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

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

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

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

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

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

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.     

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.     

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

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

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

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

Conformational cycle and ion-coupling mechanism of the Na+/hydantoin transporter Mhp1

Ion-dependent transporters of the LeuT-fold couple the uptake of physiologically essential molecules to transmembrane ion gradients. Defined by a conserved 5-helix inverted repeat that encodes common principles of ion and substrate binding, the LeuT-fold has been captured in outward-facing, occluded, and inward-facing conformations. However, fundamental questions relating to the structural basis of alternating access and coupling to ion gradients remain unanswered. Here, we used distance measurements between pairs of spin labels to define the conformational cycle of the Na⁺-coupled hydantoin symporter Mhp1 from Microbacterium liquefaciens. Our results reveal that the inward-facing and outward-facing Mhp1 crystal structures represent sampled intermediate states in solution. Here, we provide a mechanistic context for these structures, mapping them into a model of transport based on ion- and substrate-dependent conformational equilibria. In contrast to the Na⁺/leucine transporter LeuT, our results suggest that Na⁺ binding at the conserved second Na⁺ binding site does not change the energetics of the inward- and outward-facing conformations of Mhp1. Comparative analysis of ligand-dependent alternating access in LeuT and Mhp1 lead us to propose that different coupling schemes to ion gradients may define distinct conformational mechanisms within the LeuT-fold class.Secondary active transporters harness the energy of ion gradients to power the uphill movement of solutes across membranes. Mitchell (1) and others (2, 3) proposed and elaborated “alternating access” mechanisms wherein the transporter transitions between two conformational states that alternately expose the substrate binding site to the two sides of the membrane. The LeuT class of ion-coupled symporters consists of functionally distinct transporters that share a conserved scaffold of two sets of five transmembrane helices related by twofold symmetry around an axis nearly parallel to the membrane (4). Ions and substrates are bound near the middle of the membrane stabilized by electrostatic interactions with unwound regions of transmembrane helix (TM) 1 and often TM6 (4). The recurrence of this fold in transporters that play critical roles in fundamental physiological processes (5, 6) has spurred intense interest in defining the principles of alternating access.Despite rapid progress in structure determination of ion-coupled LeuT-fold transporters (7–11), extrapolation of these static snapshots to a set of conformational steps underlying alternating access (4, 7, 9–12) remains incomplete, often hindered by uncertainties in the mechanistic identities of crystal structures. Typically, transporter crystal structures are classified as inward-facing, outward-facing, or occluded on the basis of the accessibility of the substrate binding site (7–11). In a recent spectroscopic analysis of LeuT, we demonstrated that detergent selection and mutations of conserved residues appeared to stabilize conformations that were not detected in the wild-type (WT) LeuT and concurrently inhibited movement of structural elements involved in ligand-dependent alternating access (13). Therefore, although crystal structures define the structural context and identify plausible pathways of substrate binding and release, development of transport models requires confirming or assigning the mechanistic identity of these structures and framing them into ligand-dependent equilibria (14).Mhp1, an Na⁺-coupled symporter of benzyl-hydantoin (BH) from Microbacterium liquefaciens, was the first LeuT-fold member to be characterized by crystal structures purported to represent outward-facing, inward-facing, and outward-facing/occluded conformations of an alternating access cycle (8, 15). In these structures, solvent access to ligand-binding sites is defined by the relative orientation between a 4-helix bundle motif and a 4-helix scaffold motif (8). In Mhp1, alternating access between inward- and outward-facing conformations, was predicted from a computational analysis based on the inverted repeat symmetry of the LeuT fold and is referred to as the rocking-bundle model (16). The conservation of the inverted symmetry prompted proposal of the rocking-bundle mechanism as a general model for LeuT-fold transporters (16). Subsequent crystal structures of other LeuT-fold transporters (7, 9, 10) tempered this prediction because the diversity of the structural rearrangements implicit in these structures is seemingly inconsistent with a conserved conformational cycle.Another outstanding question pertains to the ion-coupling mechanism and the driving force of conformational changes. The implied ion-to-substrate stoichiometry varies across LeuT-fold ion-coupled transporters. For instance, LeuT (17) and BetP (18) require two Na⁺ ions that bind at two distinct sites referred to as Na1 and Na2 whereas Mhp1 (15) and vSGLT (19) appear to possess only the conserved Na2 site. Molecular dynamics (MD) simulations (20, 21) and electron paramagnetic resonance (EPR) analysis (13, 22) of LeuT demonstrated that Na⁺ binding favors an outward-facing conformation although it is unclear which Na⁺ site (or both) is responsible for triggering this conformational transition. Similarly, a role for Na⁺ in conformational switching has been uncovered in putative human LeuT-fold transporters, including hSGLT (23). In Mhp1, the sole Na2 site has been shown to modulate substrate affinity (15); however, its proposed involvement in gating of the intracellular side (12, 21) lacks experimental validation.Here, we used site-directed spin labeling (SDSL) (24) and double electron-electron resonance (DEER) spectroscopy (25) to elucidate the conformational changes underlying alternating access in Mhp1 and define the role of ion and substrate binding in driving transition between conformations. This methodology has been successfully applied to define coupled conformational cycles for a number of transporter classes (13, 26–32). We find that patterns of distance distributions between pairs of spin labels monitoring the intra- and extracellular sides of Mhp1 are consistent with isomerization between the crystallographic inward- and outward-facing conformations. A major finding is that this transition is driven by substrate but not Na⁺ binding. Although the amplitudes of the observed distance changes are in overall agreement with the rocking-bundle model deduced from the crystal structures of Mhp1 (8, 15) and predicted computationally (16), we present evidence that relative movement of bundle and scaffold deviate from strict rigid body. Comparative analysis of LeuT and Mhp1 alternating access reveal how the conserved LeuT fold harnesses the energy of the Na⁺ gradient through two distinct coupling mechanisms and supports divergent conformational cycles to effect substrate binding and release.     

