Sodium Channel Fragments: Contributions to Voltage Sensitivity and Ion Selectivity

The peptide strategy was employed to resolve structure-function relationships in the voltage-dependent sodium channel. Two families of motifs were studied: the four voltage sensors S4 extended with the short cytoplasmic linkers L45 and the four P-regions, between S5 and S6, each from the homologous domains of the electric eel sodium channel. Macroscopic conductance experiments conducted with synthetic S4L45s in neutral lipid planar bilayers pointed to a moderate voltage-sensitivity for repeat IV which has no proline, whereas S4L45 of repeats I and II (Pro 19) and especially of repeat III (Pro 14) were much more voltage-sensitive. The influence both of Pro and its position within the sequence was confirmed by comparing the human skeletal muscle channel isoform D4/S4 wild-type and the R4P analogue. Circular dichroism spectroscopy shows highest and lowest helicities for repeats IV and III. The conformational transition (from helix to extended, mainly beta forms), which occurs when the solvent dielectric constant increases, was broader with repeat III. These structural and functional correlates suggest alternative gating mechanisms. The different contributions of each repeat also have effects at the level of the main selectivity filter, which suggests self-recognition between the four P-regions is a key component of intact sodium channel selectivity. In addition, the P-region from domain III is significantly voltage-sensitive and molecular dynamics simulations show that the C-terminal part of P-regions is mainly helical whilst the N-terminus tends to unfold. Such specializations of the four domains both in gating and selectivity are independently confirmed in recent electrophysiological studies.     

Voltage sensors in domains III and IV, but not I and II, are immobilized by Na+ channel fast inactivation

Using site-directed fluorescent labeling, we examined conformational changes in the S4 segment of each domain of the human skeletal muscle sodium channel (hSkM1). The fluorescence signals from S4 segments in domains I and II follow activation and are unaffected as fast inactivation settles. In contrast, the fluorescence signals from S4 segments in domains III and IV show kinetic components during activation and deactivation that correlate with fast inactivation and charge immobilization. These results indicate that in hSkM1, the S4 segments in domains III and IV are responsible for voltage-sensitive conformational changes linked to fast inactivation and are immobilized by fast inactivation, while the S4 segments in domains I and II are unaffected by fast inactivation.     

Molecular action of lidocaine on the voltage sensors of sodium channels

Block of sodium ionic current by lidocaine is associated with alteration of the gating charge-voltage (Q-V) relationship characterized by a 38% reduction in maximal gating charge (Q(max)) and by the appearance of additional gating charge at negative test potentials. We investigated the molecular basis of the lidocaine-induced reduction in cardiac Na channel-gating charge by sequentially neutralizing basic residues in each of the voltage sensors (S4 segments) in the four domains of the human heart Na channel (hH1a). By determining the relative reduction in the Q(max) of each mutant channel modified by lidocaine we identified those S4 segments that contributed to a reduction in gating charge. No interaction of lidocaine was found with the voltage sensors in domains I or II. The largest inhibition of charge movement was found for the S4 of domain III consistent with lidocaine completely inhibiting its movement. Protection experiments with intracellular MTSET (a charged sulfhydryl reagent) in a Na channel with the fourth outermost arginine in the S4 of domain III mutated to a cysteine demonstrated that lidocaine stabilized the S4 in domain III in a depolarized configuration. Lidocaine also partially inhibited movement of the S4 in domain IV, but lidocaine's most dramatic effect was to alter the voltage-dependent charge movement of the S4 in domain IV such that it accounted for the appearance of additional gating charge at potentials near -100 mV. These findings suggest that lidocaine's actions on Na channel gating charge result from allosteric coupling of the binding site(s) of lidocaine to the voltage sensors formed by the S4 segments in domains III and IV.     

S1–S3 counter charges in the voltage sensor module of a mammalian sodium channel regulate fast inactivation

