     

Structural basis for membrane recruitment and allosteric activation of cytohesin family Arf GTPase exchange factors

Membrane recruitment of cytohesin family Arf guanine nucleotide exchange factors depends on interactions with phosphoinositides and active Arf GTPases that, in turn, relieve autoinhibition of the catalytic Sec7 domain through an unknown structural mechanism. Here, we show that Arf6-GTP relieves autoinhibition by binding to an allosteric site that includes the autoinhibitory elements in addition to the PH domain. The crystal structure of a cytohesin-3 construct encompassing the allosteric site in complex with the head group of phosphatidyl inositol 3,4,5-trisphosphate and N-terminally truncated Arf6-GTP reveals a large conformational rearrangement, whereby autoinhibition can be relieved by competitive sequestration of the autoinhibitory elements in grooves at the Arf6/PH domain interface. Disposition of the known membrane targeting determinants on a common surface is compatible with multivalent membrane docking and subsequent activation of Arf substrates, suggesting a plausible model through which membrane recruitment and allosteric activation could be structurally integrated.Guanine nucleotide exchange factors (GEFs) activate GTPases by catalyzing exchange of GDP for GTP (1). Because many GEFs are recruited to membranes through interactions with phospholipids, active GTPases, or other membrane-associated proteins (1–5), GTPase activation can be restricted or amplified by spatial–temporal overlap of GEFs with binding partners. GEF activity can also be controlled by autoregulatory mechanisms, which may depend on membrane recruitment (6–11). Structural relationships between these mechanisms are poorly understood.Arf GTPases function in trafficking and cytoskeletal dynamics (5, 12, 13). Membrane partitioning of a myristoylated (myr) N-terminal amphipathic helix primes Arfs for activation by Sec7 domain GEFs (14–17). Cytohesins comprise a metazoan Arf GEF family that includes the mammalian proteins cytohesin-1 (Cyth1), ARNO (Cyth2), and Grp1 (Cyth3). The Drosophila homolog steppke functions in insulin-like growth factor signaling, whereas Cyth1 and Grp1 have been implicated in insulin signaling and Glut4 trafficking, respectively (18–20). Cytohesins share a modular architecture consisting of heptad repeats, a Sec7 domain with exchange activity for Arf1 and Arf6, a PH domain that binds phosphatidyl inositol (PI) polyphosphates, and a C-terminal helix (CtH) that overlaps with a polybasic region (PBR) (21–28). The overlapping CtH and PBR will be referred to as the CtH/PBR. The phosphoinositide specificity of the PH domain is influenced by alternative splicing, which generates diglycine (2G) and triglycine (3G) variants differing by insertion of a glycine residue in the β1/β2 loop (29). Despite similar PI(4,5)P₂ (PIP₂) affinities, the 2G variant has 30-fold higher affinity for PI(3,4,5)P₃ (PIP₃) (30). In both cases, PIP₃ is required for plasma membrane (PM) recruitment (23, 26, 31–33), which is promoted by expression of constitutively active Arf6 or Arl4d and impaired by PH domain mutations that disrupt PIP₃ or Arf6 binding, or by CtH/PBR mutations (8, 34–36).Cytohesins are autoinhibited by the Sec7-PH linker and CtH/PBR, which obstruct substrate binding (8). Autoinhibition can be relieved by Arf6-GTP binding in the presence of the PIP₃ head group (8). Active myr-Arf1 and myr-Arf6 also stimulate exchange activity on PIP₂-containing liposomes (37). Whether this effect is due to relief of autoinhibition per se or enhanced membrane recruitment is not yet clear. Phosphoinositide recognition by PH domains, catalysis of nucleotide exchange by Sec7 domains, and autoinhibition in cytohesins are well characterized (8, 16, 17, 30, 38–43). How Arf-GTP binding relieves autoinhibition and promotes membrane recruitment is unknown. Here, we determine the structural basis for relief of autoinhibition and investigate potential mechanistic relationships between allosteric regulation, phosphoinositide binding, and membrane targeting.     