Hybrid-fuel bacterial flagellar motors in Escherichia coli

The bacterial flagellar motor rotates driven by an electrochemical ion gradient across the cytoplasmic membrane, either H⁺ or Na⁺ ions. The motor consists of a rotor ∼50 nm in diameter surrounded by multiple torque-generating ion-conducting stator units. Stator units exchange spontaneously between the motor and a pool in the cytoplasmic membrane on a timescale of minutes, and their stability in the motor is dependent upon the ion gradient. We report a genetically engineered hybrid-fuel flagellar motor in Escherichia coli that contains both H⁺- and Na⁺-driven stator components and runs on both types of ion gradient. We controlled the number of each type of stator unit in the motor by protein expression levels and Na⁺ concentration ([Na⁺]), using speed changes of single motors driving 1-μm polystyrene beads to determine stator unit numbers. De-energized motors changed from locked to freely rotating on a timescale similar to that of spontaneous stator unit exchange. Hybrid motor speed is simply the sum of speeds attributable to individual stator units of each type. With Na⁺ and H⁺ stator components expressed at high and medium levels, respectively, Na⁺ stator units dominate at high [Na⁺] and are replaced by H⁺ units when Na⁺ is removed. Thus, competition between stator units for spaces in a motor and sensitivity of each type to its own ion gradient combine to allow hybrid motors to adapt to the prevailing ion gradient. We speculate that a similar process may occur in species that naturally express both H⁺ and Na⁺ stator components sharing a common rotor.Molecular motors are tiny machines that perform a wide range of functions in living cells. Typically each motor generates mechanical work using a specific chemical or electrochemical energy source. Linear motors such as kinesin on microtubules or myosin on actin filaments and rotary motors such as F₁-ATPase, the soluble part of ATP-synthase, run on ATP, whereas the rotary bacterial flagellar motor embedded in the bacterial cell envelope is driven by the flux of ions across the cytoplasmic membrane (1–4). Coupling ions are known to be either protons (H⁺) or sodium ions (Na⁺) (5, 6).The bacterial flagellar motor consists of a rotor ∼50 nm in diameter surrounded by multiple stator units (7–10). Each unit contains two types of membrane proteins forming ion channels: MotA and MotB in H⁺ motors in neutrophiles (e.g., Escherichia coli and Salmonella) and PomA and PomB in Na⁺ motors in alkalophiles and Vibrio species (e.g., Vibrio alginolyticus) (1, 11). Multiple units interact with the rotor to generate torque independently in a working motor (9, 10, 12, 13). The structure and function of H⁺ and Na⁺ motors are very similar, to the extent that several functional chimeric motors have been made containing different mixtures of H⁺- and Na⁺-motor components (11). One such motor that runs on Na⁺ in E. coli combines the rotor of the H⁺-driven E. coli motor with the chimeric stator unit PomA/PotB, containing PomA from V. alginolyticus and a fusion protein between MotB from E. coli and PomB from V. alginolyticus (14).In most flagellated bacteria, motors are driven by ion-specific rotor–stator combinations. However, some species (e.g., Bacillus subtilis and Shewanella oneidensis) combine a single set of rotor genes with multiple sets of stator genes encoding both H⁺ and Na⁺ stator proteins, and it has been speculated that these stator components may interact with the rotor simultaneously, allowing a single motor to use both H⁺ and Na⁺. An appealing hypothesis that the mixture of stator components is controlled dynamically depending on the environment has arisen from the observation that the localization of both stator components depends upon Na⁺ (15). However, despite some experimental effort there is as yet no direct evidence of both H⁺ and Na⁺ stator units interacting with the same rotor (16).The rotation of single flagellar motors can be monitored in real time by light microscopy of polystyrene beads (diameter ∼1 μm) attached to truncated flagellar filaments (17). Under these conditions, the E. coli motor torque and speed are proportional to the number of stator units in both H⁺-driven MotA/MotB and Na⁺-driven PomA/PotB (17–19) motors. The maximum number of units that can work simultaneously in a single motor has been shown to be at least 11 by “resurrection” experiments, in which newly produced functional units lead to restoration of motor rotation in discrete speed increments in an E. coli strain lacking functional stator proteins (19). Stator units are not fixed permanently in a motor: Each dissociates from the motor with a typical rate of ∼2 min⁻¹, exchanging between the motor and a pool of diffusing units in the cytoplasmic membrane (20). Removal of the relevant ion gradient inactivates both H⁺ and Na⁺ stator units, most likely leading to dissociation from the motor into the membrane pool (2, 21, 22).Here we demonstrate a hybrid-fuel motor containing both H⁺-driven MotA/MotB and Na⁺-driven PomA/PotB stator components, sharing a common rotor in E. coli. We control the expression level of each stator type by induced expression from plasmids, and the affinity of Na⁺-driven stator units for the motor by external [Na⁺]. Units of each type compete for spaces around the rotor, and the motor torque is simply the sum of the independent contributions, with no evidence of direct interaction between units. Thus, we demonstrate the possibility of modularity in the E. coli flagellar motor, with ion selectivity determined by the choice of stator modules interacting with a common rotor. Our artificial hybrid motor demonstrates that species with multiple types of stator gene and a single set of rotor genes could contain natural hybrid motors that work on a similar principle (15, 16, 23).     

