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.     

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).     

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.     

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.     

Discovery of a selective NaV1.7 inhibitor from centipede venom with analgesic efficacy exceeding morphine in rodent pain models

Loss-of-function mutations in the human voltage-gated sodium channel Na_V1.7 result in a congenital indifference to pain. Selective inhibitors of Na_V1.7 are therefore likely to be powerful analgesics for treating a broad range of pain conditions. Herein we describe the identification of µ-SLPTX-Ssm6a, a unique 46-residue peptide from centipede venom that potently inhibits Na_V1.7 with an IC₅₀ of ∼25 nM. µ-SLPTX-Ssm6a has more than 150-fold selectivity for Na_V1.7 over all other human Na_V subtypes, with the exception of Na_V1.2, for which the selectivity is 32-fold. µ-SLPTX-Ssm6a contains three disulfide bonds with a unique connectivity pattern, and it has no significant sequence homology with any previously characterized peptide or protein. µ-SLPTX-Ssm6a proved to be a more potent analgesic than morphine in a rodent model of chemical-induced pain, and it was equipotent with morphine in rodent models of thermal and acid-induced pain. This study establishes µ-SPTX-Ssm6a as a promising lead molecule for the development of novel analgesics targeting Na_V1.7, which might be suitable for treating a wide range of human pain pathologies.Normal pain is a key adaptive response that serves to limit our exposure to potentially damaging or life-threatening events. In contrast, aberrant long-lasting pain transforms this adaptive response into a debilitating and often poorly managed disease. Chronic pain affects ∼20% of the population, with the incidence rising significantly in elderly cohorts (1). The economic burden of chronic pain in the United States was recently estimated to be ∼$600 billion per annum, which exceeds the combined annual cost of cancer, heart disease, and diabetes (2). There are few drugs available for treatment of chronic pain, and many of these have limited efficacy and dose-limiting side-effects.Voltage-gated sodium (Na_V) channels are integral transmembrane proteins that provide a current pathway for the rapid depolarization of excitable cells (1, 3), and they play a key role in conveying nociceptor responses to synapses in the dorsal horn (4). Humans contain nine different Na_V channel subtypes, denoted Na_V1.1 to Na_V1.9 (5, 6). In recent years, Na_V1.7 has emerged as a promising analgesic target based on several remarkable human genetic studies. Gain-of-function mutations in the SNC9A gene encoding the pore-forming α-subunit of Na_V1.7 cause severe episodic pain in inherited neuropathies, such as erythromelalgia and paroxysmal extreme pain disorder (7), whereas loss-of-function mutations in SCN9A result in a congenital indifference to pain (CIP) (8). The latter phenotype can be recapitulated in rodents via complete knockout of Na_V1.7 in all sensory and sympathetic neurons (9). Moreover, certain polymorphisms in SCN9A correlate with sensitivity to nociceptive inputs (10). Remarkably, apart from their inability to sense pain, loss of smell (anosmia) is the only other sensory impairment in individuals with CIP (11, 12). Thus, the combined genetic data suggest that subtype-selective blockers of Na_V1.7 are likely to be useful analgesics for treating a broad range of pain conditions.Centipedes are one of the oldest extant arthropods, with the fossil record dating back 430 million y (13). Centipedes were one of the first terrestrial taxa to use venom as a predation strategy, and they have adapted to capture a wide variety of prey, including insects, fish, molluscs, amphibians, reptiles, and even mammals (13, 14). The centipede venom apparatus, which is unique and bears little resemblance to that of other arthropods, evolved by modification of the first pair of walking legs into a set of pincer-like claws (forcipules) (13). Venom is secreted via a pore located near the tip of each forcipule. There are ∼3,300 extant species of centipedes, yet the venom of only a handful has been studied in any detail. We recently demonstrated that the venom of the Chinese red-headed centipede Scolopendra subspinipes mutilans is replete with unique, disulfide-rich peptides that potently modulate the activity of mammalian voltage-gated ion channels (14), and therefore we decided to explore this venom as a potential source of Na_V1.7 inhibitors. We describe the purification from this venom of a highly selective inhibitor of Na_V1.7 that is a more effective analgesic than morphine in rodent pain models.     

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.     

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.     

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.     

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.     

