L-type calcium (Ca(2+)) currents conducted by voltage-gated Ca(2+) channel CaV1.2 initiate excitation-contraction coupling in cardiomyocytes. Upon activation of ?-adrenergic receptors, phosphorylation of CaV1.2 channels by cAMP-dependent protein kinase (PKA) increases channel activity, thereby allowing more Ca(2+) entry into the cell, which leads to more forceful contraction. In vitro reconstitution studies and in vivo proteomics analysis have revealed that Ser-1700 is a key site of phosphorylation mediating this effect, but the functional role of this amino acid residue in regulation in vivo has remained uncertain. Here we have studied the regulation of calcium current and cell contraction of cardiomyocytes in vitro and cardiac function and homeostasis in vivo in a mouse line expressing the mutation Ser-1700-Ala in the CaV1.2 channel. We found that preventing phosphorylation at this site decreased the basal L-type CaV1.2 current in both neonatal and adult cardiomyocytes. In addition, the incremental increase elicited by isoproterenol was abolished in neonatal cardiomyocytes and was substantially reduced in young adult myocytes. In contrast, cellular contractility was only moderately reduced compared with wild type, suggesting a greater reserve of contractile function and/or recruitment of compensatory mechanisms. Mutant mice develop cardiac hypertrophy by the age of 3-4 mo, and maximal stress-induced exercise tolerance is reduced, indicating impaired physiological regulation in the fight-or-flight response. Our results demonstrate that phosphorylation at Ser-1700 alone is essential to maintain basal Ca(2+) current and regulation by ?-adrenergic activation. As a consequence, blocking PKA phosphorylation at this site impairs cardiovascular physiology in vivo, leading to reduced exercise capacity in the fight-or-flight response and development of cardiac hypertrophy.
Voltage-gated sodium channels mediate the initiation and propagation of action potentials in excitable cells. Transmembrane segment S4 of voltage-gated sodium channels resides in a gating pore where it senses the membrane potential and controls channel gating. Substitution of individual S4 arginine gating charges (R1-R3) with smaller amino acids allows ionic currents to flow through the mutant gating pore, and these gating pore currents are pathogenic in some skeletal muscle periodic paralysis syndromes. The voltage dependence of gating pore currents provides information about the transmembrane position of the gating charges as S4 moves in response to membrane potential. Here we studied gating pore current in mutants of the homotetrameric bacterial sodium channel NaChBac in which individual arginine gating charges were replaced by cysteine. Gating pore current was observed for each mutant channel, but with different voltage-dependent properties. Mutating the first (R1C) or second (R2C) arginine to cysteine resulted in gating pore current at hyperpolarized membrane potentials, where the channels are in resting states, but not at depolarized potentials, where the channels are activated. Conversely, the R3C gating pore is closed at hyperpolarized membrane potentials and opens with channel activation. Negative conditioning pulses revealed time-dependent deactivation of the R3C gating pore at the most hyperpolarized potentials. Our results show sequential voltage dependence of activation of gating pore current from R1 to R3 and support stepwise outward movement of the substituted cysteines through the narrow portion of the gating pore that is sealed by the arginine side chains in the wild-type channel. This pattern of voltage dependence of gating pore current is consistent with a sliding movement of the S4 helix through the gating pore. Through comparison with high-resolution models of the voltage sensor of bacterial sodium channels, these results shed light on the structural basis for pathogenic gating pore currents in periodic paralysis syndromes.
Haploinsufficiency of the voltage-gated sodium channel NaV1.1 causes Dravet syndrome, an intractable developmental epilepsy syndrome with seizure onset in the first year of life. Specific heterozygous deletion of NaV1.1 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 NaV1.1 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.
Dominant loss-of-function mutations in voltage-gated sodium channel NaV1.1 cause Dravet Syndrome, an intractable childhood-onset epilepsy. NaV1.1(+/-) Dravet Syndrome mice in C57BL/6 genetic background exhibit severe seizures, cognitive and social impairments, and premature death. Here we show that Dravet Syndrome mice in pure 129/SvJ genetic background have many fewer seizures and much less premature death than in pure C57BL/6 background. These mice also have a higher threshold for thermally induced seizures, fewer myoclonic seizures, and no cognitive impairment, similar to patients with Genetic Epilepsy with Febrile Seizures Plus. Consistent with this mild phenotype, mutation of NaV1.1 channels has much less physiological effect on neuronal excitability in 129/SvJ mice. In hippocampal slices, the excitability of CA1 Stratum Oriens interneurons is selectively impaired, while the excitability of CA1 pyramidal cells is unaffected. NaV1.1 haploinsufficiency results in increased rheobase and threshold for action potential firing and impaired ability to sustain high-frequency firing. Moreover, deletion of NaV1.1 markedly reduces the amplification and integration of synaptic events, further contributing to reduced excitability of interneurons. Excitability is less impaired in inhibitory neurons of Dravet Syndrome mice in 129/SvJ genetic background. Because specific deletion of NaV1.1 in forebrain GABAergic interneuons is sufficient to cause the symptoms of Dravet Syndrome in mice, our results support the conclusion that the milder phenotype in 129/SvJ mice is caused by lesser impairment of sodium channel function and electrical excitability in their forebrain interneurons. This mild impairment of excitability of interneurons leads to a milder disease phenotype in 129/SvJ mice, similar to Genetic Epilepsy with Febrile Seizures Plus in humans.
Eukaryotic sodium and calcium channels are made up of four linked homologous but different transmembrane domains. Bacteria express sodium channels comprised of four identical subunits, each being analogous to a single homologous domain of their eukaryotic counterparts. Key elements of primary structure are conserved between bacterial and eukaryotic sodium and calcium channels. The simple protein structure of the bacterial channels has allowed extensive structure-function probes of key regions as well as allowing determination of several X-ray crystallographic structures of these channels. The structures have revealed novel features of sodium and calcium channel pores and elucidated the structural importance of many of the conserved features of primary sequence. The structural information has also formed the basis for computational studies probing the basis for sodium and calcium selectivity and gating.