Intracellular pH reduction prevents excitotoxic and ischemic neuronal death by inhibiting NADPH oxidase

Sustained activation of N-methyl-d-aspartate (NMDA) -type glutamate receptors leads to excitotoxic neuronal death in stroke, brain trauma, and neurodegenerative disorders. Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic death. NADPH oxidase generates intracellular H⁺ along with extracellular superoxide, and the intracellular H⁺ must be released or neutralized to permit continued NADPH oxidase function. In cultured neurons, NMDA-induced superoxide production and neuronal death were prevented by intracellular acidification by as little as 0.2 pH units, induced by either lowered medium pH or by inhibiting Na⁺/H⁺ exchange. In mouse brain, superoxide production induced by NMDA injections or ischemia–reperfusion was likewise prevented by inhibiting Na⁺/H⁺ exchange and by reduced expression of the Na⁺/H⁺ exchanger-1 (NHE1). Neuronal intracellular pH and neuronal Na⁺/H⁺ exchange are thus potent regulators of excitotoxic superoxide production. These findings identify a mechanism by which cell metabolism can influence coupling between NMDA receptor activation and superoxide production.Many metabolic processes generate hydrogen ions, and hydrogen ions in turn influence cell metabolism and survival (1). Cerebral ischemia in particular produces acidosis of variable degree, depending upon blood glucose levels, degree of blood flow reduction, and other factors. Severe acidosis, below pH 6.4, exacerbates ischemic injury (2) by mechanisms involving protein denaturation, acid-sensing calcium channels, and release of ferrous iron (3–5). Conversely, lesser degrees of acidosis, in the range of 7.0–6.5, reduce both ischemic injury (6) and glutamate-induced neuronal death (7). These neuroprotective effects have been attributed to an inhibitory effect of hydrogen ions on NMDA receptor activation (8–10), but a causal link has not been demonstrated.Excessive activation of N-methyl-D-aspartate (NMDA) type glutamate receptors leads to excitotoxic cell death in stroke and other neurological disorders (11, 12). Superoxide production by NADPH oxidase is a requisite event in the process leading from NMDA receptor activation to excitotoxic cell death (13–19). NADPH oxidase exists as several isoforms, of which NOX2 is the one most abundantly expressed in CNS neurons. NOX2 is also the isoform most abundantly expressed in phagocytes, microglia, and other immune cells, in which its regulation and function have been extensively characterized (20). NOX2 is composed of three cytosolic subunits, p47^phox, p67^phox, and p40^phox, which when phosphorylated bind with two membrane-bound subunits, p22^phox and gp91^phox (the catalytic unit), to form an active transmembrane enzyme complex. The transmembrane complex generates superoxide in the extracellular space and hydrogen ions in the intracellular space: 2O₂ + NADPH → 2O₂⁻ + NADP⁺ + H⁺. In immune cells, H⁺ concentration influences the phosphorylation status of the NOX2 p47^phox subunit, and the H⁺ generated by NOX2 must be transferred to the extracellular space to sustain NOX2 activity (21–24).Together, the pH sensitivity of NOX2 and the role of NOX2 in NMDA receptor-mediated cell death suggest the possibility that reduced intracellular pH might limit neurotoxicity by dissociating NMDA receptor activation from superoxide production. Findings presented here confirm that both the superoxide production and cell death resulting from neuronal NMDA receptor activation are highly pH sensitive. We show that neurons use Na⁺/H⁺ exchange as a major route of proton efflux during NOX2 activation, and either genetic or pharmacologic inhibition of neuronal Na⁺/H⁺ exchange prevent both excitotoxic superoxide production and cell death.     