Electrochemical tuning of vertically aligned MoS2 nanofilms and its application in improving hydrogen evolution reaction

The ability to intercalate guest species into the van der Waals gap of 2D layered materials affords the opportunity to engineer the electronic structures for a variety of applications. Here we demonstrate the continuous tuning of layer vertically aligned MoS₂ nanofilms through electrochemical intercalation of Li⁺ ions. By scanning the Li intercalation potential from high to low, we have gained control of multiple important material properties in a continuous manner, including tuning the oxidation state of Mo, the transition of semiconducting 2H to metallic 1T phase, and expanding the van der Waals gap until exfoliation. Using such nanofilms after different degree of Li intercalation, we show the significant improvement of the hydrogen evolution reaction activity. A strong correlation between such tunable material properties and hydrogen evolution reaction activity is established. This work provides an intriguing and effective approach on tuning electronic structures for optimizing the catalytic activity.Layer-structured 2D materials are an interesting family of materials with strong covalent bonding within molecular layers and weak van der Waals interaction between layers. Beyond intensively studied graphene-related materials (1–4), there has been recent strong interest in other layered materials whose vertical thickness can be thinned down to less than few nanometers and horizontal width can also be reduced to nanoscale (5–9). The strong interest is driven by their interesting physical and chemical properties (2, 10) and their potential applications in transistors, batteries, topological insulators, thermoelectrics, artificial photosynthesis, and catalysis (4, 11–25).One of the unique properties of 2D layered materials is their ability to intercalate guest species into their van der Waals gaps, opening up the opportunities to tune the properties of materials. For example, the spacing between the 2D layers could be increased by intercalation such as lithium (Li) intercalated graphite or molybdenum disulfide (MoS₂) and copper intercalated bismuth selenide (26–29). The electronic structures of the host lattice, such as the charge density, anisotropic transport, oxidation state, and phase transition, may also be changed by different species intercalation (26, 27).As one of the most interesting layered materials, MoS₂ has been extensively studied in a variety of areas such as electrocatalysis (20–22, 30–36). It is known that there is a strong correlation between the electronic structure and catalytic activity of the catalysts (20, 37–41). It is intriguing to continuously tune the morphology and electronic structure of MoS₂ and explore the effects on MoS₂ hydrogen evolution reaction (HER) activity. Very recent studies demonstrated that the monolayered MoS₂ and WS₂ nanosheets with 1T metallic phase synthesized by chemical exfoliation exhibited superior HER catalytic activity to those with 2H semiconducting phase (35, 42), with a possible explanation that the strained 1T phase facilitates the hydrogen binding process during HER (42). However, it only offers two end states of materials and does not offer a continuous tuning. A systematic investigation to correlate the gradually tuned electronic structure, including oxidation state shift and semiconducting–metallic phase transition, and the corresponding HER activity is important but unexplored. We believe that the Li electrochemical intercalation method offers a unique way to tune the catalysts for optimization.In this paper, we demonstrate that the layer spacing, oxidation state, and the ratio of 2H semiconducting to 1T metallic phase of MoS₂ HER catalysts were continuously tuned by Li intercalation to different voltages vs. Li⁺/Li in nanofilms with molecular layers perpendicular to the substrates. Correspondingly, the catalytic activity for HER was observed to be continuously tuned. The lower oxidation state of Mo and 1T metallic phase of MoS₂ turn out to have better HER catalytic activities. The performance of MoS₂ catalyst on both flat and 3D electrodes was dramatically improved when it was discharged to low potentials vs. Li⁺/Li.     

Cleavage strongly influences whether soluble HIV-1 envelope glycoprotein trimers adopt a native-like conformation