The movement of positively charged S4 segments through the electric field drives the voltage-dependent gating of ion channels. Studies of prokaryotic sodium channels provide a mechanistic view of activation facilitated by electrostatic interactions of negatively charged residues in S1 and S2 segments, with positive counterparts in the S4 segment. In mammalian sodium channels, S4 segments promote domain-specific functions that include activation and several forms of inactivation. We tested the idea that S1–S3 countercharges regulate eukaryotic sodium channel functions, including fast inactivation. Using structural data provided by bacterial channels, we constructed homology models of the S1–S4 voltage sensor module (VSM) for each domain of the mammalian skeletal muscle sodium channel hNa_V1.4. These show that side chains of putative countercharges in hNa_V1.4 are oriented toward the positive charge complement of S4. We used mutagenesis to define the roles of conserved residues in the extracellular negative charge cluster (ENC), hydrophobic charge region (HCR), and intracellular negative charge cluster (INC). Activation was inhibited with charge-reversing VSM mutations in domains I–III. Charge reversal of ENC residues in domains III (E1051R, D1069K) and IV (E1373K, N1389K) destabilized fast inactivation by decreasing its probability, slowing entry, and accelerating recovery. Several INC mutations increased inactivation from closed states and slowed recovery. Our results extend the functional characterization of VSM countercharges to fast inactivation, and support the premise that these residues play a critical role in domain-specific gating transitions for a mammalian sodium channel.     

Tracking voltage-dependent conformational changes in skeletal muscle sodium channel during activation

The primary voltage sensor of the sodium channel is comprised of four positively charged S4 segments that mainly differ in the number of charged residues and are expected to contribute differentially to the gating process. To understand their kinetic and steady-state behavior, the fluorescence signals from the sites proximal to each of the four S4 segments of a rat skeletal muscle sodium channel were monitored simultaneously with either gating or ionic currents. At least one of the kinetic components of fluorescence from every S4 segment correlates with movement of gating charge. The fast kinetic component of fluorescence from sites S216C (S4 domain I), S660C (S4 domain II), and L1115C (S4 domain III) is comparable to the fast component of gating currents. In contrast, the fast component of fluorescence from the site S1436C (S4 domain IV) correlates with the slow component of gating. In all the cases, the slow component of fluorescence does not have any apparent correlation with charge movement. The fluorescence signals from sites reflecting the movement of S4s in the first three domains initiate simultaneously, whereas the fluorescence signals from the site S1436C exhibit a lag phase. These results suggest that the voltage-dependent movement of S4 domain IV is a later step in the activation sequence. Analysis of equilibrium and kinetic properties of fluorescence over activation voltage range indicate that S4 domain III is likely to move at most hyperpolarized potentials, whereas the S4s in domain I and domain II move at more depolarized potentials. The kinetics of fluorescence changes from sites near S4-DIV are slower than the activation time constants, suggesting that the voltage-dependent movement of S4-DIV may not be a prerequisite for channel opening. These experiments allow us to map structural features onto the kinetic landscape of a sodium channel during activation.     

Roles of molecular regions in determining differences between voltage dependence of activation of CaV3.1 and CaV1.2 calcium channels

Voltage-dependent calcium channels are classified into low voltage-activated and high voltage-activated channels. We have investigated the molecular basis for this difference in voltage dependence of activation by constructing chimeras between a low voltage-activated channel (Ca(V)3.1) and a high voltage-activated channel (Ca(V)1.2), focusing on steady-state activation properties. Wild type and chimeras were expressed in oocytes, and two-electrode voltage clamp recordings were made of calcium channel currents. Replacement of domains I, III, or IV of the Ca 3.1 channel with the corresponding domain of Ca(V)1.2 led (V)to high voltage-activated channels; for these constructs the current/voltage (I/V) curves were similar to those for Ca(V)1.2 wild type. However, replacement of domain II gave only a small shift to the right of the I/V curve and modulation of the activation kinetics but did not lead to a high voltage-activating channel with an I/V curve like Ca 1.2. We also investigated the role of the voltage sensor (V)S4 by replacing the S4 segment of Ca(V)3.1 with that of Ca 1.2. For domain I, there was no shift in the I/V curve (V)as compared with Ca(V)3.1, and there were relatively small shifts to the right for domains III and IV. Taken together, these results suggest that domains I, III, and IV (rather than domain II) are apparently critical for channel opening and, therefore, contribute strongly to the difference in voltage dependence of activation between Ca 3.1 and Ca(V)1.2. However, the S4 segments in domains I, (V)III, and IV did not account for this difference in voltage dependence.     

Role of S4 segments and the leucine heptad motif in the activation of an L-type calcium channel

Basic residues in the S4 segments of voltage-dependent channels and leucines within the heptad repeat motif in the S4-S5 region of Shaker potassium channels have been shown to have important influences on activation. Here we have compared the relative importance for activation of S4 arginines (mutated to neutral or negative residues) in each of the four repeats of a chimeric L-type calcium channel. Significant effects on midpoint potential and time constant of activation were produced by mutations in repeats I and III but not in repeats II and IV. Leucine or isoleucine mutations in repeats I and III had the same effect on the voltage dependence of calcium channel activation as the mutations at equivalent positions in the Shaker channel, indicating that the heptad motif plays a fundamental role in channel activation.     