On the principle of ion selectivity in Na+/H+-coupled membrane proteins: Experimental and theoretical studies of an ATP synthase rotor

Numerous membrane transporters and enzymes couple their mechanisms to the permeation of Na⁺ or H⁺, thereby harnessing the energy stored in the form of transmembrane electrochemical potential gradients to sustain their activities. The molecular and environmental factors that control and modulate the ion specificity of most of these systems are, however, poorly understood. Here, we use isothermal titration calorimetry to determine the Na⁺/H⁺ selectivity of the ion-driven membrane rotor of an F-type ATP synthase. Consistent with earlier theoretical predictions, we find that this rotor is significantly H⁺ selective, although not sufficiently to be functionally coupled to H⁺, owing to the large excess of Na⁺ in physiological settings. The functional Na⁺ specificity of this ATP synthase thus results from two opposing factors, namely its inherent chemical selectivity and the relative availability of the coupling ion. Further theoretical studies of this membrane rotor, and of two others with a much stronger and a slightly weaker H⁺ selectivity, indicate that, although the inherent selectivity of their ion-binding sites is largely set by the balance of polar and hydrophobic groups flanking a conserved carboxylic side chain, subtle variations in their structure and conformational dynamics, for a similar chemical makeup, can also have a significant contribution. We propose that the principle of ion selectivity outlined here may provide a rationale for the differentiation of Na⁺- and H⁺-coupled systems in other families of membrane transporters and enzymes.Gradients in the electrochemical potential of H⁺ or Na⁺ across biological membranes sustain a wide range of essential cellular process. The resulting proton or sodium motive forces (pmf, smf) are the predominant energy source for secondary-active membrane transporters, which mediate the uptake of many substances required by the cell (1–3), and also enable pathogenic bacteria to protect themselves from human-made antibiotics and other toxic compounds (4–6). Downhill membrane permeation of Na⁺ and H⁺ across the membrane also powers the ATP synthase (7), which produces most of cellular ATP, and energizes the rotation of bacterial flagella (8). Thus, the importance of this mode of energy transduction in cells cannot be overstated. Nevertheless, little is known about the factors that control and modulate the specificity for Na⁺ or H⁺ in most of these processes.It seems clear, although, that there is no correlation between function type and ion specificity; that is, the same process in different species can be coupled to either Na⁺ or H⁺ (2, 5–7, 9–11). Organism-specific environmental factors, such as temperature or pH, also do not provide a consistent rationale; for example, ATP synthases from thermoalkaliphilic bacteria use a H⁺ gradient despite the scarcity of H⁺ and the potentially greater degree of H⁺ leakage across the membrane at high temperatures, compared with Na⁺ (12, 13). Indeed, pmf- and smf-driven systems are often found within the same organism, and sometimes with the same or similar function; for example, malate uptake in Bacillus subtilis is mediated by the H⁺-coupled symporter CimH (14) and by the Na⁺-coupled MaeN (15). Moreover, specific membrane transporters and enzymes are sometimes coupled to both Na⁺ and H⁺, either concurrently (using multiple binding sites), such as the multidrug efflux pump NorM of Vibrio cholera (16) and the Methanosarcina acetivorans ATP synthase (17), or alternately (using a single binding