We compare the antigenicity and conformation of soluble, cleaved vs. uncleaved envelope glycoprotein (Env gp)140 trimers from the subtype A HIV type 1 (HIV-1) strain BG505. The impact of gp120–gp41 cleavage on trimer structure, in the presence or absence of trimer-stabilizing modifications (i.e., a gp120–gp41 disulfide bond and an I559P gp41 change, together designated SOSIP), was assessed. Without SOSIP changes, cleaved trimers disintegrate into their gp120 and gp41-ectodomain (gp41_ECTO) components; when only the disulfide bond is present, they dissociate into gp140 monomers. Uncleaved gp140s remain trimeric whether SOSIP substitutions are present or not. However, negative-stain electron microscopy reveals that only cleaved trimers form homogeneous structures resembling native Env spikes on virus particles. In contrast, uncleaved trimers are highly heterogeneous, adopting a variety of irregular shapes, many of which appear to be gp120 subunits dangling from a central core that is presumably a trimeric form of gp41_ECTO. Antigenicity studies with neutralizing and nonneutralizing antibodies are consistent with the EM images; cleaved, SOSIP-stabilized trimers express quaternary structure-dependent epitopes, whereas uncleaved trimers expose nonneutralizing gp120 and gp41_ECTO epitopes that are occluded on cleaved trimers. These findings have adverse implications for using soluble, uncleaved trimers for structural studies, and the rationale for testing uncleaved trimers as vaccine candidates also needs to be reevaluated.Trimeric envelope glycoprotein (Env gp) spikes on the HIV type 1 (HIV-1) surface mediate entry of the viral genome into the target cell (1, 2). When spikes interact with their cell-surface receptors, a series of conformational changes within the Env culminates in virus–cell membrane fusion. Neutralizing antibodies (NAbs) against various Env epitopes antagonize these events (2, 3). Hence, Env glycoproteins are a focus of vaccine design programs intended to induce NAbs and thereby prevent HIV-1 transmission (3, 4). Env trimers are composed of three gp120 surface glycoprotein subunits and three gp41 transmembrane glycoproteins, the six subunits all associated via noncovalent interactions (5, 6). A critical event in trimer assembly is proteolytic cleavage of the gp160 precursor into its gp120 and gp41 components, a process essential for HIV-1 entry not least because it liberates the fusion peptide (FP) at the gp41 N terminus (5, 6).Trimer-based vaccine strategies involve expressing soluble, recombinant versions of the virion-associated (i.e., native) spikes. To facilitate production and purification, the membrane-spanning and cytoplasmic domains that anchor spikes to the virion, but that are not NAb targets, are eliminated (7–12). However, the resulting proteins, known as gp140s, are highly unstable and disintegrate into their gp120 and gp41-ectodomain (gp41_ECTO) components, making them useless as immunogens. Two fundamentally different protein-engineering strategies have been used to create gp140s that can be produced and purified without falling apart (3, 4, 7–17). The most common method involves eliminating the cleavage site between gp120 and gp41_ECTO, creating uncleaved gp140s (gp140_UNC) where the two subunits remain covalently linked (7–12). Additional trimerization motifs are often added to the gp41_ECTO C terminus (10–12). Our alternative approach is based on the premise that cleavage is a fundamental feature of Env structure and involves stabilizing fully cleaved gp140s. The critical changes are an appropriately positioned disulfide bond (referred to as “SOS”) to link gp120 to gp41_ECTO covalently, and an Ile/Pro (IP) substitution at residue 559 to strengthen inter-gp41_ECTO interactions (13–17). The resulting cleaved trimers are designated SOSIP gp140s (14). Additional modifications have improved their stability, homogeneity, and antigenicity (15–17). Our current design, based on the BG505 subtype A env gene, yields SOSIP.664 trimers that mimic native, virion-associated Env spikes antigenically and when viewed by negative-stain electron microscopy (EM) (17–19).Here we show that cleavage is essential for producing stable, soluble gp140 trimers that resemble native Env spikes. EM studies reveal that purified, trimeric gp140_UNC proteins are heterogeneous and that the irregularly shaped images rarely resemble a native spike; we refer to them as “aberrant configurations” (ACs). In contrast, cleaved SOSIP gp140 trimers are homogeneous and mimic native spikes; we designate them native-like (NL) trimers. The antigenic properties of the cleaved (NL) and uncleaved (AC) trimers, assessed by surface plasmon resonance (SPR) and enzyme-linked immunoabsorbance assays (ELISA), are consistent with the EM images. Nonneutralizing gp120 and gp41_ECTO epitopes are exposed on gp140_UNC trimers but occluded on cleaved ones, whereas quaternary structure-dependent epitopes indicative of proper folding are present only on cleaved trimers. Our findings have substantial implications, because uncleaved trimers are being studied structurally and developed as vaccine candidates (3, 9, 10, 12, 20).     

