Altered closed state inactivation gating in Kv4.2 channels results in developmental and epileptic encephalopathies in human patients.

Kv4.2 subunits, encoded by KCND2, serve as the pore-forming components of voltage-gated, inactivating ISA K+ channels expressed in the brain. ISA channels inactivate without opening in response to subthreshold excitatory input, temporarily increasing neuronal excitability, the back propagation of action potentials, and Ca2+ influx into dendrites, thereby regulating mechanisms of spike timing-dependent synaptic plasticity. As previously described, a de novo variant in Kv4.2, p.Val404Met, is associated with an infant-onset developmental and epileptic encephalopathy in monozygotic twin boys. The p.Val404Met variant enhances inactivation directly from closed states, but dramatically impairs inactivation after channel opening. We now report the identification of a closely related, novel, de novo variant in Kv4.2, p.Val402Leu, in a boy with an early-onset pharmacoresistant epilepsy that evolved to an epileptic aphasia syndrome (Continuous Spike Wave during Sleep Syndrome). Like p.Val404Met, the p.Val402Leu variant increases the rate of inactivation from closed states, but significantly slows inactivation after the pore opens. Although quantitatively the p.Val402Leu mutation alters channel kinetics less dramatically than p.Val404Met, our results strongly support the conclusion that p.Val402Leu and p.Val404Met cause the clinical features seen in the affected individuals and underscore the importance of closed state inactivation in ISA channels in normal brain development and function.

Infant and adult SCA13 mutations differentially affect Purkinje cell excitability, maturation, and viability in vivo.

Mutations in KCNC3, which encodes the Kv3.3 K+ channel, cause spinocerebellar ataxia 13 (SCA13). SCA13 exists in distinct forms with onset in infancy or adulthood. Using zebrafish, we tested the hypothesis that infant- and adult-onset mutations differentially affect the excitability and viability of Purkinje cells in vivo during cerebellar development. An infant-onset mutation dramatically and transiently increased Purkinje cell excitability, stunted process extension, impaired dendritic branching and synaptogenesis, and caused rapid cell death during cerebellar development. Reducing excitability increased early Purkinje cell survival. In contrast, an adult-onset mutation did not significantly alter basal tonic firing in Purkinje cells, but reduced excitability during evoked high frequency spiking. Purkinje cells expressing the adult-onset mutation matured normally and did not degenerate during cerebellar development. Our results suggest that differential changes in the excitability of cerebellar neurons contribute to the distinct ages of onset and timing of cerebellar degeneration in infant- and adult-onset SCA13.

Kv4.2 autism and epilepsy mutation enhances inactivation of closed channels but impairs access to inactivated state after opening.

A de novo mutation in the KCND2 gene, which encodes the Kv4.2 K+ channel, was identified in twin boys with intractable, infant-onset epilepsy and autism. Kv4.2 channels undergo closed-state inactivation (CSI), a mechanism by which channels inactivate without opening during subthreshold depolarizations. CSI dynamically modulates neuronal excitability and action potential back propagation in response to excitatory synaptic input, controlling Ca2+ influx into dendrites and regulating spike timing-dependent plasticity. Here, we show that the V404M mutation specifically affects the mechanism of CSI, enhancing the inactivation of channels that have not opened while dramatically impairing the inactivation of channels that have opened. The mutation gives rise to these opposing effects by increasing the stability of the inactivated state and in parallel, profoundly slowing the closure of open channels, which according to our data, is required for CSI. The larger volume of methionine compared with valine is a major factor underlying altered inactivation gating. Our results suggest that V404M increases the strength of the physical interaction between the pore gate and the voltage sensor regardless of whether the gate is open or closed. Furthermore, in contrast to previous proposals, our data strongly suggest that physical coupling between the voltage sensor and the pore gate is maintained in the inactivated state. The state-dependent effects of V404M on CSI are expected to disturb the regulation of neuronal excitability and the induction of spike timing-dependent plasticity. Our results strongly support a role for altered CSI gating in the etiology of epilepsy and autism in the affected twins.