Voltage-gated sodium (Nav) channels initiate action potentials in brain neurons and are primary therapeutic targets for anti-epileptic drugs controlling neuronal hyperexcitability in epilepsy. The molecular mechanisms underlying abnormal Nav channel expression, localization, and function during development of epilepsy are poorly understood but can potentially result from altered posttranslational modifications (PTMs). For example, phosphorylation regulates Nav channel gating, and has been proposed to contribute to acquired insensitivity to anti-epileptic drugs exhibited by Nav channels in epileptic neurons. However, whether changes in specific brain Nav channel PTMs occur acutely in response to seizures has not been established. Here, we show changes in PTMs of the major brain Nav channel, Nav1.2, after acute kainate-induced seizures. Mass spectrometry-based proteomic analyses of Nav1.2 purified from the brains of control and seizure animals revealed a significant down-regulation of phosphorylation at nine sites, primarily located in the interdomain I-II linker, the region of Nav1.2 crucial for phosphorylation-dependent regulation of activity. Interestingly, Nav1.2 in the seizure samples contained methylated arginine (MeArg) at three sites. These MeArgs were adjacent to down-regulated sites of phosphorylation, and Nav1.2 methylation increased after seizure. Phosphorylation and MeArg were not found together on the same tryptic peptide, suggesting reciprocal regulation of these two PTMs. Coexpression of Nav1.2 with the primary brain arginine methyltransferase PRMT8 led to a surprising 3-fold increase in Nav1.2 current. Reciprocal regulation of phosphorylation and MeArg of Nav1.2 may underlie changes in neuronal Nav channel function in response to seizures and also contribute to physiological modulation of neuronal excitability.
Autism spectrum disorder (ASD) may arise from increased ratio of excitatory to inhibitory neurotransmission in the brain. Many pharmacological treatments have been tested in ASD, but only limited success has been achieved. Here we report that BTBR T(+)Itpr3(tf)/J (BTBR) mice, a model of idiopathic autism, have reduced spontaneous GABAergic neurotransmission. Treatment with low nonsedating/nonanxiolytic doses of benzodiazepines, which increase inhibitory neurotransmission through positive allosteric modulation of postsynaptic GABAA receptors, improved deficits in social interaction, repetitive behavior, and spatial learning. Moreover, negative allosteric modulation of GABAA receptors impaired social behavior in C57BL/6J and 129SvJ wild-type mice, suggesting that reduced inhibitory neurotransmission may contribute to social and cognitive deficits. The dramatic behavioral improvement after low-dose benzodiazepine treatment was subunit specific-the ?2,3-subunit-selective positive allosteric modulator L-838,417 was effective, but the ?1-subunit-selective drug zolpidem exacerbated social deficits. Impaired GABAergic neurotransmission may contribute to ASD, and ?2,3-subunit-selective positive GABAA receptor modulation may be an effective treatment.
The CaV1.1 and CaV1.2 voltage-gated calcium channels initiate excitation-contraction coupling in skeletal and cardiac myocytes, excitation-transcription coupling in neurons, and many other cellular processes. Up-regulation of their activity by the ?-adrenergic-PKA signaling pathway increases these physiological responses. PKA up-regulation of CaV1.2 activity can be reconstituted in a transfected cell system expressing CaV1.2?1800 truncated at the in vivo proteolytic processing site, the distal C-terminal domain (DCT; CaV1.2[1801-2122]), the auxiliary ?2? and ? subunits of CaV1.2 channels, and A-kinase anchoring protein-15 (AKAP15), which binds to a site in the DCT. AKAP79/150 binds to the same site in the DCT as AKAP15. Here we report that AKAP79 is ineffective in supporting up-regulation of CaV1.2 channel activity by PKA, even though it binds to the same site in the DCT and inhibits the up-regulation of CaV1.2 channel activity supported by AKAP15. Mutation of the calcineurin-binding site in AKAP79 (AKAP79?PIX) allows it to support PKA-dependent up-regulation of CaV1.2 channel activity, suggesting that calcineurin bound to AKAP79 rapidly dephosphorylates CaV1.2 channels, thereby preventing their regulation by PKA. Both AKAP15 and AKAP79?PIX exert their regulatory effects on CaV1.2 channels in transfected cells by interaction with the modified leucine zipper motif in the DCT. Our results introduce an unexpected mode of differential regulation by AKAPs, in which binding of different AKAPs at a single site can competitively confer differential regulatory effects on the target protein by their association with different signaling proteins.
L-type Ca(2+) currents conducted by CaV1.2 channels initiate excitation-contraction coupling in the heart. Their activity is increased by ?-adrenergic/cAMP signaling via phosphorylation by PKA in the fight-or-flight response, but the sites of regulation are unknown. We describe the functional role of phosphorylation of Ser1700 and Thr1704-sites of phosphorylation by PKA and casein kinase II at the interface between the proximal and distal C-terminal regulatory domains. Mutation of both residues to Ala in STAA mice reduced basal L-type Ca(2+) currents, due to a small decrease in expression and a substantial decrease in functional activity. The increase in L-type Ca(2+) current caused by isoproterenol was markedly reduced at physiological levels of stimulation (3-10 nM). Maximal increases in calcium current at nearly saturating concentrations of isoproterenol (100 nM) were also significantly reduced, but the mutation effects were smaller, suggesting that alternative regulatory mechanisms are engaged at maximal levels of stimulation. The ?-adrenergic increase in cell contraction was also diminished. STAA ventricular myocytes exhibited arrhythmic contractions in response to isoproterenol, and up to 20% of STAA cells failed to sustain contractions when stimulated at 1 Hz. STAA mice have reduced exercise capacity, and cardiac hypertrophy is evident at 3 mo. We conclude that phosphorylation of Ser1700 and Thr1704 is essential for regulation of basal activity of CaV1.2 channels and for up-regulation by ?-adrenergic signaling at physiological levels of stimulation. Disruption of phosphorylation at those sites leads to impaired cardiac function in vivo, as indicated by reduced exercise capacity and cardiac hypertrophy.
Voltage-gated sodium channels undergo slow inactivation during repetitive depolarizations, which controls the frequency and duration of bursts of action potentials and prevents excitotoxic cell death. Although homotetrameric bacterial sodium channels lack the intracellular linker-connecting homologous domains III and IV that causes fast inactivation of eukaryotic sodium channels, they retain the molecular mechanism for slow inactivation. Here, we examine the functional properties and slow inactivation of the bacterial sodium channel NavAb expressed in insect cells under conditions used for structural studies. NavAb activates at very negative membrane potentials (V1/2 of approximately -98 mV), and it has both an early phase of slow inactivation that arises during single depolarizations and reverses rapidly, and a late use-dependent phase of slow inactivation that reverses very slowly. Mutation of Asn49 to Lys in the S2 segment in the extracellular negative cluster of the voltage sensor shifts the activation curve ?75 mV to more positive potentials and abolishes the late phase of slow inactivation. The gating charge R3 interacts with Asn49 in the crystal structure of NavAb, and mutation of this residue to Cys causes a similar positive shift in the voltage dependence of activation and block of the late phase of slow inactivation as mutation N49K. Prolonged depolarizations that induce slow inactivation also cause hysteresis of gating charge movement, which results in a requirement for very negative membrane potentials to return gating charges to their resting state. Unexpectedly, the mutation N49K does not alter hysteresis of gating charge movement, even though it prevents the late phase of slow inactivation. Our results reveal an important molecular interaction between R3 in S4 and Asn49 in S2 that is crucial for voltage-dependent activation and for late slow inactivation of NavAb, and they introduce a NavAb mutant that enables detailed functional studies in parallel with structural analysis.