Transition paths,diffusive processes,and preequilibria of protein folding

Fundamental relationships between the thermodynamics and kinetics of protein folding were investigated using chain models of natural proteins with diverse folding rates by extensive comparisons between the distribution of conformations in thermodynamic equilibrium and the distribution of conformations sampled along folding trajectories. Consistent with theory and single-molecule experiment, duration of the folding transition paths exhibits only a weak correlation with overall folding time. Conformational distributions of folding trajectories near the overall thermodynamic folding/unfolding barrier show significant deviations from preequilibrium. These deviations, the distribution of transition path times, and the variation of mean transition path time for different proteins can all be rationalized by a diffusive process that we modeled using simple Monte Carlo algorithms with an effective coordinate-independent diffusion coefficient. Conformations in the initial stages of transition paths tend to form more nonlocal contacts than typical conformations with the same number of native contacts. This statistical bias, which is indicative of preferred folding pathways, should be amenable to future single-molecule measurements. We found that the preexponential factor defined in the transition state theory of folding varies from protein to protein and that this variation can be rationalized by our Monte Carlo diffusion model. Thus, protein folding physics is different in certain fundamental respects from the physics envisioned by a simple transition-state picture. Nonetheless, transition state theory can be a useful approximate predictor of cooperative folding speed, because the height of the overall folding barrier is apparently a proxy for related rate-determining physical properties.Protein folding is an intriguing phenomenon at the interface of physics and biology. In the early days of folding kinetics studies, folding was formulated almost exclusively in terms of mass-action rate equations connecting the folded, unfolded, and possibly, one or a few intermediate states (1, 2). With the advent of site-directed mutagenesis, the concept of free energy barriers from transition state theory (TST) (3) was introduced to interpret mutational data (4), and subsequently, it was adopted for the Φ-value analysis (5). Since the 1990s, the availability of more detailed experimental data (6), in conjunction with computational development of coarse-grained chain models, has led to an energy landscape picture of folding (7–15). This perspective emphasizes the diversity of microscopic folding trajectories, and it conceptualizes folding as a diffusive process (16–25) akin to the theory of Kramers (26).For two-state-like folding, the transition path (TP), i.e., the sequence of kinetic events that leads directly from the unfolded state to the folded state (27, 28), constitutes only a tiny fraction of a folding trajectory that spends most of the time diffusing, seemingly unproductively, in the vicinity of the free energy minimum of the unfolded state. The development of ultrafast laser spectroscopy (29, 30) and single-molecule (27, 28, 31) techniques have made it possible to establish upper bounds on the transition path time (t_TP) ranging from <200 and <10 μs by earlier (27) and more recent (28), respectively, direct single-molecule FRET to <2 μs (30) by bulk relaxation measurements. Consistent with these observations, recent extensive atomic simulations have also provided estimated t_TP values of the order of ∼1 μs (32, 33). These advances offer exciting prospects of characterizing the productive events along folding TPs.It is timely, therefore, to further the theoretical investigation of TP-related questions (19). To this end, we used coarse-grained C_α models (14) to perform extensive simulations of the folding trajectories of small proteins with 56- to 86-aa residues. These tractable models are useful, because despite significant progress, current atomic models cannot provide the same degree of sampling coverage for proteins of comparable sizes (32, 33). In addition to structural insights, this study provides previously unexplored vantage points to compare the diffusion and TST pictures of folding. Deviations of folding behaviors from TST predictions are not unexpected, because TST is mostly applicable to simple gas reactions; however, the nature and extent of the deviations have not been much explored. Our explicit-chain simulation data conform well to the diffusion picture but not as well to TST. In particular, the preexponential factors of the simulated folding rates exhibit a small but appreciable variation that depends on native topology. These findings and others reported below underscore the importance of single-molecule measurements (13, 27, 28, 31, 34, 35) in assessing the merits of proposed scenarios and organizing principles of folding (7–25, 36, 37).     

Tumor-derived hydrogen sulfide,produced by cystathionine-β-synthase,stimulates bioenergetics,cell proliferation,and angiogenesis in colon cancer

The physiological functions of hydrogen sulfide (H₂S) include vasorelaxation, stimulation of cellular bioenergetics, and promotion of angiogenesis. Analysis of human colon cancer biopsies and patient-matched normal margin mucosa revealed the selective up-regulation of the H₂S-producing enzyme cystathionine-β-synthase (CBS) in colon cancer, resulting in an increased rate of H₂S production. Similarly, colon cancer-derived epithelial cell lines (HCT116, HT-29, LoVo) exhibited selective CBS up-regulation and increased H₂S production, compared with the nonmalignant colonic mucosa cells, NCM356. CBS localized to the cytosol, as well as the mitochondrial outer membrane. ShRNA-mediated silencing of CBS or its pharmacological inhibition with aminooxyacetic acid reduced HCT116 cell proliferation, migration, and invasion; reduced endothelial cell migration in tumor/endothelial cell cocultures; and suppressed mitochondrial function (oxygen consumption, ATP turnover, and respiratory reserve capacity), as well as glycolysis. Treatment of nude mice with aminooxyacetic acid attenuated the growth of patient-derived colon cancer xenografts and reduced tumor blood flow. Similarly, CBS silencing of the tumor cells decreased xenograft growth and suppressed neovessel density, suggesting a role for endogenous H₂S in tumor angiogenesis. In contrast to CBS, silencing of cystathionine-γ-lyase (the expression of which was unchanged in colon cancer) did not affect tumor growth or bioenergetics. In conclusion, H₂S produced from CBS serves to (i) maintain colon cancer cellular bioenergetics, thereby supporting tumor growth and proliferation, and (ii) promote angiogenesis and vasorelaxation, consequently providing the tumor with blood and nutritients. The current findings identify CBS-derived H₂S as a tumor growth factor and anticancer drug target.The endogenous gasotransmitter hydrogen sulfide (H₂S) is a stimulator of vasorelaxation (1–3), angiogenesis (3–5), and cellular bioenergetics (6, 7). H₂S is generated from l-cysteine by two pyridoxal-5′-phospate–dependent enzymes, cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE), and by the combined action of cysteine aminotransferase and 3-mercaptopyruvate sulfurtransferase (3-MST) (8–10). H₂S exerts its cellular actions via multiple mechanisms (1–15), including activation of potassium channels (1–3), stimulation of kinase pathways (4, 11, 12), and inhibition of phosphodiesterases (3, 15).Both ATP generation and angiogenesis are vital factors for the growth and proliferation of tumors (16–19). Using human colon cancer tissues and cancer-derived cell lines, we have now conducted a series of in vitro and in vivo studies to explore whether endogenous, tumor cell-derived H₂S plays a role as a tumor-derived survival factor. The results show that CBS is selectively overexpressed in colon cancer, and that H₂S produced by it serves to maintain the tumor''s cellular bioenergetics and to promote tumor angiogenesis.     