Altered thalamocortical rhythmicity and connectivity in mice lacking CaV3.1 T-type Ca2+ channels in unconsciousness

In unconscious status (e.g., deep sleep and anesthetic unconsciousness) where cognitive functions are not generated there is still a significant level of brain activity present. Indeed, the electrophysiology of the unconscious brain is characterized by well-defined thalamocortical rhythmicity. Here we address the ionic basis for such thalamocortical rhythms during unconsciousness. In particular, we address the role of Ca_V3.1 T-type Ca²⁺ channels, which are richly expressed in thalamic neurons. Toward this aim, we examined the electrophysiological and behavioral phenotypes of mice lacking Ca_V3.1 channels (Ca_V3.1 knockout) during unconsciousness induced by ketamine or ethanol administration. Our findings indicate that Ca_V3.1 KO mice displayed attenuated low-frequency oscillations in thalamocortical loops, especially in the 1- to 4-Hz delta band, compared with control mice (Ca_V3.1 WT). Intriguingly, we also found that Ca_V3.1 KO mice exhibited augmented high-frequency oscillations during unconsciousness. In a behavioral measure of unconsciousness dynamics, Ca_V3.1 KO mice took longer to fall into the unconscious state than controls. In addition, such unconscious events had a shorter duration than those of control mice. The thalamocortical interaction level between mediodorsal thalamus and frontal cortex in Ca_V3.1 KO mice was significantly lower, especially for delta band oscillations, compared with that of Ca_V3.1 WT mice, during unconsciousness. These results suggest that the Ca_V3.1 channel is required for the generation of a given set of thalamocortical rhythms during unconsciousness. Further, that thalamocortical resonant neuronal activity supported by this channel is important for the control of vigilance states.Thalamocortical interactive rhythmic activities are well-defined physiological correlates of both conscious and unconscious conditions (1, 2). From a functional perspective, abnormal slow cortical rhythms and their synchronized network dynamics are omnipresent correlates of unconscious states, such as coma and general anesthesia (3, 4). Moreover, a dynamic alteration of coherence as well as coupling/uncoupling in thalamocortical circuits also can be characterized as likely correlates of unconsciousness (3–5).Since the discovery of low threshold, T-type Ca²⁺ channels (6, 7) and the subsequent studies of intrinsic electrophysiological properties in the thalamic neurons (8, 9), T-type Ca²⁺ channels have been implicated in many physiological and pathological brain states (for a review, see ref. 10). The ionic conductances they support have been shown to generate synchronized oscillatory activity in thalamocortical circuits through calcium-dependent low-threshold spikes (LTSs). Indeed, these LTSs, generated by “deinactivation” of T-type Ca²⁺ channels, underlie thalamic burst firing. This activity is reflected as high-amplitude low-frequency oscillations in electroencephalography, and its presence is recognized as spike-wave-discharges, low-frequency rhythms (<1 Hz slow, delta and theta rhythms), as well as by spindle-generated rhythmicity (10).Recent molecular genetic studies coupled with electrophysiological and behavioral approaches confirmed the classical view that Ca_V3.1 channels play a central role in the generation of thalamocortical rhythms, such as 3- to 4-Hz spike-wave discharge during absence seizures (11, 12). Regarding the role of Ca_V3.1 in slow wave sleep, however, mice with such genetic deletions present electrophysiological consequences that are inconsistent with the above generalization, even with behavioral phenotypes exhibiting fragmented sleep. Indeed, mice with a global Ca_V3.1 deletion showed reduced delta rhythm (13) in contrast to the increased delta rhythms found in mice with thalamus-restricted deletion of Ca_V3.1 (14). In addition, there is clear evidence that thalamic T-type Ca²⁺ channels support the generation of spindle oscillations (15). However, recently published work proposes that sleep spindles are sustained in mice lacking Ca_V3.1 channels (16). These results differ from the classical view and raise the need to examine further the role of thalamic Ca_V3.1 channels in the generation of thalamocortical rhythms.Here we addressed the issue of whether Ca_V3.1 channels are important for the generation of low-frequency thalamocortical rhythms during unconsciousness. Spectral analysis of EEG recordings from Ca_V3.1 KO mice indicates a shift away from low frequency, with an increase probability toward the high-frequency rhythmic components. There is also a significant alteration of thalamocortical dynamic interactions.     

T-type channel blockade impairs long-term potentiation at the parallel fiber–Purkinje cell synapse and cerebellar learning