site), such as the Escherichia coli melibiose permease (18) and some membrane-integral pyrophosphatases (19).At the molecular level, the architecture of pmf- and smf-driven membrane proteins within the same family has consistently been found to be largely conserved (20–28). Thus, it appears as if the Na⁺ or H⁺ specificity of these systems is dictated by localized variations in their amino acid sequence, rather than by major structural or mechanistic adaptations. This conclusion leads to an intriguing dilemma. Under most physiological conditions, the concentration of Na⁺ exceeds that of protons by many orders of magnitude (for example, a millionfold in mitochondria, or a billionfold in alkaline environments). Thus, in membrane-protein families with members that are driven by the smf or the pmf, the latter must have evolved amino acid adaptions that result in an extreme H⁺ selectivity, so as to counter the large Na⁺ concentration excess. Conversely, specific coupling to the smf would not actually require a strong Na⁺ selectivity; weak H⁺ selectivity or nonselectivity would be sufficient for Na⁺-coupling, physiologically. How can variation changes at the amino acid level result in such extreme variations in the H⁺ selectivity of a protein structure, spanning 10 or more orders of magnitude?In previous theoretical studies, we have addressed this question for the family of ion-motive ATPases (17, 29, 30), which comprises eukaryotic and prokaryotic ATP synthases, as well as vacuolar ion pumps. In these multicomponent enzymes, a membrane-embedded substructure known as the rotor ring can revolve around its axis, relative to the rest of the protein’s membrane domain. This rotational motion enables the ring to capture Na⁺ or H⁺ ions as they enter the protein via an access channel, and to shuttle them to a separate exit channel, in a sequential manner. Because the entry and exit channels are not colinear, there is a strict correspondence between the direction of ion permeation and the sense of rotation of the ring (which in turn determines the type of activity of the catalytic sector of the enzyme, i.e., ATP synthesis or hydrolysis).The principle of ion selectivity emerging from the abovementioned computational studies posits that rotor rings are universally H⁺ selective, owing to the fact that ion binding is consistently mediated by a conserved carboxylic side chain (the intrinsic H⁺/Na⁺ selectivity of a carboxylic group in solution is 10,000-fold), and that this inherent H⁺ selectivity is enhanced or suppressed by multiple orders of magnitude, depending on the balance between hydrophobic and polar groups lining the ion-binding sites (aside from the key carboxyl side chain); geometric factors may additionally fine-tune the selectivity of these sites, for a given chemical composition.In this study, we challenge the validity of this theory through experimental measurements and further computational analyses. Specifically, we set out to experimentally determine the thermodynamic ion selectivity of a rotor ring from a Na⁺-driven ATP synthase, and to compare this ring with others physiologically driven by either Na⁺ or H⁺. We find that the results of this analysis are qualitatively and quantitatively consistent with the theory of ion selectivity proposed previously, and provide novel insights into the influence of conformational factors.     

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.     

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.     

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).     

From the Cover: Widespread convergence in toxin resistance by predictable molecular evolution