Allosteric activation of M4 muscarinic receptors improve behavioral and physiological alterations in early symptomatic YAC128 mice

Mutations that lead to Huntington’s disease (HD) result in increased transmission at glutamatergic corticostriatal synapses at early presymptomatic stages that have been postulated to set the stage for pathological changes and symptoms that are observed at later ages. Based on this, pharmacological interventions that reverse excessive corticostriatal transmission may provide a novel approach for reducing early physiological changes and motor symptoms observed in HD. We report that activation of the M₄ subtype of muscarinic acetylcholine receptor reduces transmission at corticostriatal synapses and that this effect is dramatically enhanced in presymptomatic YAC128 HD and BACHD relative to wild-type mice. Furthermore, chronic administration of a novel highly selective M₄ positive allosteric modulator (PAM) beginning at presymptomatic ages improves motor and synaptic deficits in 5-mo-old YAC128 mice. These data raise the exciting possibility that selective M₄ PAMs could provide a therapeutic strategy for the treatment of HD.Huntington’s disease (HD) is a rare and fatal neurodegenerative disease caused by an expansion of a CAG triplet repeat in Htt, the gene that encodes for the protein huntingtin (1, 2). HD is characterized by a prediagnostic phase that includes subtle changes in personality, cognition, and motor function, followed by a more severe symptomatic stage initially characterized by hyperkinesia (chorea), motor incoordination, deterioration of cognitive abilities, and psychiatric symptoms. At later stages of disease progression, patients experience dystonia, rigidity, and bradykinesia, and ultimately death (3–7). The cortex and striatum are the most severely affected brain regions in HD and, interestingly, an increasing number of reports suggest that alterations in cortical and striatal physiology are present in prediagnostic individuals and in young HD mice (6–16).Striatal spiny projection neurons (SPNs) receive large glutamatergic inputs from the cortex and thalamus, as well as dopaminergic innervation from the substantia nigra. In the healthy striatum, the interplay of these neurotransmitters coordinates the activity of SPNs and striatal interneurons, regulating motor planning and execution as well as cognition and motivation (17, 18). Htt mutations lead to an early increase in striatal glutamatergic transmission, which begins during the asymptomatic phase of HD (12–14) and could contribute to synaptic changes observed in later stages of HD (19, 20). Based on this, pharmacological agents that reduce excitatory transmission in the striatum could reduce or prevent the progression of alterations in striatal synaptic function and behavior observed in symptomatic stages of HD.Muscarinic acetylcholine receptors (mAChRs), particularly M₄, can inhibit transmission at corticostriatal synapses (21–25). Therefore, it is possible that selective activation of specific mAChR subtypes could normalize excessive corticostriatal transmission in HD. Interestingly, previous studies also suggest that HD is associated with alterations of striatal cholinergic markers, including mAChRs (26–29). We now provide exciting new evidence that M₄-mediated control of corticostriatal transmission is increased in young asymptomatic HD mice and that M₄ positive allosteric modulators (PAMs) may represent a new treatment strategy for normalizing early changes in corticostriatal transmission and reducing the progression of HD.