Ca_V3.1 T-type channels are abundant at the cerebellar synapse between parallel fibers and Purkinje cells where they contribute to synaptic depolarization. So far, no specific physiological function has been attributed to these channels neither as charge carriers nor more specifically as Ca²⁺ carriers. Here we analyze their incidence on synaptic plasticity, motor behavior, and cerebellar motor learning, comparing WT animals and mice where T-type channel function has been abolished either by gene deletion or by acute pharmacological blockade. At the cellular level, we show that Ca_V3.1 channels are required for long-term potentiation at parallel fiber–Purkinje cell synapses. Moreover, basal simple spike discharge of the Purkinje cell in KO mice is modified. Acute or chronic T-type current blockade results in impaired motor performance in particular when a good body balance is required. Because motor behavior integrates reflexes and past memories of learned behavior, this suggests impaired learning. Indeed, subjecting the KO mice to a vestibulo-ocular reflex phase reversal test reveals impaired cerebellum-dependent motor learning. These data identify a role of low-voltage activated calcium channels in synaptic plasticity and establish a role for Ca_V3.1 channels in cerebellar learning.Neurotransmission at the parallel fiber (PF) and Purkinje cell (PC) synapse plays a pivotal role in cerebellar motor learning probably involving bidirectional changes of its strength (1–3). Unlike in the hippocampus, postsynaptic Ca²⁺ signaling at PF–PC spines may not be dominated by ionotropic glutamatergic receptors, as postsynaptic N-methyl-D-aspartate receptors (NMDARs) are not prominently present at this site and AMPA receptors are predominantly impermeable for calcium ions (4, 5). PCs bear different voltage-dependent Ca channels including P/Q-type (6–8) and T-type channels (9, 10). The spines of PCs contain a high density of Ca_V3.1 T-type channels (11), which can be readily activated by typical bursts of PF activity that occur during sensory stimulation (12–14). To date, the function of the PF to PC synapse plays a pivotal role in cerebellar motor learning, probably involving bidirectional changes of its strength (1–3). Unlike in the hippocampus, T-type channels during PF–PC plasticity induction and cerebellar learning has not been explored.In cerebellar PCs, the elevation of Ca²⁺ in the spine has been suggested to control directly the sign of the changes in synaptic weights (15). Long-term depression (LTD) induction requires conjunctive stimulation of the climbing fibers (CFs) and PFs, which triggers a large supralinear calcium entry mediated by mGluR1, inositol triphosphate (IP3) receptors and voltage-gated calcium channels (16–19). In contrast, long-term potentiation (LTP) develops after PF stimulation only and requires a moderate [Ca²⁺]_i elevation (15). Here, we evaluated the hypothesis that Ca_V3.1 T-type channel activation is essential for LTP and LTP-dependent motor learning.We first looked at PF–PC plasticity of T-type channel blockade/deletion, and then investigated both in vitro and in vivo the dynamics of PC activity as well as the motor behavior of both wild-type and Ca_V3.1 KO mice. Because, in our experiments, motor behavior appears to be impaired in tests requiring a refined body balance, we have analyzed vestibulo-ocular reflex (VOR) adaptation, a learning paradigm more specifically dependent on vestibulo-cerebellar function. We show all three processes to be impaired after T-type channel functional inactivation. We propose that T-type calcium channels contribute to the definition of the learning rules in the cerebellar cortex.     

RING finger protein 121 facilitates the degradation and membrane localization of voltage-gated sodium channels