Voltage-gated sodium channels are responsible for the rising phase of the action potential in cardiac muscle. Previously, both TTX-sensitive neuronal sodium channels (NaV1.1, NaV1.2, NaV1.3, NaV1.4 and NaV1.6) and the TTX-resistant cardiac sodium channel (NaV1.5) have been detected in cardiac myocytes, but relative levels of protein expression of the isoforms were not determined. Using a quantitative approach, we analyzed z-series of confocal microscopy images from individual mouse myocytes stained with either anti-NaV1.1, anti-NaV1.2, anti-NaV1.3, anti-NaV1.4, anti-NaV1.5, or anti-NaV1.6 antibodies and calculated the relative intensity of staining for these sodium channel isoforms. Our results indicate that the TTX-sensitive channels represented approximately 23% of the total channels, whereas the TTX-resistant NaV1.5 channel represented 77% of the total channel staining in mouse ventricular myocytes. These ratios are consistent with previous electrophysiological studies in mouse ventricular myocytes. NaV1.5 was located at the cell surface, with high density at the intercalated disc, but was absent from the transverse (t)-tubular system, suggesting that these channels support surface conduction and inter-myocyte transmission. Low-level cell surface staining of NaV1.4 and NaV1.6 channels suggest a minor role in surface excitation and conduction. Conversely, NaV1.1 and NaV1.3 channels are localized to the t-tubules and are likely to support t-tubular transmission of the action potential to the myocyte interior. This quantitative immunocytochemical approach for assessing sodium channel density and localization provides a more precise view of the relative importance and possible roles of these individual sodium channel protein isoforms in mouse ventricular myocytes and may be applicable to other species and cardiac tissue types.
Voltage-gated calcium (CaV) channels catalyse rapid, highly selective influx of Ca(2+) into cells despite a 70-fold higher extracellular concentration of Na(+). How CaV channels solve this fundamental biophysical problem remains unclear. Here we report physiological and crystallographic analyses of a calcium selectivity filter constructed in the homotetrameric bacterial NaV channel NaVAb. Our results reveal interactions of hydrated Ca(2+) with two high-affinity Ca(2+)-binding sites followed by a third lower-affinity site that would coordinate Ca(2+) as it moves inward. At the selectivity filter entry, Site 1 is formed by four carboxyl side chains, which have a critical role in determining Ca(2+) selectivity. Four carboxyls plus four backbone carbonyls form Site 2, which is targeted by the blocking cations Cd(2+) and Mn(2+), with single occupancy. The lower-affinity Site 3 is formed by four backbone carbonyls alone, which mediate exit into the central cavity. This pore architecture suggests a conduction pathway involving transitions between two main states with one or two hydrated Ca(2+) ions bound in the selectivity filter and supports a knock-off mechanism of ion permeation through a stepwise-binding process. The multi-ion selectivity filter of our CaVAb model establishes a structural framework for understanding the mechanisms of ion selectivity and conductance by vertebrate CaV channels.
Voltage-gated sodium channels composed of a pore-forming ? subunit and auxiliary ? subunits are responsible for the upstroke of the action potential in cardiac muscle. However, their localization and expression patterns in human myocardium have not yet been clearly defined. We used immunohistochemical methods to define the level of expression and the subcellular localization of sodium channel ? and ? subunits in human atrial myocytes. Nav1.2 channels are located in highest density at intercalated disks where ?1 and ?3 subunits are also expressed. Nav1.4 and the predominant Nav1.5 channels are located in a striated pattern on the cell surface at the z-lines together with ?2 subunits. Nav1.1, Nav1.3, and Nav1.6 channels are located in scattered puncta on the cell surface in a pattern similar to ?3 and ?4 subunits. Nav1.5 comprised approximately 88% of the total sodium channel staining, as assessed by quantitative immunohistochemistry. Functional studies using whole cell patch-clamp recording and measurements of contractility in human atrial cells and tissue showed that TTX-sensitive (non-Nav1.5) ? subunit isoforms account for up to 27% of total sodium current in human atrium and are required for maximal contractility. Overall, our results show that multiple sodium channel ? and ? subunits are differentially localized in subcellular compartments in human atrial myocytes, suggesting that they play distinct roles in initiation and conduction of the action potential and in excitation-contraction coupling. TTX-sensitive sodium channel isoforms, even though expressed at low levels relative to TTX-sensitive Nav1.5, contribute substantially to total cardiac sodium current and are required for normal contractility. This article is part of a Special Issue entitled "Na(+) Regulation in Cardiac Myocytes".
Seizures remain uncontrolled in 30% of patients with epilepsy, even with concurrent use of multiple drugs, and uncontrolled seizures result in increased morbidity and mortality. An extreme example is Dravet syndrome (DS), an infantile-onset severe epilepsy caused by heterozygous loss of function mutations in SCN1A, the gene encoding the brain type-I voltage-gated sodium channel NaV1.1. Studies in Scn1a heterozygous knockout mice demonstrate reduced excitability of GABAergic interneurons, suggesting that enhancement of GABA signaling may improve seizure control and comorbidities. We studied the efficacy of two GABA-enhancing drugs, clonazepam and tiagabine, alone and in combination, against thermally evoked myoclonic and generalized tonic-clonic seizures. Clonazepam, a positive allosteric modulator of GABA-A receptors, protected against myoclonic and generalized tonic-clonic seizures. Tiagabine, a presynaptic GABA reuptake inhibitor, was protective against generalized tonic-clonic seizures but only minimally protective against myoclonic seizures and enhanced myoclonic seizure susceptibility at high doses. Combined therapy with clonazepam and tiagabine was synergistic against generalized tonic-clonic seizures but was additive against myoclonic seizures. Toxicity determined by rotorod testing was additive for combination therapy. The synergistic actions of clonazepam and tiagabine gave enhanced seizure protection and reduced toxicity, suggesting that combination therapy may be well tolerated and effective for seizures in DS.