In Vivo Analysis of Potassium Channelopathies: Loose Patch Recording of Purkinje Cell Firing in Living, Awake Zebrafish.

Zebrafish is a lower vertebrate model organism that facilitates integrative analysis of the in vivo effects of potassium and other ion channel mutations at the molecular, cellular, developmental, circuit, systems, and behavioral levels of analysis. Here, we describe a method for extracellular, loose patch electrophysiological recording of electrical activity in cerebellar Purkinje cells in living, awake zebrafish, with the goal of investigating pathological mechanisms underlying channelopathies or other diseases that disrupt cerebellar function. Purkinje cell excitability and a functional cerebellar circuit develop rapidly in zebrafish and show strong conservation with the mammalian cerebellum.

Spinocerebellar ataxia type 19/22 mutations alter heterocomplex Kv4.3 channel function and gating in a dominant manner.

The dominantly inherited cerebellar ataxias are a heterogeneous group of neurodegenerative disorders caused by Purkinje cell loss in the cerebellum. Recently, we identified loss-of-function mutations in the KCND3 gene as the cause of spinocerebellar ataxia type 19/22 (SCA19/22), revealing a previously unknown role for the voltage-gated potassium channel, Kv4.3, in Purkinje cell survival. However, how mutant Kv4.3 affects wild-type Kv4.3 channel functioning remains unknown. We provide evidence that SCA19/22-mutant Kv4.3 exerts a dominant negative effect on the trafficking and surface expression of wild-type Kv4.3 in the absence of its regulatory subunit, KChIP2. Notably, this dominant negative effect can be rescued by the presence of KChIP2. We also found that all SCA19/22-mutant subunits either suppress wild-type Kv4.3 current amplitude or alter channel gating in a dominant manner. Our findings suggest that altered Kv4.3 channel localization and/or functioning resulting from SCA19/22 mutations may lead to Purkinje cell loss, neurodegeneration and ataxia.

Exome sequencing identifies de novo gain of function missense mutation in KCND2 in identical twins with autism and seizures that slows potassium channel inactivation.

Numerous studies and case reports show comorbidity of autism and epilepsy, suggesting some common molecular underpinnings of the two phenotypes. However, the relationship between the two, on the molecular level, remains unclear. Here, whole exome sequencing was performed on a family with identical twins affected with autism and severe, intractable seizures. A de novo variant was identified in the KCND2 gene, which encodes the Kv4.2 potassium channel. Kv4.2 is a major pore-forming subunit in somatodendritic subthreshold A-type potassium current (ISA) channels. The de novo mutation p.Val404Met is novel and occurs at a highly conserved residue within the C-terminal end of the transmembrane helix S6 region of the ion permeation pathway. Functional analysis revealed the likely pathogenicity of the variant in that the p.Val404Met mutant construct showed significantly slowed inactivation, either by itself or after equimolar coexpression with the wild-type Kv4.2 channel construct consistent with a dominant effect. Further, the effect of the mutation on closed-state inactivation was evident in the presence of auxiliary subunits that associate with Kv4 subunits to form ISA channels in vivo. Discovery of a functionally relevant novel de novo variant, coupled with physiological evidence that the mutant protein disrupts potassium current inactivation, strongly supports KCND2 as the causal gene for epilepsy in this family. Interaction of KCND2 with other genes implicated in autism and the role of KCND2 in synaptic plasticity provide suggestive evidence of an etiological role in autism.

Rapid development of Purkinje cell excitability, functional cerebellar circuit, and afferent sensory input to cerebellum in zebrafish.