Following their synthesis in the endoplasmic reticulum (ER), voltage-gated sodium channels (Na_V) are transported to the membranes of excitable cells, where they often cluster, such as at the axon initial segment of neurons. Although the mechanisms by which Na_V channels form and maintain clusters have been extensively examined, the processes that govern their transport and degradation have received less attention. Our entry into the study of these processes began with the isolation of a new allele of the zebrafish mutant alligator, which we found to be caused by mutations in the gene encoding really interesting new gene (RING) finger protein 121 (RNF121), an E3-ubiquitin ligase present in the ER and cis-Golgi compartments. Here we demonstrate that RNF121 facilitates two opposing fates of Na_V channels: (i) ubiquitin-mediated proteasome degradation and (ii) membrane localization when coexpressed with auxiliary Na_Vβ subunits. Collectively, these results indicate that RNF121 participates in the quality control of Na_V channels during their synthesis and subsequent transport to the membrane.Voltage-gated sodium channels (Na_V) are large (∼230 kDa) multipass transmembrane proteins (1). The Na_V channel family is comprised of nine members (Na_V1.1–Na_V1.9), whose activity typically underlies the rising phase of action potentials in excitable cells. In excitable cells, Na_V channels form complexes with auxiliary β subunits (Na_Vβ_1–4) in the Golgi apparatus (2), a process that enhances the kinetics and membrane localization of Na_V channels (3, 4). In addition to these roles, several Na_Vβ subunits also function as cell adhesion molecules independent of Na_V channels (5). At the axon initial segment (AIS) and nodes of Ranvier of neurons, Na_V channels form clusters that facilitate the generation and propagation of action potentials. Although the molecular basis of Na_V clustering at these sites has been extensively studied (6), the transport of Na_V channels to these sites has been less explored. For instance, to date, only the annexin II light chain (p11) has been shown to associate with and facilitate the transport of Na_V1.8 to the plasma membrane (7). Furthermore, subsequent efforts revealed that p11 acts only on Na_V1.8 (8). Thus, the transport of other Na_V channels remains unclear.In zebrafish, several studies have explored the contribution of Na_V channels and their auxiliary Na_Vβ subunits through the use of forward and reverse genetics. In brief, impairments in Na_V1.1, Na_V1.6a, and Na_Vβ_1b have been shown to diminish touch-evoked escape responses and Na_V channel activity in Rohon–Beard (RB) sensory neurons (9–11). In addition, two other mutants identified in forward genetic screens have been shown to affect Na_V channel activity indirectly. The first, pigu, arises from a mutation in a GPI-transamidase necessary for the proper localization of Na_V channels (12). Although the genetic locus of the second mutation, macho (13, 14), has yet to be identified, rough mapping indicates that it lies within a region lacking both Na_V channels and auxiliary Na_Vβ subunits. Collectively, these results indicate that the characterization of touch-unresponsive zebrafish mutants is an efficient strategy to gain insight into the trafficking and function of Na_V channels.In this study, we identified a touch-unresponsive zebrafish mutant (mi500), which was found to be a new allele of the molecularly unidentified motor mutant alligator (13). Electrophysiological analysis revealed that Na_V channel activity was severely diminished throughout the sensorimotor circuit in mutants. Further characterization uncovered that Na_V channels were not localized at the AIS in mutant RBs, but instead seem to be accumulated within the endoplasmic reticulum (ER) and cis-Golgi compartments. Meiotic mapping and sequence analysis showed that the alligator locus encodes really interesting new gene (RING) finger protein 121 (RNF121), an ER- and cis-Golgi–resident E3-ubiquitin ligase that mediates the ubiquitination of Na_V1.6. We found that RNF121 promotes the degradation and membrane transport of Na_V1.6. Furthermore, overexpression of Na_V1.6 worsened the touch response in rnf121-knockdown larvae, suggesting that an excess amount of Na_V exerts proteotoxicity. These findings suggest that the proper transport of Na_V channels is attributable to RNF121-mediated quality control of Na_V channels within the ER and Golgi apparatus.     