Sudden unexpected death in epilepsy (SUDEP) is the most common cause of death in intractable epilepsies, but physiological mechanisms that lead to SUDEP are unknown. Dravet syndrome (DS) is an infantile-onset intractable epilepsy caused by heterozygous loss-of-function mutations in the SCN1A gene, which encodes brain type-I voltage-gated sodium channel NaV1.1. We studied the mechanism of premature death in Scn1a heterozygous KO mice and conditional brain- and cardiac-specific KOs. Video monitoring demonstrated that SUDEP occurred immediately following generalized tonic-clonic seizures. A history of multiple seizures was a strong risk factor for SUDEP. Combined video-electroencephalography-electrocardiography revealed suppressed interictal resting heart-rate variability and episodes of ictal bradycardia associated with the tonic phases of generalized tonic-clonic seizures. Prolonged atropine-sensitive ictal bradycardia preceded SUDEP. Similar studies in conditional KO mice demonstrated that brain, but not cardiac, KO of Scn1a produced cardiac and SUDEP phenotypes similar to those found in DS mice. Atropine or N-methyl scopolamine treatment reduced the incidence of ictal bradycardia and SUDEP in DS mice. These findings suggest that SUDEP is caused by apparent parasympathetic hyperactivity immediately following tonic-clonic seizures in DS mice, which leads to lethal bradycardia and electrical dysfunction of the ventricle. These results have important implications for prevention of SUDEP in DS patients.
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 3(10)-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.
CaV2.1 channels, which conduct P/Q-type Ca2+ currents, initiate synaptic transmission at most synapses in the central nervous system. Ca2+/calmodulin-dependent facilitation and inactivation of these channels contributes to short-term facilitation and depression of synaptic transmission, respectively. Other calcium sensor proteins displace calmodulin (CaM) from its binding site, differentially regulate CaV2.1 channels, and contribute to the diversity of short-term synaptic plasticity. The neuronal calcium sensor protein visinin-like protein 2 (VILIP-2) inhibits inactivation and enhances facilitation of CaV2.1 channels. Here we examine the molecular determinants for differential regulation of CaV2.1 channels by VILIP-2 and CaM by construction and functional analysis of chimeras in which the functional domains of VILIP-2 are substituted in CaM. Our results show that the N-terminal domain, including its myristoylation site, the central ?-helix, and the C-terminal lobe containing EF-hands 3 and 4 of VILIP-2 are sufficient to transfer its regulatory properties to CaM. This regulation by VILIP-2 requires binding to the IQ-like domain of CaV2.1 channels. Our results identify the essential molecular determinants of differential regulation of CaV2.1 channels by VILIP-2 and define the molecular code that these proteins use to control short-term synaptic plasticity.
Voltage-gated Na(+) channels initiate action potentials during electrical signaling in excitable cells. Opening and closing of the pore of voltage-gated ion channels are mechanically linked to voltage-driven outward movement of the positively charged S4 transmembrane segment in their voltage sensors. Disulfide locking of cysteine residues substituted for the outermost T0 and R1 gating-charge positions and a conserved negative charge (E43) at the extracellular end of the S1 segment of the bacterial Na(+) channel NaChBac detects molecular interactions that stabilize the resting state of the voltage sensor and define its conformation. Upon depolarization, the more inward gating charges R2 and R3 engage in these molecular interactions as the S4 segment moves outward to its intermediate and activated states. The R4 gating charge does not disulfide-lock with E43, suggesting an outer limit to its transmembrane movement. These molecular interactions reveal how the S4 gating charges are stabilized in the resting state and how their outward movement is catalyzed by interaction with negatively charged residues to effect pore opening and initiate electrical signaling.
Presynaptic Ca(V)2.1 channels, which conduct P/Q-type Ca(2+) currents, initiate synaptic transmission at most synapses in the central nervous system. Regulation of Ca(V)2.1 channels by CaM contributes significantly to short term facilitation and rapid depression of synaptic transmission. Short term synaptic plasticity is diverse in form and function at different synapses, yet CaM is ubiquitously expressed. Differential regulation of Ca(V)2.1 channels by CaM-like Ca(2+) sensor (CaS) proteins differentially affects short term synaptic facilitation and rapid synaptic depression in transfected sympathetic neuron synapses. Here, we define the molecular determinants for differential regulation of Ca(V)2.1 channels by the CaS protein calcium-binding protein-1 (CaBP1) by analysis of chimeras in which the unique structural domains of CaBP1 are inserted into CaM. Our results show that the N-terminal domain, including its myristoylation site, and the second EF-hand, which is inactive in Ca(2+) binding, are the key molecular determinants of differential regulation of Ca(V)2.1 channels by CaBP1. These findings give insight into the molecular code by which CaS proteins differentially regulate Ca(V)2.1 channel function and provide diversity of form and function of short term synaptic plasticity.
The ?-scorpions toxins bind to the resting state of Na(+) channels and inhibit fast inactivation by interaction with a receptor site formed by domains I and IV. Mutants T1560A, F1610A, and E1613A in domain IV had lower affinities for Leiurus quinquestriatus hebraeus toxin II (LqhII), and mutant E1613R had ~73-fold lower affinity. Toxin dissociation was accelerated by depolarization and increased by these mutations, whereas association rates at negative membrane potentials were not changed. These results indicate that Thr1560 in the S1-S2 loop, Phe1610 in the S3 segment, and Glu1613 in the S3-S4 loop in domain IV participate in toxin binding. T393A in the SS2-S6 loop in domain I also had lower affinity for LqhII, indicating that this extracellular loop may form a secondary component of the receptor site. Analysis with the Rosetta-Membrane algorithm resulted in a model of LqhII binding to the voltage sensor in a resting state, in which amino acid residues in an extracellular cleft formed by the S1-S2 and S3-S4 loops in domain IV interact with two faces of the wedge-shaped LqhII molecule. The conserved gating charges in the S4 segment are in an inward position and form ion pairs with negatively charged amino acid residues in the S2 and S3 segments of the voltage sensor. This model defines the structure of the resting state of a voltage sensor of Na(+) channels and reveals its mode of interaction with a gating modifier toxin.