The zebrafish has significant advantages for studying the morphological development of the brain. However, little is known about the functional development of the zebrafish brain. We used patch clamp electrophysiology in live animals to investigate the emergence of excitability in cerebellar Purkinje cells, functional maturation of the cerebellar circuit, and establishment of sensory input to the cerebellum. Purkinje cells are born at 3 days post-fertilization (dpf). By 4 dpf, Purkinje cells spontaneously fired action potentials in an irregular pattern. By 5 dpf, the frequency and regularity of tonic firing had increased significantly and most cells fired complex spikes in response to climbing fiber activation. Our data suggest that, as in mammals, Purkinje cells are initially innervated by multiple climbing fibers that are winnowed to a single input. To probe the development of functional sensory input to the cerebellum, we investigated the response of Purkinje cells to a visual stimulus consisting of a rapid change in light intensity. At 4 dpf, sudden darkness increased the rate of tonic firing, suggesting that afferent pathways carrying visual information are already active by this stage. By 5 dpf, visual stimuli also activated climbing fibers, increasing the frequency of complex spiking. Our results indicate that the electrical properties of zebrafish and mammalian Purkinje cells are highly conserved and suggest that the same ion channels, Nav1.6 and Kv3.3, underlie spontaneous pacemaking activity. Interestingly, functional development of the cerebellum is temporally correlated with the emergence of complex, visually-guided behaviors such as prey capture. Because of the rapid formation of an electrically-active cerebellum, optical transparency, and ease of genetic manipulation, the zebrafish has great potential for functionally mapping cerebellar afferent and efferent pathways and for investigating cerebellar control of motor behavior.

Spinocerebellar ataxia type 13 mutation that is associated with disease onset in infancy disrupts axonal pathfinding during neuronal development.

Spinocerebellar ataxia type 13 (SCA13) is an autosomal dominant disease caused by mutations in the Kv3.3 voltage-gated potassium (K(+)) channel. SCA13 exists in two forms: infant onset is characterized by severe cerebellar atrophy, persistent motor deficits and intellectual disability, whereas adult onset is characterized by progressive ataxia and progressive cerebellar degeneration. To test the hypothesis that infant- and adult-onset mutations have differential effects on neuronal development that contribute to the age at which SCA13 emerges, we expressed wild-type Kv3.3 or infant- or adult-onset mutant proteins in motor neurons in the zebrafish spinal cord. We characterized the development of CaP (caudal primary) motor neurons at ∼36 and ∼48 hours post-fertilization using confocal microscopy and 3D digital reconstruction. Exogenous expression of wild-type Kv3.3 had no significant effect on CaP development. In contrast, CaP neurons expressing the infant-onset mutation made frequent pathfinding errors, sending long, abnormal axon collaterals into muscle territories that are normally innervated exclusively by RoP (rostral primary) or MiP (middle primary) motor neurons. This phenotype might be directly relevant to infant-onset SCA13 because interaction with inappropriate synaptic partners might trigger cell death during brain development. Importantly, pathfinding errors were not detected in CaP neurons expressing the adult-onset mutation. However, the adult-onset mutation tended to increase the complexity of the distal axonal arbor. From these results, we speculate that infant-onset SCA13 is associated with marked changes in the development of Kv3.3-expressing cerebellar neurons, reducing their health and viability early in life and resulting in the withered cerebellum seen in affected children.

Altered Kv3.3 channel gating in early-onset spinocerebellar ataxia type 13.

Mutations in Kv3.3 cause spinocerebellar ataxia type 13 (SCA13). Depending on the causative mutation, SCA13 is either a neurodevelopmental disorder that is evident in infancy or a progressive neurodegenerative disease that emerges during adulthood. Previous studies did not clarify the relationship between these distinct clinical phenotypes and the effects of SCA13 mutations on Kv3.3 function. The F448L mutation alters channel gating and causes early-onset SCA13. R420H and R423H suppress Kv3 current amplitude by a dominant negative mechanism. However, R420H results in the adult form of the disease whereas R423H produces the early-onset, neurodevelopmental form with significant clinical overlap with F448L. Since individuals with SCA13 have one wild type and one mutant allele of the Kv3.3 gene, we analysed the properties of tetrameric channels formed by mixtures of wild type and mutant subunits. We report that one R420H subunit and at least one R423H subunit can co-assemble with the wild type protein to form active channels. The functional properties of channels containing R420H and wild type subunits strongly resemble those of wild type alone. In contrast, channels containing R423H and wild type subunits show significantly altered gating, including a hyperpolarized shift in the voltage dependence of activation, slower activation, and modestly slower deactivation. Notably, these effects resemble the modified gating seen in channels containing a mixture of F448L and wild type subunits, although the F448L subunit slows deactivation more dramatically than the R423H subunit. Our results suggest that the clinical severity of R423H reflects its dual dominant negative and dominant gain of function effects. However, as shown by R420H, reducing current amplitude without altering gating does not result in infant onset disease. Therefore, our data strongly suggest that changes in Kv3.3 gating contribute significantly to an early age of onset in SCA13.