Individual IKs channels at the surface of mammalian cells contain two KCNE1 accessory subunits

KCNE1 (E1) β-subunits assemble with KCNQ1 (Q1) voltage-gated K⁺ channel α-subunits to form I_Kslow (I_Ks) channels in the heart and ear. The number of E1 subunits in I_Ks channels has been an issue of ongoing debate. Here, we use single-molecule spectroscopy to demonstrate that surface I_Ks channels with human subunits contain two E1 and four Q1 subunits. This stoichiometry does not vary. Thus, I_Ks channels in cells with elevated levels of E1 carry no more than two E1 subunits. Cells with low levels of E1 produce I_Ks channels with two E1 subunits and Q1 channels with no E1 subunits—channels with one E1 do not appear to form or are restricted from surface expression. The plethora of models of cardiac function, transgenic animals, and drug screens based on variable E1 stoichiometry do not reflect physiology.Voltage-gated potassium (K_V) channels include four α-subunits that form a single, central ion conduction pathway with four peripheral voltage sensors (1–3). Incorporation of accessory β-subunits modifies the function of K_V channels to suit the diverse requirements of different tissues. KCNE genes encode minK-related peptides (MiRPs) (4–6), β-subunits with a single transmembrane span that assemble with a wide array of K_V α-subunits (7, 8) to control surface expression, voltage dependence, and kinetics of gating transitions, unitary conductance, ion selectivity, and pharmacology of the resultant channel complexes (4, 9–15). I_Kslow (I_Ks) channels in the heart and inner ear are formed by the α-subunit encoded by KCNQ1 (called Q1, K_VLQT1, K_V7.1, or KCNQ1) and the β-subunit encoded by KCNE1 (called E1, mink, or KCNE1) (16, 17). Inherited mutations in Q1 and E1 are associated with cardiac arrhythmia and deafness.The number of E1 subunits in I_Ks channels has been a longstanding matter of disagreement. We first argued for two E1 subunits per channel based on the suppression of current by an E1 mutant (18). Subsequently, we reached the same conclusion by determining the total number of channels using radiolabeled charybdotoxin (CTX), a scorpion toxin that blocks channels when one molecule binds in the external conduction pore vestibule, and an antibody-based luminescence assay to tally E1 subunits (19). Morin and Kobertz (20) used iterative chemical linkage between CTX in the pore and E1, and they also assigned two accessory subunits to >95% of I_Ks channels without gathering evidence for variation in subunit valence. Furthermore, when we formed I_Ks channels from separate E1 and Q1 subunits and compared them with channels enforced via genetic encoding to contain two or four E1 subunits (19), we observed the natural I_Ks channels to have the same gating attributes, small-molecule pharmacology, and CTX on and off rates (a reflection of pore vestibule structure) as channels encoded with two E1 subunits but not those with four. These findings support the conclusion that two E1 subunits are necessary, sufficient, and the normal number in I_Ks channels.In contrast, others have argued that I_Ks channels have variable stoichiometry with one to four E1 subunits, or even more (21–24). Recently, Nakajo et al. (25) applied single-particle spectroscopy to the question; this powerful “gold-standard” tool has been a valuable strategy to assess the subunit composition of ion channels (26–28) and should be expected to improve on prior investigations conducted on populations of I_Ks channels and subject, therefore, to the simplifying assumptions that attend macroscopic studies (29). Nakajo et al. (25) reported a variable number of E1 subunits, from one to four, in I_Ks channels studied in Xenopus laevis oocytes. The impact of this result has been striking because it has engendered new models of cardiac physiology, altered models of I_Ks channel biosynthesis and function, stimulated the use of transgenic animals artificially enforced to express I_Ks channels with four E1 subunits (by expression of a fused E1–Q1 subunit), and prompted cardiac drug design based on the assumption that I_Ks channels can form with one E1 subunit (23, 30–32).We were concerned that the conclusions of Nakajo et al. (25) were in error because they appraised only a limited fraction of particles that were immobile in the oocyte membrane; counted E1 and Q1 asynchronously rather than simultaneously (increasing the risk that particles moved into or out of the field of view); and studied Q1 and E1 appended not only with the fluorescent proteins (FP) required to count subunits by photobleaching but also with a common trafficking motif that suppressed channel mobility by interacting with an overexpressed anchoring protein, thereby risking nonnatural aggregation of subunits.Here, to resolve mobility problems and obviate the need for modification of subunits with targeting motifs, we describe and perform single-fluorescent-particle photobleaching at the surface of live mammalian cells, demonstrating three spectroscopic counting approaches: standard, asynchronous subunit counting; simultaneous, two-color subunit counting; and toxin-directed, simultaneous, two-color photobleaching. To analyze the data, we use two statistical approaches—one to assess the degree of colocalization of objects in dual-color images (33) and the other to infer stoichiometry from single-molecule photobleaching (34). These methods also allow determination of the surface density of assemblies of defined subunit composition and are therefore useful to assess the formation and life cycle of membrane protein complexes.We report that single I_Ks channels at the surface of mammalian cells contain two E1 subunits—no more and no less. This finding refutes the single-particle studies of Nakajo et al. (25) in oocytes and macroscopic studies (21–24, 30–32), arguing that forcing cells to express excess E1 produces I_Ks channels containing more than two E1 subunits and that low levels of E1 yields I_Ks channels with less than two E1 subunits. Not once did we observe an I_Ks channel with three or four E1 subunits. Moreover, simultaneous, two-color subunit counting revealed that low amounts of E1 relative to Q1 [ratios like those reported in human cardiac ventricle (35, 36)] produced two types of channels on the cell surface: I_Ks channels (with two E1 subunits) and Q1 channels (with no E1 subunits). Finally, E1 was shown to increase in I_Ks channel surface expression threefold, as we predicted based on assessment of I_Ks channel unitary conductance (11), whereas few E1 subunits were on the surface outside of I_Ks channels, even when E1 was expressed alone. This finding indicates that E1 does not travel to the surface readily on its own, that two E1 subunits facilitate I_Ks channel trafficking to the surface (or enhance surface residence time compared with Q1 channels), and that I_Ks channels with only one E1 subunit do not form, do not reach the surface, or are rapidly recycled.     