Voltage-gated sodium (Na(v)) channels are the molecular targets of ?-scorpion toxins, which shift the voltage dependence of activation to more negative membrane potentials by a voltage sensor-trapping mechanism. Molecular determinants of ?-scorpion toxin (CssIV) binding and action on rat brain sodium channels are located in the S1-S2 (IIS1-S2) and S3-S4 (IIS3-S4) extracellular linkers of the voltage-sensing module in domain II. In IIS1-S2, mutations of two amino acid residues (Glu(779) and Pro(782)) significantly altered the toxin effect by reducing binding affinity. In IIS3-S4, six positions surrounding the key binding determinant, Gly(845), define a hot spot of high-impact residues. Two of these substitutions (A841N and L846A) reduced voltage sensor trapping. The other three substitutions (N842R, V843A, and E844N) increased voltage sensor trapping. These bidirectional effects suggest that the IIS3-S4 loop plays a primary role in determining both toxin affinity and efficacy. A high resolution molecular model constructed with the Rosetta-Membrane modeling system reveals interactions of amino acid residues in sodium channels that are crucial for toxin action with residues in CssIV that are required for its effects. In this model, the wedge-shaped CssIV inserts between the IIS1-S2 and IIS3-S4 loops of the voltage sensor, placing key amino acid residues in position to interact with binding partners in these extracellular loops. These results provide new molecular insights into the voltage sensor-trapping model of toxin action and further define the molecular requirements for the development of antagonists that can prevent or reverse toxicity of scorpion toxins.
Voltage-gated sodium (Na(V)) channels initiate electrical signalling in excitable cells and are the molecular targets for drugs and disease mutations, but the structural basis for their voltage-dependent activation, ion selectivity and drug block is unknown. Here we report the crystal structure of a voltage-gated Na(+) channel from Arcobacter butzleri (NavAb) captured in a closed-pore conformation with four activated voltage sensors at 2.7?Å resolution. The arginine gating charges make multiple hydrophilic interactions within the voltage sensor, including unanticipated hydrogen bonds to the protein backbone. Comparisons to previous open-pore potassium channel structures indicate that the voltage-sensor domains and the S4-S5 linkers dilate the central pore by pivoting together around a hinge at the base of the pore module. The NavAb selectivity filter is short, ?4.6?Å wide, and water filled, with four acidic side chains surrounding the narrowest part of the ion conduction pathway. This unique structure presents a high-field-strength anionic coordination site, which confers Na(+) selectivity through partial dehydration via direct interaction with glutamate side chains. Fenestrations in the sides of the pore module are unexpectedly penetrated by fatty acyl chains that extend into the central cavity, and these portals are large enough for the entry of small, hydrophobic pore-blocking drugs. This structure provides the template for understanding electrical signalling in excitable cells and the actions of drugs used for pain, epilepsy and cardiac arrhythmia at the atomic level.
Regulation of CaV1.2 channels in cardiac myocytes by the ?-adrenergic pathway requires a signaling complex in which the proteolytically processed distal C-terminal domain acts as an autoinhibitor of channel activity and mediates up-regulation by the ?-adrenergic receptor and PKA bound to A-kinase anchoring protein 15 (AKAP15). We examined the significance of this distal C-terminal signaling complex for CaV1.2 and CaV1.3 channels in neurons. AKAP15 co-immunoprecipitates with CaV1.2 and CaV1.3 channels. AKAP15 has overlapping localization with CaV1.2 and CaV1.3 channels in cell bodies and proximal dendrites and is closely co-localized with CaV1.2 channels in punctate clusters. The neuronal AKAP MAP2B, which also interacts with CaV1.2 and CaV1.3 channels, has complementary localization to AKAP15, suggesting different functional roles in calcium channel regulation. Studies with mice that lack the distal C-terminal domain of CaV1.2 channels (CaV1.2?DCT) reveal that AKAP15 interacts with neuronal CaV1.2 channels via their C terminus in vivo and is co-localized in punctate clusters of CaV1.2 channels via that interaction. CaV1.2?DCT neurons have reduced L-type calcium current, indicating that the distal C-terminal domain is required for normal functional expression in vivo. Deletion of the distal C-terminal domain impairs calcium-dependent signaling from CaV1.2 channels to the nucleus, as shown by reduction in phosphorylation of the cAMP response element-binding protein. Our results define AKAP signaling complexes of CaV1.2 and CaV1.3 channels in brain and reveal three previously unrecognized functional roles for the distal C terminus of neuronal CaV1.2 channels in vivo: increased functional expression, anchoring of AKAP15 and PKA, and initiation of excitation-transcription coupling.
L-type calcium currents conducted by CaV1.2 channels initiate excitation-contraction coupling in cardiac and vascular smooth muscle. In the heart, the distal portion of the C terminus (DCT) is proteolytically processed in vivo and serves as a noncovalently associated autoinhibitor of CaV1.2 channel activity. This autoinhibitory complex, with A-kinase anchoring protein-15 (AKAP15) bound to the DCT, is hypothesized to serve as the substrate for ?-adrenergic regulation in the fight-or-flight response. Mice expressing CaV1.2 channels with the distal C terminus deleted (DCT-/-) develop cardiac hypertrophy and die prematurely after E15. Cardiac hypertrophy and survival rate were improved by drug treatments that reduce peripheral vascular resistance and hypertension, consistent with the hypothesis that CaV1.2 hyperactivity in vascular smooth muscle causes hypertension, hypertrophy, and premature death. However, in contrast to expectation, L-type Ca2+ currents in cardiac myocytes from DCT-/- mice were dramatically reduced due to decreased cell-surface expression of CaV1.2 protein, and the voltage dependence of activation and the kinetics of inactivation were altered. CaV1.2 channels in DCT-/- myocytes fail to respond to activation of adenylyl cyclase by forskolin, and the localized expression of AKAP15 is reduced. Therefore, we conclude that the DCT of CaV1.2 channels is required in vivo for normal vascular regulation, cell-surface expression of CaV1.2 channels in cardiac myocytes, and ?-adrenergic stimulation of L-type Ca2+ currents in the heart.
During the fight-or-flight response, the sympathetic nervous system stimulates L-type calcium ion (Ca2+) currents conducted by Ca(V)1 channels through activation of ?-adrenergic receptors, adenylyl cyclase, and phosphorylation by adenosine 3,5-monophosphate-dependent protein kinase [also known as protein kinase A (PKA)], increasing contractility of skeletal and cardiac muscles. We reconstituted this regulation of cardiac Ca(V)1.2 channels in non-muscle cells by forming an autoinhibitory signaling complex composed of Ca(V)1.2?1800 (a form of the channel truncated at the in vivo site of proteolytic processing), its noncovalently associated distal carboxyl-terminal domain, the auxiliary ???? and ?(2b) subunits, and A-kinase anchoring protein 15 (AKAP15). A factor of 3.6 range of Ca(V)1.2 channel activity was observed from a minimum in the presence of protein kinase inhibitors to a maximum upon activation of adenylyl cyclase. Basal Ca(V)1.2 channel activity in unstimulated cells was regulated by phosphorylation of serine-1700 and threonine-1704, two residues located at the interface between the distal and the proximal carboxyl-terminal regulatory domains, whereas further stimulation of channel activity through the PKA signaling pathway only required phosphorylation of serine-1700. Our results define a conceptual framework for Ca(V)1.2 channel regulation and identify sites of phosphorylation that regulate channel activity.