R1 in the Shaker S4 occupies the gating charge transfer center in the resting state.

During voltage-dependent activation in Shaker channels, four arginine residues in the S4 segment (R1-R4) cross the transmembrane electric field. It has been proposed that R1-R4 movement is facilitated by a "gating charge transfer center" comprising a phenylalanine (F290) in S2 plus two acidic residues, one each in S2 and S3. According to this proposal, R1 occupies the charge transfer center in the resting state, defined as the conformation in which S4 is maximally retracted toward the cytoplasm. However, other evidence suggests that R1 is located extracellular to the charge transfer center, near I287 in S2, in the resting state. To investigate the resting position of R1, we mutated I287 to histidine (I287H), paired it with histidine mutations of key voltage sensor residues, and determined the effect of extracellular Zn(2+) on channel activity. In I287H+R1H, Zn(2+) generated a slow component of activation with a maximum amplitude (A(slow,max)) of ∼56%, indicating that only a fraction of voltage sensors can bind Zn(2+) at a holding potential of -80 mV. A(slow,max) decreased after applying either depolarizing or hyperpolarizing prepulses from -80 mV. The decline of A(slow,max) after negative prepulses indicates that R1 moves inward to abolish ion binding, going beyond the point where reorientation of the I287H and R1H side chains would reestablish a binding site. These data support the proposal that R1 occupies the charge transfer center upon hyperpolarization. Consistent with this, pairing I287H with A359H in the S3-S4 loop generated a Zn(2+)-binding site. At saturating concentrations, A(slow,max) reached 100%, indicating that Zn(2+) traps the I287H+A359H voltage sensor in an absorbing conformation. Transferring I287H+A359H into a mutant background that stabilizes the resting state significantly enhanced Zn(2+) binding at -80 mV. Our results strongly support the conclusion that R1 occupies the gating charge transfer center in the resting conformation.

Spinocerebellar ataxia type 13 mutant potassium channel alters neuronal excitability and causes locomotor deficits in zebrafish.

Whether changes in neuronal excitability can cause neurodegenerative disease in the absence of other factors such as protein aggregation is unknown. Mutations in the Kv3.3 voltage-gated K(+) channel cause spinocerebellar ataxia type 13 (SCA13), a human autosomal-dominant disease characterized by locomotor impairment and the death of cerebellar neurons. Kv3.3 channels facilitate repetitive, high-frequency firing of action potentials, suggesting that pathogenesis in SCA13 is triggered by changes in electrical activity in neurons. To investigate whether SCA13 mutations alter excitability in vivo, we expressed the human dominant-negative R420H mutant subunit in zebrafish. The disease-causing mutation specifically suppressed the excitability of Kv3.3-expressing, fast-spiking motor neurons during evoked firing and fictive swimming and, in parallel, decreased the precision and amplitude of the startle response. The dominant-negative effect of the mutant subunit on K(+) current amplitude was directly responsible for the reduced excitability and locomotor phenotype. Our data provide strong evidence that changes in excitability initiate pathogenesis in SCA13 and establish zebrafish as an excellent model system for investigating how changes in neuronal activity impair locomotor control and cause cell death.

Frequency of KCNC3 DNA variants as causes of spinocerebellar ataxia 13 (SCA13).