Alteration of channel characteristics by exchange of pore-forming regions between two structurally related Ca2+ Channels

Several types of structurally homologous high voltage-gated Ca²⁺ channels (L-, P-and N-type) have been identified via biochemical, pharmacological and electrophysiological techniques. Among these channels, the cardiac L-type and the brain BI-2 Ca²⁺ channel display significantly different biophysical properties. The BI-2 channel exhibits more rapid voltage-dependent current activation and inactivation and smaller single-channel conductance compared to the L-type Ca²⁺ channel. To examine the molecular basis for the functional differences between the two structurally related Ca²⁺ channels, we measured macroscopic and single-channel currents from oocytes injected with wild-type and various chimeric channel ₁ subunit cRNAs. The results show that a chimeric channel in which the segment between S5-SS2 in repeat IV of the cardiac L-type Ca²⁺ channel, was replaced by the corresponding region of the BI-2 channel, exhibited macroscopic current activation and inactivation time-courses and single-channel conductance, characteristic of the BI-2 Ca²⁺ channel. The voltage-dependence of steady-state inactivation was not affected by the replacement. Chimeras, in which the SS2-S6 segment in repeat III or IV of the cardiac channel was replaced by the corresponding BI-2 sequence, exhibited altered macroscopic current kinetics without changes in single-channel conductance. These results suggest that part of the S5-SS2 segment plays a critical role in determining voltage-dependent current activation and inactivation and single-channel conductance and that the SS2-S6 segment may control voltage-dependent kinetics of the Ca²⁺ channel.     

Sodium Channel Activation Gating Is Affected by Substitutions of Voltage Sensor Positive Charges in All Four Domains

The role of the voltage sensor positive charges in the activation and deactivation gating of the rat brain IIA sodium channel was investigated by mutating the second and fourth conserved positive charges in the S4 segments of all four homologous domains. Both charge-neutralizing (by glutamine substitution) and -conserving mutations were constructed in a cDNA encoding the sodium channel α subunit that had fast inactivation removed by the incorporation of the IFMQ3 mutation in the III–IV linker (West, J.W., D.E. Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A. Catterall. 1992. Proc. Natl. Acad. Sci. USA. 89:10910–10914.). A total of 16 single and 2 double mutants were constructed and analyzed with respect to voltage dependence and kinetics of activation and deactivation. The most significant effects were observed with substitutions of the fourth positive charge in each domain. Neutralization of the fourth positive charge in domain I or II produced the largest shifts in the voltage dependence of activation, both in the positive direction. This change was accompanied by positive shifts in the voltage dependence of activation and deactivation kinetics. Combining the two mutations resulted in an even larger positive shift in half-maximal activation and a significantly reduced gating valence, together with larger positive shifts in the voltage dependence of activation and deactivation kinetics. In contrast, neutralization of the fourth positive charge in domain III caused a negative shift in the voltage of half-maximal activation, while the charge-conserving mutation resulted in a positive shift. Neutralization of the fourth charge in domain IV did not shift the half-maximal voltage of activation, but the conservative substitution produced a positive shift. These data support the idea that both charge and structure are determinants of function in S4 voltage sensors. Overall, the data supports a working model in which all four S4 segments contribute to voltage-dependent activation of the sodium channel.     

Sodium Channel Inactivation Is Altered by Substitution of Voltage Sensor Positive Charges