Voltage-gated sodium channels carry the major inward current responsible for action potential depolarization in excitable cells as well as providing additional inward current that modulates overall excitability. Both their expression and function is under tight control of protein phosphorylation by specific kinases and phosphatases and this control is particular to each type of sodium channel. This article examines the impact and mechanism of phosphorylation for isoforms where it has been studied in detail in an attempt to delineate common features as well as differences.
Scorpion ?-toxin 4 from Centruroides suffusus suffusus (Css4) enhances the activation of voltage-gated sodium channels through a voltage sensor trapping mechanism by binding the activated state of the voltage sensor in domain II and stabilizing it in its activated conformation. Here we describe the antagonist and partial agonist properties of a mutant derivative of this toxin. Substitution of seven different amino acid residues for Glu(15) in Css4 yielded toxin derivatives with both increased and decreased affinities for binding to neurotoxin receptor site 4 on sodium channels. Css4(E15R) is unique among this set of mutants in that it retained nearly normal binding affinity but lost its functional activity for modification of sodium channel gating in our standard electrophysiological assay for voltage sensor trapping. More detailed analysis of the functional effects of Css4(E15R) revealed weak voltage sensor trapping activity, which was very rapidly reversed upon repolarization and therefore was not observed in our standard assay of toxin effects. This partial agonist activity of Css4(E15R) is observed clearly in voltage sensor trapping assays with brief (5 ms) repolarization between the conditioning prepulse and the test pulse. The effects of Css4(E15R) are fit well by a three-step model of toxin action involving concentration-dependent toxin binding to its receptor site followed by depolarization-dependent activation of the voltage sensor and subsequent voltage sensor trapping. Because it is a partial agonist with much reduced efficacy for voltage sensor trapping, Css4(E15R) can antagonize the effects of wild-type Css4 on sodium channel activation and can prevent paralysis by Css4 when injected into mice. Our results define the first partial agonist and antagonist activities for scorpion toxins and open new avenues of research toward better understanding of the structure-function relationships for toxin action on sodium channel voltage sensors and toward potential toxin-based therapeutics to prevent lethality from scorpion envenomation.
Hypokalemic periodic paralysis and normokalemic periodic paralysis are caused by mutations of the gating charge-carrying arginine residues in skeletal muscle Na(V)1.4 channels, which induce gating pore current through the mutant voltage sensor domains. Inward sodium currents through the gating pore of mutant R666G are only approximately 1% of central pore current, but substitution of guanidine for sodium in the extracellular solution increases their size by 13- +/- 2-fold. Ethylguanidine is permeant through the R666G gating pore at physiological membrane potentials but blocks the gating pore at hyperpolarized potentials. Guanidine is also highly permeant through the proton-selective gating pore formed by the mutant R666H. Gating pore current conducted by the R666G mutant is blocked by divalent cations such as Ba(2+) and Zn(2+) in a voltage-dependent manner. The affinity for voltage-dependent block of gating pore current by Ba(2+) and Zn(2+) is increased at more negative holding potentials. The apparent dissociation constant (K(d)) values for Zn(2+) block for test pulses to -160 mV are 650 +/- 150 microM, 360 +/- 70 microM, and 95.6 +/- 11 microM at holding potentials of 0 mV, -80 mV, and -120 mV, respectively. Gating pore current is blocked by trivalent cations, but in a nearly voltage-independent manner, with an apparent K(d) for Gd(3+) of 238 +/- 14 microM at -80 mV. To test whether these periodic paralyses might be treated by blocking gating pore current, we screened several aromatic and aliphatic guanidine derivatives and found that 1-(2,4-xylyl)guanidinium can block gating pore current in the millimolar concentration range without affecting normal Na(V)1.4 channel function. Together, our results demonstrate unique permeability of guanidine through Na(V)1.4 gating pores, define voltage-dependent and voltage-independent block by divalent and trivalent cations, respectively, and provide initial support for the concept that guanidine-based gating pore blockers could be therapeutically useful.
Electrical signaling in biology depends upon a unique electromechanical transduction process mediated by the S4 segments of voltage-gated ion channels. These transmembrane segments are driven outward by the force of the electric field on positively charged amino acid residues termed "gating charges," which are positioned at three-residue intervals in the S4 transmembrane segment, and this movement is coupled to opening of the pore. Here, we use the disulfide-locking method to demonstrate sequential ion pair formation between the fourth gating charge in the S4 segment (R4) and two acidic residues in the S2 segment during activation. R4 interacts first with E70 at the intracellular end of the S2 segment and then with D60 near the extracellular end. Analysis with the Rosetta Membrane method reveals the 3-D structures of the gating pore as these ion pairs are formed sequentially to catalyze the S4 transmembrane movement required for voltage-dependent activation. Our results directly demonstrate sequential ion pair formation that is an essential feature of the sliding helix model of voltage sensor function but is not compatible with the other widely discussed gating models.
L-type Ca(2+) currents conducted by Ca(v)1.2 channels initiate excitation-contraction coupling in cardiac myocytes. Intracellular Mg(2+) (Mg(i)) inhibits the ionic current of Ca(v)1.2 channels. Because Mg(i) is altered in ischemia and heart failure, its regulation of Ca(v)1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg(i) on voltage-dependent inactivation (VDI) of Ca(v)1.2 channels using Na(+) as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca(2+)-dependent inactivation process. We confirmed that increased Mg(i) reduces peak ionic currents and increases VDI of Ca(v)1.2 channels in ventricular myocytes and in transfected cells when measured with Na(+) as permeant ion. The increased rate and extent of VDI caused by increased Mg(i) were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg(i) to the EF-hand (K(D) approximately 0.9 mM) near the resting level of Mg(i) in ventricular myocytes. VDI was more rapid for L-type Ca(2+) currents in ventricular myocytes than for Ca(v)1.2 channels in transfected cells. Coexpression of Ca(v)beta(2b) subunits and formation of an autoinhibitory complex of truncated Ca(v)1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca(v)1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg(i) binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca(v)1.2 channels by Mg(i), the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.