BACKGROUND: Gain-of function or dominant-negative mutations in the voltage-gated potassium channel KCNC3 (Kv3.3) were recently identified as a cause of autosomal dominant spinocerebellar ataxia. Our objective was to describe the frequency of mutations associated with KCNC3 in a large cohort of index patients with sporadic or familial ataxia presenting to three US ataxia clinics at academic medical centers. METHODOLOGY: DNA sequence analysis of the coding region of the KCNC3 gene was performed in 327 index cases with ataxia. Analysis of channel function was performed by expression of DNA variants in Xenopus oocytes. PRINCIPAL FINDINGS: Sequence analysis revealed two non-synonymous substitutions in exon 2 and five intronic changes, which were not predicted to alter splicing. We identified another pedigree with the p.Arg423His mutation in the highly conserved S4 domain of this channel. This family had an early-onset of disease and associated seizures in one individual. The second coding change, p.Gly263Asp, subtly altered biophysical properties of the channel, but was unlikely to be disease-associated as it occurred in an individual with an expansion of the CAG repeat in the CACNA1A calcium channel. CONCLUSIONS: Mutations in KCNC3 are a rare cause of spinocerebellar ataxia with a frequency of less than 1%. The p.Arg423His mutation is recurrent in different populations and associated with early onset. In contrast to previous p.Arg423His mutation carriers, we now observed seizures and mild mental retardation in one individual. This study confirms the wide phenotypic spectrum in SCA13.

Neural circuit activity in freely behaving zebrafish (Danio rerio).

Examining neuronal network activity in freely behaving animals is advantageous for probing the function of the vertebrate central nervous system. Here, we describe a simple, robust technique for monitoring the activity of neural circuits in unfettered, freely behaving zebrafish (Danio rerio). Zebrafish respond to unexpected tactile stimuli with short- or long-latency escape behaviors, which are mediated by distinct neural circuits. Using dipole electrodes immersed in the aquarium, we measured electric field potentials generated in muscle during short- and long-latency escapes. We found that activation of the underlying neural circuits produced unique field potential signatures that are easily recognized and can be repeatedly monitored. In conjunction with behavioral analysis, we used this technique to track changes in the pattern of circuit activation during the first week of development in animals whose trigeminal sensory neurons were unilaterally ablated. One day post-ablation, the frequency of short- and long-latency responses was significantly lower on the ablated side than on the intact side. Three days post-ablation, a significant fraction of escapes evoked by stimuli on the ablated side was improperly executed, with the animal turning towards rather than away from the stimulus. However, the overall response rate remained low. Seven days post-ablation, the frequency of escapes increased dramatically and the percentage of improperly executed escapes declined. Our results demonstrate that trigeminal ablation results in rapid reconfiguration of the escape circuitry, with reinnervation by new sensory neurons and adaptive changes in behavior. This technique is valuable for probing the activity, development, plasticity and regeneration of neural circuits under natural conditions.

Functional effects of spinocerebellar ataxia type 13 mutations are conserved in zebrafish Kv3.3 channels.

BACKGROUND: The zebrafish has been suggested as a model system for studying human diseases that affect nervous system function and motor output. However, few of the ion channels that control neuronal activity in zebrafish have been characterized. Here, we have identified zebrafish orthologs of voltage-dependent Kv3 (KCNC) K+ channels. Kv3 channels have specialized gating properties that facilitate high-frequency, repetitive firing in fast-spiking neurons. Mutations in human Kv3.3 cause spinocerebellar ataxia type 13 (SCA13), an autosomal dominant genetic disease that exists in distinct neurodevelopmental and neurodegenerative forms. To assess the potential usefulness of the zebrafish as a model system for SCA13, we have characterized the functional properties of zebrafish Kv3.3 channels with and without mutations analogous to those that cause SCA13. RESULTS: The zebrafish genome (release Zv8) contains six Kv3 family members including two Kv3.1 genes (kcnc1a and kcnc1b), one Kv3.2 gene (kcnc2), two Kv3.3 genes (kcnc3a and kcnc3b), and one Kv3.4 gene (kcnc4). Both Kv3.3 genes are expressed during early development. Zebrafish Kv3.3 channels exhibit strong functional and structural homology with mammalian Kv3.3 channels. Zebrafish Kv3.3 activates over a depolarized voltage range and deactivates rapidly. An amino-terminal extension mediates fast, N-type inactivation. The kcnc3a gene is alternatively spliced, generating variant carboxyl-terminal sequences. The R335H mutation in the S4 transmembrane segment, analogous to the SCA13 mutation R420H, eliminates functional expression. When co-expressed with wild type, R335H subunits suppress Kv3.3 activity by a dominant negative mechanism. The F363L mutation in the S5 transmembrane segment, analogous to the SCA13 mutation F448L, alters channel gating. F363L shifts the voltage range for activation in the hyperpolarized direction and dramatically slows deactivation. CONCLUSIONS: The functional properties of zebrafish Kv3.3 channels are consistent with a role in facilitating fast, repetitive firing of action potentials in neurons. The functional effects of SCA13 mutations are well conserved between human and zebrafish Kv3.3 channels. The high degree of homology between human and zebrafish Kv3.3 channels suggests that the zebrafish will be a useful model system for studying pathogenic mechanisms in SCA13.