The question about whether evolution is unpredictable and stochastic or intermittently constrained along predictable pathways is the subject of a fundamental debate in biology, in which understanding convergent evolution plays a central role. At the molecular level, documented examples of convergence are rare and limited to occurring within specific taxonomic groups. Here we provide evidence of constrained convergent molecular evolution across the metazoan tree of life. We show that resistance to toxic cardiac glycosides produced by plants and bufonid toads is mediated by similar molecular changes to the sodium-potassium-pump (Na⁺/K⁺-ATPase) in insects, amphibians, reptiles, and mammals. In toad-feeding reptiles, resistance is conferred by two point mutations that have evolved convergently on four occasions, whereas evidence of a molecular reversal back to the susceptible state in varanid lizards migrating to toad-free areas suggests that toxin resistance is maladaptive in the absence of selection. Importantly, resistance in all taxa is mediated by replacements of 2 of the 12 amino acids comprising the Na⁺/K⁺-ATPase H1–H2 extracellular domain that constitutes a core part of the cardiac glycoside binding site. We provide mechanistic insight into the basis of resistance by showing that these alterations perturb the interaction between the cardiac glycoside bufalin and the Na⁺/K⁺-ATPase. Thus, similar selection pressures have resulted in convergent evolution of the same molecular solution across the breadth of the animal kingdom, demonstrating how a scarcity of possible solutions to a selective challenge can lead to highly predictable evolutionary responses.Convergent evolution is the process by which phenotypic similarities evolve independently among disparate species (1–3). Although convergence is sometimes distinguished from parallel evolution, both are part of a continuum, which often makes attempting to distinguish between them problematic and potentially misleading (4); here, we simply use the term “convergence” throughout. Convergence has strong bearing on a fundamental and heated debate on the predictability of evolution. Epitomized by the writings of Gould (5) and Conway Morris (6), this debate centers on whether evolution is stochastic and unpredictable (5) or subject to constraints that limit the available options for evolution, resulting in frequent convergence and a degree of predictability (6). However, evidence of convergence at the genetic level, where similar molecular changes confer the same change in phenotype, is currently limited to only a few taxonomically restricted examples (7–11).Cardiac glycosides offer an ideal model system to investigate the extent to which evolution can be constrained to predictable changes throughout the animal kingdom. These organic compounds are highly toxic molecules that inhibit the sodium-potassium-pump (Na⁺/K⁺-ATPase), which disrupts ion transport and thereby perturbs membrane potentials, often resulting in lethal cardiotoxicity (12, 13). Cardiac glycosides are produced independently by a number of plants and bufonid toads as secondary metabolites and are used for defense against natural enemies. For example, milkweed, foxglove, and oleander plants produce “cardenolides” (e.g., ouabain) that protect against herbivorous insects (13), whereas bufonid toads secrete structurally and functionally similar defensive compounds, “bufotoxins” (e.g., bufalin), from their parotoid glands (14).Despite the toxicity of these molecules, natural resistance exists in many different herbivores and predators and is predominately mediated by molecular alterations to the H1–H2 extracellular domain of the Na⁺/K⁺-ATPase, resulting in target-site insensitivity to cardiac glycosides (9, 15, 16). For example, we recently showed that two amino acid replacements in the Na⁺/K⁺-ATPase α3 subunit (previously denoted α1) are responsible for a 3,000-fold increased resistance to bufalin in toad-feeding African and Asian varanid lizards, compared with toad toxin-susceptible Australian varanids (17). However, until now, comparisons of the molecular basis of resistance and mechanisms of action across the diverse range of resistant taxa reported (9, 15, 17, 18) have been lacking. Here we present an analysis of convergent molecular changes to the Na⁺/K⁺-ATPase H1–H2 domain in cardiac glycoside-resistant invertebrates and vertebrates. Our results support the view that molecular evolution in the animal kingdom can be heavily constrained, resulting in convergent processes canalizing evolution along highly predictable pathways.     

Arginine oscillation explains Na+ independence in the substrate/product antiporter CaiT