Voltage-gated sodium channels are composed of pore-forming alpha- and auxiliary beta-subunits and are responsible for the rapid depolarization of cardiac action potentials. Recent evidence indicates that neuronal tetrodotoxin (TTX) sensitive sodium channel alpha-subunits are expressed in the heart in addition to the predominant cardiac TTX-resistant Na(v)1.5 sodium channel alpha-subunit. These TTX-sensitive isoforms are preferentially localized in the transverse tubules of rodents. Since neonatal cardiomyocytes have yet to develop transverse tubules, we determined the complement of sodium channel subunits expressed in these cells. Neonatal rat ventricular cardiomyocytes were stained with antibodies specific for individual isoforms of sodium channel alpha- and beta-subunits. alpha-actinin, a component of the z-line, was used as an intracellular marker of sarcomere boundaries. TTX-sensitive sodium channel alpha-subunit isoforms Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4 and Na(v)1.6 were detected in neonatal rat heart but at levels reduced compared to the predominant cardiac alpha-subunit isoform, Na(v)1.5. Each of the beta-subunit isoforms (beta1-beta4) was also expressed in neonatal cardiac cells. In contrast to adult cardiomyocytes, the alpha-subunits are distributed in punctate clusters across the membrane surface of neonatal cardiomyocytes; no isoform-specific subcellular localization is observed. Voltage clamp recordings in the absence and presence of 20 nM TTX provided functional evidence for the presence of TTX-sensitive sodium current in neonatal ventricular myocardium which represents between 20 and 30% of the current, depending on membrane potential and experimental conditions. Thus, as in the adult heart, a range of sodium channel alpha-subunits are expressed in neonatal myocytes in addition to the predominant TTX-resistant Na(v)1.5 alpha-subunit and they contribute to the total sodium current.
Heterozygous loss-of-function mutations in the alpha subunit of the type I voltage-gated sodium channel Na(V)1.1 cause severe myoclonic epilepsy in infancy (SMEI), an infantile-onset epileptic encephalopathy characterized by normal development followed by treatment-refractory febrile and afebrile seizures and psychomotor decline. Mice with SMEI (mSMEI), created by heterozygous deletion of Na(V)1.1 channels, develop seizures and ataxia. Here we investigated the temperature and age dependence of seizures and interictal epileptiform spike-and-wave activity in mSMEI. Combined video-EEG monitoring demonstrated that mSMEI had seizures induced by elevated body core temperature but wild-type mice were unaffected. In the 3 age groups tested, no postnatal day (P)17-18 mSMEI had temperature-induced seizures, but nearly all P20-22 and P30-46 mSMEI had myoclonic seizures followed by generalized seizures caused by elevated core body temperature. Spontaneous seizures were only observed in mice older than P32, suggesting that mSMEI become susceptible to temperature-induced seizures before spontaneous seizures. Interictal spike activity was seen at normal body temperature in most P30-46 mSMEI but not in P20-22 or P17-18 mSMEI, indicating that interictal epileptic activity correlates with seizure susceptibility. Most P20-22 mSMEI had interictal spike activity with elevated body temperature. Our results define a critical developmental transition for susceptibility to seizures in SMEI, demonstrate that body temperature elevation alone is sufficient to induce seizures, and reveal a close correspondence between human and mouse SMEI in the striking temperature and age dependence of seizure frequency and severity and in the temperature dependence and frequency of interictal epileptiform spike activity.
Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca(V)2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca(2+)/CaM stimulus. Autophosphorylated CaMKII can bind the Ca(V)2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca(V)2.1 channels induces Ca(2+)-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca(V)2.1 channels by binding of Ca(2+)/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca(V)2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca(V)2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.
Increases in intracellular Mg(2+) (Mg(2+)(i)), as observed in transient cardiac ischemia, decrease L-type Ca(2+) current of mammalian ventricular myocytes (VMs). However, cardiac ischemia is associated with an increase in sympathetic tone, which could stimulate L-type Ca(2+) current. Therefore, the effect of Mg(2+)(i) on L-type Ca(2+) current in the context of increased sympathetic tone was unclear. We tested the impact of increased Mg(2+)(i) on the ?-adrenergic stimulation of L-type Ca(2+) current. Exposure of acutely dissociated adult VMs to higher Mg(2+)(i) concentrations decreased isoproterenol stimulation of the L-type Ca(2+) current from 75 ± 13% with 0.8 mM Mg(2+)(i) to 20 ± 8% with 2.4 mM Mg(2+)(i). We activated this signaling cascade at different steps to determine the site or sites of Mg(2+)(i) action. Exposure of VMs to increased Mg(2+)(i) attenuated the stimulation of L-type Ca(2+) current induced by activation of adenylyl cyclase with forskolin, inhibition of cyclic nucleotide phosphodiesterases with isobutylmethylxanthine, and inhibition of phosphoprotein phosphatases I and IIA with calyculin A. These experiments ruled out significant effects of Mg(2+)(i) on these upstream steps in the signaling cascade and suggested that Mg(2+)(i) acts directly on Ca(V)1.2 channels. One possible site of action is the EF-hand in the proximal C-terminal domain, just downstream in the signaling cascade from the site of regulation of Ca(V)1.2 channels by protein phosphorylation on the C terminus. Consistent with this hypothesis, Mg(2+)(i) had no effect on enhancement of Ca(V)1.2 channel activity by the dihydropyridine agonist (S)-BayK8644, which activates Ca(V)1.2 channels by binding to a site formed by the transmembrane domains of the channel. Collectively, our results suggest that, in transient ischemia, increased Mg(2+)(i) reduces stimulation of L-type Ca(2+) current by the ?-adrenergic receptor by directly acting on Ca(V)1.2 channels in a cell-autonomous manner, effectively decreasing the metabolic stress imposed on VMs until blood flow can be reestablished.
Modulation of P/Q-type Ca(2+) currents through presynaptic voltage-gated calcium channels (Ca(V)2.1) by binding of Ca(2+)/calmodulin contributes to short-term synaptic plasticity. Ca(2+)-binding protein-1 (CaBP1) and Visinin-like protein-2 (VILIP-2) are neurospecific calmodulin-like Ca(2+) sensor proteins that differentially modulate Ca(V)2.1 channels, but how they contribute to short-term synaptic plasticity is unknown. Here, we show that activity-dependent modulation of presynaptic Ca(V)2.1 channels by CaBP1 and VILIP-2 has opposing effects on short-term synaptic plasticity in superior cervical ganglion neurons. Expression of CaBP1, which blocks Ca(2+)-dependent facilitation of P/Q-type Ca(2+) current, markedly reduced facilitation of synaptic transmission. VILIP-2, which blocks Ca(2+)-dependent inactivation of P/Q-type Ca(2+) current, reduced synaptic depression and increased facilitation under conditions of high release probability. These results demonstrate that activity-dependent regulation of presynaptic Ca(V)2.1 channels by differentially expressed Ca(2+) sensor proteins can fine-tune synaptic responses to trains of action potentials and thereby contribute to the diversity of short-term synaptic plasticity.