Transfer of ion binding site from ether-a-go-go to Shaker: Mg2+ binds to resting state to modulate channel opening.

In ether-à-go-go (eag) K(+) channels, extracellular divalent cations bind to the resting voltage sensor and thereby slow activation. Two eag-specific acidic residues in S2 and S3b coordinate the bound ion. Residues located at analogous positions are approximately 4 A apart in the x-ray structure of a Kv1.2/Kv2.1 chimera crystallized in the absence of a membrane potential. It is unknown whether these residues remain in proximity in Kv1 channels at negative voltages when the voltage sensor domain is in its resting conformation. To address this issue, we mutated Shaker residues I287 and F324, which correspond to the binding site residues in eag, to aspartate and recorded ionic and gating currents in the presence and absence of extracellular Mg(2+). In I287D+F324D, Mg(2+) significantly increased the delay before ionic current activation and slowed channel opening with no readily detectable effect on closing. Because the delay before Shaker opening reflects the initial phase of voltage-dependent activation, the results indicate that Mg(2+) binds to the voltage sensor in the resting conformation. Supporting this conclusion, Mg(2+) shifted the voltage dependence and slowed the kinetics of gating charge movement. Both the I287D and F324D mutations were required to modulate channel function. In contrast, E283, a highly conserved residue in S2, was not required for Mg(2+) binding. Ion binding affected activation by shielding the negatively charged side chains of I287D and F324D. These results show that the engineered divalent cation binding site in Shaker strongly resembles the naturally occurring site in eag. Our data provide a novel, short-range structural constraint for the resting conformation of the Shaker voltage sensor and are valuable for evaluating existing models for the resting state and voltage-dependent conformational changes that occur during activation. Comparing our data to the chimera x-ray structure, we conclude that residues in S2 and S3b remain in proximity throughout voltage-dependent activation.

KCNC3: phenotype, mutations, channel biophysics-a study of 260 familial ataxia patients.

We recently identified KCNC3, encoding the Kv3.3 voltage-gated potassium channel, as the gene mutated in SCA13. One g.10684G>A (p.Arg420His) mutation caused late-onset ataxia resulting in a nonfunctional channel subunit with dominant-negative properties. A French early-onset pedigree with mild mental retardation segregated a g.10767T>C (p.Phe448Leu) mutation. This mutation changed the relative stability of the channel's open conformation. Coding exons were amplified and sequenced in 260 autosomal-dominant ataxia index cases of European descent. Functional analyses were performed using expression in Xenopus oocytes. The previously identified p.Arg420His mutation occurred in three families with late-onset ataxia. A novel mutation g.10693G>A (p.Arg423His) was identified in two families with early-onset. In one pedigree, a novel g.10522G>A (p.Arg366His) sequence variant was seen in one index case but did not segregate with affected status in the respective family. In a heterologous expression system, the p.Arg423His mutation exhibited dominant-negative properties. The p.Arg420His mutation, which results in a nonfunctional channel subunit, was recurrent and associated with late-onset progressive ataxia. In two families the p.Arg423His mutation was associated with early-onset slow-progressive ataxia. Despite a phenotype reminiscent of the p.Phe448Leu mutation, segregating in a large early-onset French pedigree, the p.Arg423His mutation resulted in a nonfunctional subunit with a strong dominant-negative effect.