Structural interactions of a voltage sensor toxin with lipid membranes

Protein toxins from tarantula venom alter the activity of diverse ion channel proteins, including voltage, stretch, and ligand-activated cation channels. Although tarantula toxins have been shown to partition into membranes, and the membrane is thought to play an important role in their activity, the structural interactions between these toxins and lipid membranes are poorly understood. Here, we use solid-state NMR and neutron diffraction to investigate the interactions between a voltage sensor toxin (VSTx1) and lipid membranes, with the goal of localizing the toxin in the membrane and determining its influence on membrane structure. Our results demonstrate that VSTx1 localizes to the headgroup region of lipid membranes and produces a thinning of the bilayer. The toxin orients such that many basic residues are in the aqueous phase, all three Trp residues adopt interfacial positions, and several hydrophobic residues are within the membrane interior. One remarkable feature of this preferred orientation is that the surface of the toxin that mediates binding to voltage sensors is ideally positioned within the lipid bilayer to favor complex formation between the toxin and the voltage sensor.Protein toxins from venomous organisms have been invaluable tools for studying the ion channel proteins they target. For example, in the case of voltage-activated potassium (Kv) channels, pore-blocking scorpion toxins were used to identify the pore-forming region of the channel (1, 2), and gating modifier tarantula toxins that bind to S1–S4 voltage-sensing domains have helped to identify structural motifs that move at the protein–lipid interface (3–5). In many instances, these toxin–channel interactions are highly specific, allowing them to be used in target validation and drug development (6–8).Tarantula toxins are a particularly interesting class of protein toxins that have been found to target all three families of voltage-activated cation channels (3, 9–12), stretch-activated cation channels (13–15), as well as ligand-gated ion channels as diverse as acid-sensing ion channels (ASIC) (16–21) and transient receptor potential (TRP) channels (22, 23). The tarantula toxins targeting these ion channels belong to the inhibitor cystine knot (ICK) family of venom toxins that are stabilized by three disulfide bonds at the core of the molecule (16, 17, 24–31). Although conventional tarantula toxins vary in length from 30 to 40 aa and contain one ICK motif, the recently discovered double-knot toxin (DkTx) that specifically targets TRPV1 channels contains two separable lobes, each containing its own ICK motif (22, 23).One unifying feature of all tarantula toxins studied thus far is that they act on ion channels by modifying the gating properties of the channel. The best studied of these are the tarantula toxins targeting voltage-activated cation channels, where the toxins bind to the S3b–S4 voltage sensor paddle motif (5, 32–36), a helix-turn-helix motif within S1–S4 voltage-sensing domains that moves in response to changes in membrane voltage (37–41). Toxins binding to S3b–S4 motifs can influence voltage sensor activation, opening and closing of the pore, or the process of inactivation (4, 5, 36, 42–46). The tarantula toxin PcTx1 can promote opening of ASIC channels at neutral pH (16, 18), and DkTx opens TRPV1 in the absence of other stimuli (22, 23), suggesting that these toxin stabilize open states of their target channels.For many of these tarantula toxins, the lipid membrane plays a key role in the mechanism of inhibition. Strong membrane partitioning has been demonstrated for a range of toxins targeting S1–S4 domains in voltage-activated channels (27, 44, 47–50), and for GsMTx4 (14, 50), a tarantula toxin that inhibits opening of stretch-activated cation channels in astrocytes, as well as the cloned stretch-activated Piezo1 channel (13, 15). In experiments on stretch-activated channels, both the d- and l-enantiomers of GsMTx4 are active (14, 50), implying that the toxin may not bind directly to the channel. In addition, both forms of the toxin alter the conductance and lifetimes of gramicidin channels (14), suggesting that the toxin inhibits stretch-activated channels by perturbing the interface between the membrane and the channel. In the case of Kv channels, the S1–S4 domains are embedded in the lipid bilayer and interact intimately with lipids (48, 51, 52) and modification in the lipid composition can dramatically alter gating of the channel (48, 53–56). In one study on the gating of the Kv2.1/Kv1.2 paddle chimera (53), the tarantula toxin VSTx1 was proposed to inhibit Kv channels by modifying the forces acting between the channel and the membrane. Although these studies implicate a key role for the membrane in the activity of Kv and stretch-activated channels, and for the action of tarantula toxins, the influence of the toxin on membrane structure and dynamics have not been directly examined. The goal of the present study was to localize a tarantula toxin in membranes using structural approaches and to investigate the influence of the toxin on the structure of the lipid bilayer.     

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.     

Rab3-interacting molecules 2α and 2β promote the abundance of voltage-gated CaV1.3 Ca2+ channels at hair cell active zones