Most secondary-active transporters transport their substrates using an electrochemical ion gradient. In contrast, the carnitine transporter (CaiT) is an ion-independent, l-carnitine/γ-butyrobetaine antiporter belonging to the betaine/carnitine/choline transporter family of secondary transporters. Recently determined crystal structures of CaiT from Escherichia coli and Proteus mirabilis revealed an inverted five-transmembrane-helix repeat similar to that in the amino acid/Na⁺ symporter LeuT. The ion independence of CaiT makes it unique in this family. Here we show that mutations of arginine 262 (R262) make CaiT Na⁺-dependent. The transport activity of R262 mutants increased by 30–40% in the presence of a membrane potential, indicating substrate/Na⁺ cotransport. Structural and biochemical characterization revealed that R262 plays a crucial role in substrate binding by stabilizing the partly unwound TM1′ helix. Modeling CaiT from P. mirabilis in the outward-open and closed states on the corresponding structures of the related symporter BetP reveals alternating orientations of the buried R262 sidechain, which mimic sodium binding and unbinding in the Na⁺-coupled substrate symporters. We propose that a similar mechanism is operative in other Na⁺/H⁺-independent transporters, in which a positively charged amino acid replaces the cotransported cation. The oscillation of the R262 sidechain in CaiT indicates how a positive charge triggers the change between outward-open and inward-open conformations as a unifying critical step in LeuT-type transporters.The carnitine/γ-butyrobetaine antiporter CaiT belongs to the betaine/carnitine/choline transporter family of secondary transporters that transfer substrates containing a quaternary ammonium group (1, 2) in and out of the cell. In Escherichia coli and other enterobacteria, such as Proteus mirabilis, carnitine is taken up by CaiT and converted to γ-butyrobetaine via the reaction intermediate crotonobetaine (3–5), which serves as an external electron acceptor under anaerobic growth conditions (4). Biochemical studies of E. coli CaiT (EcCaiT) have shown that it is a constitutively active, Na⁺/H⁺-independent antiporter (6).Crystal structures of CaiT from P. mirabilis (PmCaiT) and E. coli (EcCaiT) were recently determined with and without bound substrate (7, 8). These structures revealed a trimeric assembly of CaiT, as previously found (9). The protein was in an inward-facing conformation with two substrate molecules bound per EcCaiT monomer: one in the central transport site and another in an external binding site (7). Fluorescent binding assays with the protein reconstituted into liposomes indicated that substrate binding was cooperative. This suggested a regulatory role for the external binding site, which was proposed to increase substrate affinity and initiate substrate transport (7). Strikingly, the crystal structures revealed that CaiT adopts a fold similar to that of the LeuT-type transporters (7, 10, 11). This places it in the amino acid–polyamine-organocation (APC) superfamily (12), which shares a conserved architecture of two inverted repeats of five transmembrane (TM) helices each, implying common mechanistic principles. Among the APC transporters, the leucine transporter LeuT from the neurotransmitter/sodium symporter family (10), the betaine transporter BetP from the betaine/choline/carnitine transporter family (13), the benzyl-hydantoin transporter Mhp1 of the nucleobase/cation symport 1 family, and the Na⁺/galactose symporter vSGLT of the solute/sodium symporter family are substrate/sodium symporters (14, 15). Although the substrate to sodium stoichiometry varies for each Na⁺-dependent LeuT-type transporter, most of them possess a conserved sodium-binding site (Na2 site) at which the binding and dissociation of a sodium ion is proposed to facilitate structural changes that lead to substrate transport (16–18). Although an additional sodium-binding site (Na1 site) exists in transporters such as LeuT and BetP, the position or even the presence of this site is not strictly conserved among the Na⁺-dependent LeuT-type transporters (14, 15, 19–21).A small number of LeuT-type transporters, namely, AdiC (arginine/agmatine antiporter), ApcT (broad-specificity amino acid transporter), and CaiT, are Na⁺-independent (6, 22–25). The crystal structure of ApcT revealed that a lysine residue (K158) occupies a position equivalent to the Na2 site in LeuT. This lysine is proposed to undergo a protonation/deprotonation event that leads to conformational changes facilitating substrate transport (25). In CaiT, a methionine residue (M331) occupies a position equivalent to Na1 in LeuT, whereas a positively charged arginine residue (R262) occupies the Na2 site (7). Previously, we have shown that mutating M331 reduces transport activity but does not induce Na⁺ dependence in CaiT (7). Here we report that point mutations of R262 render CaiT inactive. Strikingly, the transport activity was partially restored by the addition of sodium, thus making these mutants Na⁺-dependent. Unlike wild-type, the transport activity of R262 mutants increased by 30–40% when a membrane potential was applied, suggesting that Na⁺ and substrate were cotransported. To find out whether and how the mutation affects the Na2 site, we determined the crystal structure of CaiT R262E. Although we did not find any major changes in the mutant protein, comparison with the substrate-bound EcCaiT wild-type protein revealed that the γ-butyrobetaine substrate adopts a different orientation at the central binding site, in which it directly interacts with the unwound part of TM1′. (To make the nomenclature consistent, we adopt the same helix numbering as in LeuT. Because CaiT has two extra helices at the N terminus in comparison with LeuT, TM3 in CaiT corresponds to TM1 in LeuT and is denoted as TM1′, and the remainder of the CaiT helices follow suit.) Because R262 is known to play a role in stabilizing the unwound part of TM1′ (7), we propose that R262 is crucial for substrate binding, similar to Na⁺ in the Na2 site of LeuT and BetP (17, 19). Indeed, our fluorescent binding assays using R262 mutants showed markedly decreased substrate affinity. Modeling CaiT in various conformations with BetP as a template revealed that R262 undergoes an oscillatory movement, contributing to different hydrogen bond networks in each conformation. We suggest that this movement of the positively charged R262 sidechain in CaiT mimics Na⁺ binding/unbinding in Na⁺-dependent LeuT-type transporters and plays a central role in the transport mechanism.     