Voltage-dependent conformational changes of KVAP S4 segment in bacterial membrane environment.

The nature and magnitude of voltage sensor conformational changes during ion channel activation are controversial. We have analyzed the topology of the K(V)AP voltage sensor domain in the absence and presence of a hyperpolarized voltage using native, right-side out membrane vesicles from E. coli. This approach does not disrupt the normal membrane environment of the channel protein and does not involve detergent solubilization. We found that voltage-dependent conformational changes are focused in the N-terminal half of the K(V)AP S4 segment, in excellent agreement with results obtained with Shaker. Homologous residues in the K(V)AP and Shaker S4 segments are transferred from the extracellular to the intracellular compartment upon hyperpolarization. Taken together with X-ray structures indicating that the K(V)AP S4 segment is outwardly displaced at 0 mV compared to S4 in a mammalian Shaker channel, our results are consistent with the idea that S4 moves further during voltage-dependent activation in K(V)AP than in Shaker.

Differences between ion binding to eag and HERG voltage sensors contribute to differential regulation of activation and deactivation gating.

HERG (KCNH2) and ether-à-go-go (eag) (KCNH1) are members of the same subfamily of voltage-gated K+ channels. In eag, voltage-dependent activation is significantly slowed by extracellular divalent cations. To exert this effect, ions bind to a site located between transmembrane segments S2 and S3 in the voltage sensor domain where they interact with acidic residues that are conserved only among members of the eag subfamily. In HERG channels, extracellular divalent ions significantly accelerate deactivation. To investigate the ionbinding site in HERG, acidic residues in S2 and S3 were neutralized singly or in pairs to alanine, and the functional effects of extracellular Mg(2+) were characterized in Xenopus oocytes. To modulate deactivation kinetics in HERG, divalent cations interact with eag subfamily-specific acidic residues (D460 and D509) and also with an acidic residue in S2 (D456) that is widely conserved in the voltage-gated channel superfamily. In contrast, the analogous widely-conserved residue does not contribute to the ion-binding site that modulates activation kinetics in eag. We propose that structural differences between the ion-binding sites in the eag and HERG voltage sensors contribute to the differential regulation of activation and deactivation gating in these channels. A previously proposed model for S4 conformational changes during voltagedependent activation can account for the differential regulation of gating seen in eag and HERG.

Distance measurements reveal a common topology of prokaryotic voltage-gated ion channels in the lipid bilayer.

Voltage-dependent ion channels are fundamental to the physiology of excitable cells because they underlie the generation and propagation of the action potential and excitation-contraction coupling. To understand how ion channels work, it is important to determine their structures in different conformations in a membrane environment. The validity of the crystal structure for the prokaryotic K(+) channel, K(V)AP, has been questioned based on discrepancies with biophysical data from functional eukaryotic channels, underlining the need for independent structural data under native conditions. We investigated the structural organization of two prokaryotic voltage-gated channels, NaChBac and K(V)AP, in liposomes by using luminescence resonance energy transfer. We describe here a transmembrane packing representation of the voltage sensor and pore domains of the prokaryotic Na channel, NaChBac. We find that NaChBac and K(V)AP share a common arrangement in which the structures of the Na and K selective pores and voltage-sensor domains are conserved. The packing arrangement of the voltage-sensing region as determined by luminescence resonance energy transfer differs significantly from that of the K(V)AP crystal structure, but resembles that of the eukaryotic K(V)1.2 crystal structure. However, the voltage-sensor domain in prokaryotic channels is closer to the pore domain than in the K(V)1.2 structure. Our results indicate that prokaryotic and eukaryotic channels that share similar functional properties have similar helix arrangements, with differences arising likely from the later introduction of additional structural elements.