Ca²⁺ influx triggers the fusion of synaptic vesicles at the presynaptic active zone (AZ). Here we demonstrate a role of Ras-related in brain 3 (Rab3)–interacting molecules 2α and β (RIM2α and RIM2β) in clustering voltage-gated Ca_V1.3 Ca²⁺ channels at the AZs of sensory inner hair cells (IHCs). We show that IHCs of hearing mice express mainly RIM2α, but also RIM2β and RIM3γ, which all localize to the AZs, as shown by immunofluorescence microscopy. Immunohistochemistry, patch-clamp, fluctuation analysis, and confocal Ca²⁺ imaging demonstrate that AZs of RIM2α-deficient IHCs cluster fewer synaptic Ca_V1.3 Ca²⁺ channels, resulting in reduced synaptic Ca²⁺ influx. Using superresolution microscopy, we found that Ca²⁺ channels remained clustered in stripes underneath anchored ribbons. Electron tomography of high-pressure frozen synapses revealed a reduced fraction of membrane-tethered vesicles, whereas the total number of membrane-proximal vesicles was unaltered. Membrane capacitance measurements revealed a reduction of exocytosis largely in proportion with the Ca²⁺ current, whereas the apparent Ca²⁺ dependence of exocytosis was unchanged. Hair cell-specific deletion of all RIM2 isoforms caused a stronger reduction of Ca²⁺ influx and exocytosis and significantly impaired the encoding of sound onset in the postsynaptic spiral ganglion neurons. Auditory brainstem responses indicated a mild hearing impairment on hair cell-specific deletion of all RIM2 isoforms or global inactivation of RIM2α. We conclude that RIM2α and RIM2β promote a large complement of synaptic Ca²⁺ channels at IHC AZs and are required for normal hearing.Tens of Ca_V1.3 Ca²⁺ channels are thought to cluster within the active zone (AZ) membrane underneath the presynaptic density of inner hair cells (IHCs) (1–4). They make up the key signaling element, coupling the sound-driven receptor potential to vesicular glutamate release (5–7). The mechanisms governing the number of Ca²⁺ channels at the AZ as well as their spatial organization relative to membrane-tethered vesicles are not well understood. Disrupting the presynaptic scaffold protein Bassoon diminishes the numbers of Ca²⁺ channels and membrane-tethered vesicles at the AZ (2, 8). However, the loss of Bassoon is accompanied by the loss of the entire synaptic ribbon, which makes it challenging to distinguish the direct effects of gene disruption from secondary effects (9).Among the constituents of the cytomatrix of the AZ, RIM1 and RIM2 proteins are prime candidates for the regulation of Ca²⁺ channel clustering and function (10, 11). The family of RIM proteins has seven identified members (RIM1α, RIM1β, RIM2α, RIM2β, RIM2γ, RIM3γ, and RIM4γ) encoded by four genes (RIM1–RIM4). All isoforms contain a C-terminal C₂ domain but differ in the presence of additional domains. RIM1 and RIM2 interact with Ca²⁺ channels, most other proteins of the cytomatrix of the AZ, and synaptic vesicle proteins. They interact directly with the auxiliary β (Ca_Vβ) subunits (12, 13) and pore-forming Ca_Vα subunits (14, 15). In addition, RIMs are indirectly linked to Ca²⁺ channels via RIM-binding protein (14, 16, 17). A regulation of biophysical channel properties has been demonstrated in heterologous expression systems for RIM1 (12) and RIM2 (13).A role of RIM1 and RIM2 in clustering Ca²⁺ channels at the AZ was demonstrated by analysis of RIM1/2-deficient presynaptic terminals of cultured hippocampal neurons (14), auditory neurons in slices (18), and Drosophila neuromuscular junction (19). Because α-RIMs also bind the vesicle-associated protein Ras-related in brain 3 (Rab3) via the N-terminal zinc finger domain (20), they are also good candidates for molecular coupling of Ca²⁺ channels and vesicles (18, 21, 22). Finally, a role of RIMs in priming of vesicles for fusion is the subject of intense research (18, 21–27). RIMs likely contribute to priming via disinhibiting Munc13 (26) and regulating vesicle tethering (27). Here, we studied the expression and function of RIM in IHCs. We combined molecular, morphologic, and physiologic approaches for the analysis of RIM2α knockout mice [RIM2α SKO (28); see Methods] and of hair cell-specific RIM1/2 knockout mice (RIM1/2 cDKO). We demonstrate that RIM2α and RIM2β are present at IHC AZs of hearing mice, positively regulate the number of synaptic Ca_V1.3 Ca²⁺ channels, and are required for normal hearing.     

Basal and β-adrenergic regulation of the cardiac calcium channel CaV1.2 requires phosphorylation of serine 1700