Effects of lymphocyte profile on development of EBV-induced lymphoma subtypes in humanized mice

Epstein-Barr virus (EBV) infection causes both Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphoma (NHL). The present study reveals that EBV-induced HL and NHL are intriguingly associated with a repopulated immune cell profile in humanized mice. Newborn immunodeficient NSG mice were engrafted with human cord blood CD34⁺ hematopoietic stem cells (HSCs) for a 8- or 15-wk reconstitution period (denoted ^8whN and ^15whN, respectively), resulting in human B-cell and T-cell predominance in peripheral blood cells, respectively. Further, novel humanized mice were established via engraftment of hCD34⁺ HSCs together with nonautologous fetal liver-derived mesenchymal stem cells (MSCs) or MSCs expressing an active notch ligand DLK1, resulting in mice skewed with human B or T cells, respectively. After EBV infection, whereas NHL developed more frequently in B-cell–predominant humanized mice, HL was seen in T-cell–predominant mice (P = 0.0013). Whereas human splenocytes from NHL-bearing mice were positive for EBV-associated NHL markers (hBCL2⁺, hCD20⁺, hKi67⁺, hCD20⁺/EBNA1⁺, and EBER⁺) but negative for HL markers (LMP1⁻, EBNA2⁻, and hCD30⁻), most HL-like tumors were characterized by the presence of malignant Hodgkin’s Reed–Sternberg (HRS)-like cells, lacunar RS (hCD30⁺, hCD15⁺, IgJ⁻, EBER⁺/hCD30⁺, EBNA1⁺/hCD30⁺, LMP⁺/EBNA2⁻, hCD68⁺, hBCL2⁻, hCD20^-/weak, Phospho STAT6⁺), and mummified RS cells. This study reveals that immune cell composition plays an important role in the development of EBV-induced B-cell lymphoma.Epstein Barr virus (EBV) infects human B lymphocytes and epithelial cells in >90% of the human population (1, 2). EBV infection is widely associated with the development of diverse human disorders that include Hodgkin’s lymphoma (HL) and non-Hodgkin’s lymphomas (NHL), including diffused large B-cell lymphoma (DLBCL), follicular B-cell lymphoma (FBCL), endemic Burkitt’s lymphoma (BL), and hemophagocytic lymphohistiocytosis (HLH) (3).HL is a malignant lymphoid neoplasm most prevalent in adolescents and young adults (4–6). Hodgkin/Reed–Sternberg (HRS) cells are the sole malignant cells of HL. HRS cells are characterized by CD30⁺/CD15⁺/BCL6⁻/CD20^+/− markers and appear large and multinucleated owing to multiple nuclear divisions without cytokinesis. Although HRS cells are malignant in the body, surrounding inflammatory cells greatly outnumber them. These reactive nonmalignant inflammatory cells, including lymphocytes, histiocytes, eosinophils, fibroblasts, neutrophils, and plasma cells, compose the vast majority of the tumor mass. The presence of HRS cells in the context of this inflammatory cellular background is a critical hallmark of the HL diagnosis (4). Approximately 50% of HL cases are EBV-associated (EBVaHL) (7–11). EBV-positive HRS cells express EBV latent membrane protein (LMP) 1 (LMP1), LMP2A, LMP2B, and EBV nuclear antigen (EBNA) 1 (EBNA1), but lack EBNA2 (latency II marker) (12). LMP1 is consistently expressed in all EBV-associated cases of classical HL (13, 14). LMP1 mimics activated CD40 receptors, induces NF-κB, and allows cells to become malignant while escaping apoptosis (15).The etiologic role of EBV in numerous disorders has been studied in humanized mouse models in diverse experimental conditions. Humanized mouse models recapitulate key characteristics of EBV infection-associated disease pathogenesis (16–24). Different settings have given rise to quite distinct phenotypes, including B-cell type NHL (DLBCL, FBCL, and unspecified B-cell lymphomas), natural killer/T cell lymphoma (NKTCL), nonmalignant lymphoproliferative disorder (LPD), extremely rare HL, HLH, and arthritis (16–24). Despite considerable efforts (16–24), EBVaHL has not been properly produced in the humanized mouse setting model, owing to inappropriate animal models and a lack of in-depth analyses. After an initial report of infected humanized mice, HRS-like cells appeared to be extremely rare in the spleens of infected humanized mice; however, the findings were inconclusive (18). Here we report direct evidence of EBVaHL or HL-like neoplasms in multiple humanized mice in which T cells were predominant over B cells. Our study demonstrates that EBV-infected humanized mice display additional EBV-associated pathogenesis, including DLBCL and hemophagocytic lymphohistiocytosis (16, 17).