The role of the voltage sensor positive charges in fast and slow inactivation of the rat brain IIA sodium channel was investigated by mutating the second and fourth conserved positive charges in the S4 segments of all four homologous domains. Both charge-neutralizing mutations (by glutamine substitution) and charge-conserving mutations were constructed in a cDNA encoding the sodium channel α subunit. To determine if fast inactivation altered the effects of the mutations on slow inactivation, the mutations were also constructed in a channel that had fast inactivation removed by the incorporation of the IFMQ3 mutation in the III–IV linker (West, J.W., D.E. Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A. Catterall. 1992. Proc. Natl. Acad. Sci. USA. 89:10910– 10914). Most of the mutations shifted the v_1/2 of fast inactivation in the negative direction, with the largest effects resulting from mutations in domains I and II. These shifts were in the opposite direction compared with those observed for activation. The effects of the mutations on slow inactivation depended on whether fast inactivation was intact or not. When fast inactivation was eliminated, most of the mutations resulted in positive shifts in the v_1/2 of slow inactivation. The largest effects again resulted from mutations in domains I and II. When fast inactivation was intact, the mutations in domains II and III resulted in negative shifts in the v_1/2 of slow inactivation. Neutralization of the fourth charge in domain I or II resulted in the appearance of a second component in the voltage dependence of slow inactivation that was only observable when fast inactivation was intact. These results suggest the S4 regions of all four domains of the sodium channel are involved in the voltage dependence of inactivation, but to varying extents. Fast inactivation is not strictly coupled to activation, but it derives some independent voltage sensitivity from the charges in the S4 domains. Finally, there is an interaction between the fast and slow inactivation processes.     

Hinge-bending motions in the pore domain of a bacterial voltage-gated sodium channel

Computational methods and experimental data are used to provide structural models for NaChBac, the homo-tetrameric voltage-gated sodium channel from the bacterium Bacillus halodurans, with a closed and partially open pore domain. Molecular dynamic (MD) simulations on membrane-bound homo-tetrameric NaChBac structures, each comprising six helical transmembrane segments (labeled S1 through S6), reveal that the shape of the lumen, which is defined by the bundle of four alpha-helical S6 segments, is modulated by hinge bending motions around the S6 glycine residues. Mutation of these glycine residues into proline and alanine affects, respectively, the structure and conformational flexibility of the S6 bundle. In the closed channel conformation, a cluster of stacked phenylalanine residues from the four S6 helices hinders diffusion of water molecules and Na(+) ions. Activation of the voltage sensor domains causes destabilization of the aforementioned cluster of phenylalanines, leading to a more open structure. The conformational change involving the phenylalanine cluster promotes a kink in S6, suggesting that channel gating likely results from the combined action of hinge-bending motions of the S6 bundle and concerted reorientation of the aromatic phenylalanine side-chains.     

Glutamate substitution in repeat IV alters divalent and monovalent cation permeation in the heart Ca2+ channel.

In voltage-gated ion channels, residues responsible for ion selectivity were identified in the pore-lining SS1-SS2 segments. Negatively charged glutamate residues (E393, E736, E1145, and E1446) found in each of the four repeats of the alpha 1C subunit were identified as the major determinant of selectivity in Ca2+ channels. Neutralization of glutamate residues by glutamine in repeat I (E393Q), repeat III (E1145Q), and repeat IV (E1446Q) decreased the channel affinity for calcium ions 10-fold from the wild-type channel. In contrast, neutralization of glutamate residues in repeat II failed to significantly alter Ca2+ affinity. Likewise, mutation of neighboring residues in E1149K and D1450N did not affect the channel affinity, further supporting the unique role of glutamate residues E1145 in repeat III and E1446 in repeat IV in determining Ca2+ selectivity. Conservative mutations E1145D and E1446D preserved high-affinity Ca2+ binding, which suggests that the interaction between Ca2+ and the pore ligand sites is predominantly electrostatic and involves charge neutralization. Mutational analysis of E1446 showed additionally that polar residues could achieve higher Ca2+ affinity than small hydrophobic residues could. The role of high-affinity calcium binding sites in channel permeation was investigated at the single-channel level. Neutralization of glutamate residue in repeats I, II, and III did not affect single-channel properties measured with 115 mM BaCl2. However, mutation of the high-affinity binding site E1446 was found to significantly affect the single-channel conductance for Ba2+ and Li+, providing strong evidence that E1446 is located in the narrow region of the channel outer mouth. Side-chain substitutions at 1446 in repeat IV were used to probe the nature of divalent cation-ligand interaction and monovalent cation-ligand interaction in the calcium channel pore. Monovalent permeation was found to be inversely proportional to the volume of the side chain at position 1446, with small neutral residues such as alanine and glycine producing higher Li+ currents than the wild-type channel. This suggests that steric hindrance is a major determinant for monovalent cation conductance. Divalent permeation was more complex. Ba2+ single-channel conductance decreased when small neutral residues such as glycine were replaced by bulkier ones such as glutamine. However, negatively charged amino acids produced single-channel conductance higher than predicted from the size of their side chain. Hence, negatively charged residues at position 1446 in repeat IV are required for divalent cation permeation.     

On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel.

Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains I, II, III, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the mu 1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain III of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(III) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.     

Analysis of protease-sensitive regions in the skeletal muscle sodium channel in vitro and implications for channel tertiary structure.

The tertiary structure of the rat skeletal muscle sodium channel was probed in vitro by determining regions of sensitivity to V-8 protease, trypsin, and chymotrypsin. Resultant channel fragments were identified with antibodies to defined sequences distributed along the primary structure. The temporal pattern of proteolysis was followed with channel protein in either detergent-phospholipid micelles or membrane fragments as well as with channel exposed to sodium dodecyl sulfate. Proteolysis in micelles and membranes occurred in discrete, reproducible steps that were similar in both systems. Although the size of intermediates varied slightly, their sequence of appearance was similar for all enzymes, suggesting that the observed pattern was determined by the relative accessibility of selected sites in the tertiary structure. No major change in channel organization appeared to occur after solubilization of membranes in nonionic detergents. Highly accessible sites in the native structure included the carboxyl terminus and the region linking the second and third internal repeat domains, while the amino terminus and the repeat domains themselves were relatively resistant to proteolysis unless the protein was denatured. Kinetically, interdomain II-III was the most readily cleaved; interdomains I-II and especially III-IV were less easily accessible. While domains I and IV appeared to remain intact throughout our experiments, limit fragments for epitopes associated with domains II and III suggest that cleavage eventually occurs at sites between the putative S5 and S6 helices in these domains.     

A Double Tyrosine Motif in the Cardiac Sodium Channel Domain III-IV Linker Couples Calcium-dependent Calmodulin Binding to Inactivation Gating

Voltage-gated sodium channels maintain the electrical cadence and stability of neurons and muscle cells by selectively controlling the transmembrane passage of their namesake ion. The degree to which these channels contribute to cellular excitability can be managed therapeutically or fine-tuned by endogenous ligands. Intracellular calcium, for instance, modulates sodium channel inactivation, the process by which sodium conductance is negatively regulated. We explored the molecular basis for this effect by investigating the interaction between the ubiquitous calcium binding protein calmodulin (CaM) and the putative sodium channel inactivation gate composed of the cytosolic linker between homologous channel domains III and IV (DIII-IV). Experiments using isothermal titration calorimetry show that CaM binds to a novel double tyrosine motif in the center of the DIII-IV linker in a calcium-dependent manner, N-terminal to a region previously reported to be a CaM binding site. An alanine scan of aromatic residues in recombinant DIII-DIV linker peptides shows that whereas multiple side chains contribute to CaM binding, two tyrosines (Tyr¹⁴⁹⁴ and Tyr¹⁴⁹⁵) play a crucial role in binding the CaM C-lobe. The functional relevance of these observations was then ascertained through electrophysiological measurement of sodium channel inactivation gating in the presence and absence of calcium. Experiments on patch-clamped transfected tsA201 cells show that only the Y1494A mutation of the five sites tested renders sodium channel steady-state inactivation insensitive to cytosolic calcium. The results demonstrate that calcium-dependent calmodulin binding to the sodium channel inactivation gate double tyrosine motif is required for calcium regulation of the cardiac sodium channel.     

Coupling interactions between voltage sensors of the sodium channel as revealed by site-specific measurements

The voltage-sensing S4 segments in the sodium channel undergo conformational rearrangements in response to changes in the electric field. However, it remains unclear whether these structures move independently or in a coordinated manner. Previously, site-directed fluorescence measurements were shown to track S4 transitions in each of the four domains. Here, using a similar technique, we provide direct evidence of coupling interactions between voltage sensors in the sodium channel. Pairwise interactions between S4s were evaluated by comparing site-specific conformational changes in the presence and absence of a gating perturbation in a distal domain. Reciprocity of effect, a fundamental property of thermodynamically coupled systems, was measured by generating converse mutants. The magnitude of a local gating perturbation induced by a remote S4 mutation depends on the coupling strength and the relative equilibrium positions of the two voltage sensors. In general, our data indicates that the movement of all four voltage sensors in the sodium channel are coupled to a varying extent. Moreover, a gating perturbation in S4-DI has the largest effect on the activation of S4-DIV and vice versa, demonstrating an energetic linkage between S4-DI and S4-DIV. This result suggests a physical mechanism by which the activation and inactivation process may be coupled in voltage-gated sodium channels. In addition, we propose that cooperative interactions between voltage sensors may be the mechanistic basis for the fast activation kinetics of the sodium channel.     