Haploinsufficiency of the SCN1A gene encoding voltage-gated sodium channel Na(V)1.1 causes Dravets syndrome, a childhood neuropsychiatric disorder including recurrent intractable seizures, cognitive deficit and autism-spectrum behaviours. The neural mechanisms responsible for cognitive deficit and autism-spectrum behaviours in Dravets syndrome are poorly understood. Here we report that mice with Scn1a haploinsufficiency exhibit hyperactivity, stereotyped behaviours, social interaction deficits and impaired context-dependent spatial memory. Olfactory sensitivity is retained, but novel food odours and social odours are aversive to Scn1a(+/-) mice. GABAergic neurotransmission is specifically impaired by this mutation, and selective deletion of Na(V)1.1 channels in forebrain interneurons is sufficient to cause these behavioural and cognitive impairments. Remarkably, treatment with low-dose clonazepam, a positive allosteric modulator of GABA(A) receptors, completely rescued the abnormal social behaviours and deficits in fear memory in the mouse model of Dravets syndrome, demonstrating that they are caused by impaired GABAergic neurotransmission and not by neuronal damage from recurrent seizures. These results demonstrate a critical role for Na(V)1.1 channels in neuropsychiatric functions and provide a potential therapeutic strategy for cognitive deficit and autism-spectrum behaviours in Dravets syndrome.
Activation of voltage-gated sodium (Na(v)) channels initiates and propagates action potentials in electrically excitable cells. ?-Scorpion toxins, including toxin IV from Centruroides suffusus suffusus (CssIV), enhance activation of Na(V) channels. CssIV stabilizes the voltage sensor in domain II in its activated state via a voltage-sensor trapping mechanism. Amino acid residues required for the action of CssIV have been identified in the S1-S2 and S3-S4 extracellular loops of domain II. The extracellular loops of domain III are also involved in toxin action, but individual amino acid residues have not been identified. We used site-directed mutagenesis and voltage clamp recording to investigate amino acid residues of domain III that are involved in CssIV action. In the IIISS2-S6 loop, five substitutions at four positions altered voltage-sensor trapping by CssIV(E15A). Three substitutions (E1438A, D1445A, and D1445Y) markedly decreased voltage-sensor trapping, whereas the other two substitutions (N1436G and L1439A) increased voltage-sensor trapping. These bidirectional effects suggest that residues in IIISS2-S6 make both positive and negative interactions with CssIV. N1436G enhanced voltage-sensor trapping via increased binding affinity to the resting state, whereas L1439A increased voltage-sensor trapping efficacy. Based on these results, a three-dimensional model of the toxin-channel interaction was developed using the Rosetta modeling method. These data provide additional molecular insight into the voltage-sensor trapping mechanism of toxin action and define a three-point interaction site for ?-scorpion toxins on Na(V) channels. Binding of ?- and ?-scorpion toxins to two distinct, pseudo-symmetrically organized receptor sites on Na(V) channels acts synergistically to modify channel gating and paralyze prey.
In excitable cells, voltage-gated sodium (Na(V)) channels activate to initiate action potentials and then undergo fast and slow inactivation processes that terminate their ionic conductance. Inactivation is a hallmark of Na(V) channel function and is critical for control of membrane excitability, but the structural basis for this process has remained elusive. Here we report crystallographic snapshots of the wild-type Na(V)Ab channel from Arcobacter butzleri captured in two potentially inactivated states at 3.2?Å resolution. Compared to previous structures of Na(V)Ab channels with cysteine mutations in the pore-lining S6 helices (ref. 4), the S6 helices and the intracellular activation gate have undergone significant rearrangements: one pair of S6 helices has collapsed towards the central pore axis and the other S6 pair has moved outward to produce a striking dimer-of-dimers configuration. An increase in global structural asymmetry is observed throughout our wild-type Na(V)Ab models, reshaping the ion selectivity filter at the extracellular end of the pore, the central cavity and its residues that are analogous to the mammalian drug receptor site, and the lateral pore fenestrations. The voltage-sensing domains have also shifted around the perimeter of the pore module in wild-type Na(V)Ab, compared to the mutant channel, and local structural changes identify a conserved interaction network that connects distant molecular determinants involved in Na(V) channel gating and inactivation. These potential inactivated-state structures provide new insights into Na(V) channel gating and novel avenues to drug development and therapy for a range of debilitating Na(V) channelopathies.
We have identified an asynchronously activated Ca(2+) current through voltage-gated Ca(2+) (Ca(V))-2.1 and Ca(V)2.2 channels, which conduct P/Q- and N-type Ca(2+) currents that initiate neurotransmitter release. In nonneuronal cells expressing Ca(V)2.1 or Ca(V)2.2 channels and in hippocampal neurons, prolonged Ca(2+) entry activates a Ca(2+) current, I(Async), which is observed on repolarization and decays slowly with a half-time of 150-300 ms. I(Async) is not observed after L-type Ca(2+) currents of similar size conducted by Ca(V)1.2 channels. I(Async) is Ca(2+)-selective, and it is unaffected by changes in Na(+), K(+), Cl(-), or H(+) or by inhibitors of a broad range of ion channels. During trains of repetitive depolarizations, I(Async) increases in a pulse-wise manner, providing Ca(2+) entry that persists between depolarizations. In single-cultured hippocampal neurons, trains of depolarizations evoke excitatory postsynaptic currents that show facilitation followed by depression accompanied by asynchronous postsynaptic currents that increase steadily during the train in parallel with I(Async). I(Async) is much larger for slowly inactivating Ca(V)2.1 channels containing ?(2a)-subunits than for rapidly inactivating channels containing ?(1b)-subunits. I(Async) requires global rises in intracellular Ca(2+), because it is blocked when Ca(2+) is chelated by 10 mM EGTA in the patch pipette. Neither mutations that prevent Ca(2+) binding to calmodulin nor mutations that prevent calmodulin regulation of Ca(V)2.1 block I(Async). The rise of I(Async) during trains of stimuli, its decay after repolarization, its dependence on global increases of Ca(2+), and its enhancement by ?(2a)-subunits all resemble asynchronous release, suggesting that I(Async) is a Ca(2+) source for asynchronous neurotransmission.
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