L-type calcium (Ca²⁺) currents conducted by voltage-gated Ca²⁺ channel Ca_V1.2 initiate excitation–contraction coupling in cardiomyocytes. Upon activation of β-adrenergic receptors, phosphorylation of Ca_V1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca²⁺ entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700–Ala in the Ca_V1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type Ca_V1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3–4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the fight-or-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca²⁺ current and regulation by β-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the fight-or-flight response and development of cardiac hypertrophy.Upon membrane depolarization, Ca_V1.2 channels conduct L-type calcium (Ca²⁺) current into cardiomyocytes and initiate excitation–contraction coupling (1, 2). Ca²⁺ influx through Cav1.2 channels activates Ca²⁺ release from the sarcoplasmic reticulum, which leads to contraction of myofilaments. As the initiator of excitation–contraction coupling, Ca²⁺ influx via Ca_V1.2 channels is tightly regulated. Under conditions of fear, stress, and exercise, the sympathetic nervous system activates the fight-or-flight response, in which the marked increase in contractile force of the heart is caused by epinephrine and norepinephrine acting through β-adrenergic receptors, activation of adenylyl cyclase, increased cAMP, activation of cAMP-dependent protein kinase (PKA), and phosphorylation of the Ca_V1.2 channel (1, 3). Phosphorylation of the Ca_V1.2 channel leads to a threefold to fourfold increase in peak current amplitude in mammalian cardiomyocytes. Regulation of the Ca_V1.2 channel by the cAMP signaling pathway is altered in cardiac hypertrophy and heart failure (4–6). Under those pathological conditions, responsiveness of Ca_V1.2 channel activity to β-adrenergic receptors and PKA activation is severely blunted, resulting in diminished contractile reserve and impaired fight-or-flight response (6, 7). Enormous effort has been devoted to understanding how β-adrenergic regulation of the Ca_V1.2 channel is achieved, but the exact molecular mechanisms remain unresolved.Ca_V1.2 channels contain multiple subunits, including a pore-forming α₁1.2 subunit (also designated α_1C), β and α₂δ subunits that modulate expression of Ca_V1.2 at the cell surface, and possibly γ subunits (8). The closely related Ca_V1.1 and Ca_V1.2 channels in skeletal and cardiac muscle, respectively, are both proteolytically processed near the center of their large C-terminal domains (9, 10), and the distal C terminus (dCT) remains associated noncovalently with the proximal C terminus (pCT) and serves as a potent autoinhibitor (11, 12). Regulation of Ca_V1.2 channels by PKA was reconstituted in nonmuscle cells with a dynamic range of threefold to fourfold similar to native cardiomyocytes by building the autoinhibitory Ca_V1.2 complex through cotransfection of each of its components (13). Successful reconstitution required an A Kinase Anchoring Protein (AKAP), which recruits PKA to the dCT (13–15). Deletion of the dCT in vivo results in loss of regulation of the L-type Ca²⁺ current by the β-adrenergic pathway and embryonic death from heart failure (16, 17). These results suggest that the autoinhibited Ca_V1.2 signaling complex serves as the substrate for β-adrenergic regulation, and disruption of this complex leads to heart failure.PKA is responsible for phosphorylation of the Ca_V1.2 channel in response to β-adrenergic stimulation in cardiac myocytes (18–22). Although multiple PKA sites have been identified in α₁ subunits by in vitro phosphorylation (10, 23), none of these sites is required for regulation of Ca_V1.2 channels in vivo. For example, PKA-dependent phosphorylation of S1928 is prominent in transfected cells and cardiomyocytes (10, 24), but its phosphorylation has little or no effect on β-adrenergic up-regulation of cardiac Ca_V1.2 channel activity in transfected cells or cardiomyocytes (13, 25, 26). Two sites in the C terminus of the skeletal muscle Ca_V1.1 channel are phosphorylated in vivo as assessed by mass spectrometry (S1575 and T1579), and phosphorylation of S1575 is increased by β-adrenergic stimulation (27). These sites are conserved in cardiac Ca_V1.2 channels as S1700 and T1704, and phosphoproteomics analysis revealed β-adrenergic–stimulated phosphorylation of S1700 by PKA (28). S1700 and T1704 reside at the interface between the pCT and dCT. In studies of the Ca_V1.2 signaling complex reconstituted in nonmuscle cells, phosphorylation of both sites was required for normal basal channel activity, whereas only S1700 was essential for PKA stimulation (13). Mutation of S1700 and T1704 to Ala in STAA mice reduced basal activity and Ca_V1.2 channel regulation by the β-adrenergic pathway in cardiomyocytes (29). To further dissect the contribution of S1700, we studied a mutant mouse line expressing Ca_V1.2 channel with the S1700A mutation (SA mice). Our results demonstrate that this single phosphorylation site is required for normal regulation of Ca_V1.2 channels, contraction of cardiac myocytes, exercise capacity, and cardiac homeostasis.     

Prokaryotic NavMs channel as a structural and functional model for eukaryotic sodium channel antagonism

Voltage-gated sodium channels are important targets for the development of pharmaceutical drugs, because mutations in different human sodium channel isoforms have causal relationships with a range of neurological and cardiovascular diseases. In this study, functional electrophysiological studies show that the prokaryotic sodium channel from Magnetococcus marinus (NavMs) binds and is inhibited by eukaryotic sodium channel blockers in a manner similar to the human Na_v1.1 channel, despite millions of years of divergent evolution between the two types of channels. Crystal complexes of the NavMs pore with several brominated blocker compounds depict a common antagonist binding site in the cavity, adjacent to lipid-facing fenestrations proposed to be the portals for drug entry. In silico docking studies indicate the full extent of the blocker binding site, and electrophysiology studies of NavMs channels with mutations at adjacent residues validate the location. These results suggest that the NavMs channel can be a valuable tool for screening and rational design of human drugs.Nine highly homologous human voltage-gated sodium channel isoforms have been identified (1). They are composed of single polypeptide chains containing four pseudorepeated domains (designated DI to DIV), each of which is composed of six transmembrane helical segments (S1 to S6); the pore region is formed from S5 to S6, including the intervening loop and selectivity filter (SF), from all four domains. Prokaryotic sodium channels, in contrast, are homotetramers of four identical polypeptide chains, each of which is equivalent to, and homologous with, one of the eukaryotic domains. Although there are as yet no crystal structures of eukaryotic sodium channels, crystal structures of several prokaryotic sodium channels in different conformational states have been determined, including ones with closed (2), partially (3) and fully (4) open pores, and two potentially inactivated forms (5, 6). Mutations in human sodium channels (hNa_vs) have been linked to channelopathies such as epilepsy, cardiac arrhythmia, and chronic pain syndromes; consequently sodium channel blockers have been developed as anticonvulsant, antiarrhythmic, and local anesthetic drugs (7–10). Several eukaryotic calcium channel blocker drugs have previously been found to bind and block prokaryotic sodium channels (11–13).     

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.