Cysteine Mapping in the Ion Selectivity and Toxin Binding Region of the Cardiac Na+ Channel Pore

Aqueous exposure of critical residues in the selectivity region of voltage gated Na⁺ channels was studied by cysteine-scanning mutagenesis at three positions in each of the SS2 segments of domains III (D3) and IV (D4) of the human heart Na⁺ channel. Ionic currents were modified by charged cysteine-specific methanethiosulfonate (MTS) reagents, (2-aminoethyl)methanethiosulfonate (MTSEA⁺) and (2-sulfonatoethyl)methanethiosulfonate (MTSES⁻) in all six of the Cys-substituted channels, including Trp → Cys substitutions at homologous positions in D3 and D4 that were predicted in secondary structure models to have buried side chains. Furthermore, in the absence of MTS modification, each of the Cys mutants showed a reduction in tetrodotoxin (TTX) block by a factor >10². Cysteine substitution without MTS modification abolished the alkali metal ion selectivity in K1418C (D3), but not in A1720C (the corresponding position in D4) suggesting that the lysine but not the alanine side chains contribute to selectivity even though both were exposed. Neither position responded to MTSES⁻ suggesting that these residues occupy either a size- or charge-restricted region of the pore. By contrast, MTSES⁻ markedly increased, and MTSEA⁺ markedly decreased conductance of D1713C (D4) suggesting that the acidic side chain of Asp¹⁷¹³ acts electrostatically in an unrestricted region. These results suggest that Lys¹⁴¹⁸ lies in a restricted region favorable to cations, whereas Asp¹⁷¹³ is at a more peripheral location in the Na⁺ channel pore. Received: 8 May 1996/Revised: 15 August 1996     

Molecular cloning of a novel form (two-repeat) protein related to voltage-gated sodium and calcium channels

Voltage-gated Ca and Na channels share similar structure: four homologous domains (I-IV), each with six transmembrane segments (S1-S6). They may be formed by two rounds of duplication of a single channel domain similar to voltage-activated potassium channels. However, the channels with the intermediate structure, namely, two-domain channels have not yet been identified. We report here the cloning of a novel protein from rat kidney that contains two domains (I-II), each with S1-S6 segments that are found in voltage-gated Ca and Na channels. Because of unusual structure, the protein was named two-pore channel 1 (TPC1). TPC1 encodes 819 amino acids with two conserved positively charged voltage sensor segments (S4) but the pore segments are not conserved. Northern blot analysis showed that TPC1 mRNA (5 kb) was expressed widely. It was expressed at relatively high level in kidney, liver, and lung. Immunohistochemistry of kidney revealed that TPC1 was expressed at inner medullary collecting ducts. In expression studies, no functional currents could be detected in CHO cells and Xenopus oocytes. Based on its primary structure, we propose that TPC1 might be a predecessor of the conventional four repeat voltage-gated Ca and Na channels and will give insights into the evolution of ion channels.     

Implication of segment S45 in the permeation pathway of voltage-dependent sodium channels

A 34-mer peptide, encompassing the S4 and S45 segments of domain IV of the electric eel voltage-dependent sodium channel, was synthesized in order to test the potential implication of S45 in the gating or permeation pathway. The secondary structure of peptide S4–S45 assessed by circular dichroism was found mainly helical, both in organic solvents and in lipid vesicles, especially negatively-charged ones. The macroscopic conductance properties of neutral and negatively-charged Montal-Mueller planar lipid bilayers doped with S4–S45 were studied and compared with those of S4. With regard to voltage-dependence, the most efficient system was S4–S45 in neutral bilayers. Voltage thresholds for exponential conductance development were found to correlate with the background or leak conductance. Assuming that the latter reflects interfacial peptide concentration, the mean apparent number of monomers per conducting aggregate could be estimated to be 3–5. In single-channel experiments, the most probable events had amplitudes of 8 pS and 5 pS in neutral and negatively-charged bilayers respectively. Ionic selectivity under salt gradients conditions, both at macroscopic and single-channel levels, was in favour of sodium ions (P_Na/P_K = 3). These properties compare favourably to previous reports dealing with peptide modelling transmembrane segments of voltage-dependent ionic channels. Specifically, when compared to S4 alone, the reduced unit conductance and the increased selectivity for sodium support the implication of the S45 region in the inner lining of the open configuration of sodium channels. Correspondence to: H. Duclohier