A putative two pore channel AtTPC1 mediates Ca(2+) flux in Arabidopsis leaf cells

The gene encoding voltage-gated channel with high affinity for Ca(2+) permeation has not been cloned from plants. In the present study, we isolated a full-length cDNA encoding a putative Ca(2+ )channel (AtTPC1) from Arabidopsis. AtTPC1 has two conserved homologous domains, both of which contain six transmembrane segments (S1-S6) and a pore loop (P) between S5 and S6 in each domain, and has the highest homology with the two pore channel TPC1 recently cloned from rat. The overall structure is similar to the half of the general structure of alpha-subunits of voltage-activated Ca(2+) channels from animals. AtTPC1 rescued the Ca(2+) uptake activity of a yeast mutant cch1. Sucrose-induced luminescence, which reflects a cytosolic free Ca(2+) increase in aequorin-expressing Arabidopsis leaves, was enhanced by overexpression of AtTPC1 and suppressed by antisense expression of it. Sucrose-H(+) symporters AtSUC1 and 2, which depolarize membrane potential of cells receiving sucrose, also depressed a Ca(2+) increase by their antisense expression. These results suggest that AtTPC1 mediates a voltage-activated Ca(2+ )influx in Arabidopsis leaf cells.     

A hot spot for the interaction of gating modifier toxins with voltage-dependent ion channels

The gating modifier toxins are a large family of protein toxins that modify either activation or inactivation of voltage-gated ion channels. omega-Aga-IVA is a gating modifier toxin from spider venom that inhibits voltage-gated Ca(2+) channels by shifting activation to more depolarized voltages. We identified two Glu residues near the COOH-terminal edge of S3 in the alpha(1A) Ca(2+) channel (one in repeat I and the other in repeat IV) that align with Glu residues previously implicated in forming the binding sites for gating modifier toxins on K(+) and Na(+) channels. We found that mutation of the Glu residue in repeat I of the Ca(2+) channel had no significant effect on inhibition by omega-Aga-IVA, whereas the equivalent mutation of the Glu in repeat IV disrupted inhibition by the toxin. These results suggest that the COOH-terminal end of S3 within repeat IV contributes to forming a receptor for omega-Aga-IVA. The strong predictive value of previous mapping studies for K(+) and Na(+) channel toxins argues for a conserved binding motif for gating modifier toxins within the voltage-sensing domains of voltage-gated ion channels.     

Coupling between voltage sensors and activation gate in voltage-gated K+ channels

Current through voltage-gated K+ channels underlies the action potential encoding the electrical signal in excitable cells. The four subunits of a voltage-gated K+ channel each have six transmembrane segments (S1-S6), whereas some other K+ channels, such as eukaryotic inward rectifier K+ channels and the prokaryotic KcsA channel, have only two transmembrane segments (M1 and M2). A voltage-gated K+ channel is formed by an ion-pore module (S5-S6, equivalent to M1-M2) and the surrounding voltage-sensing modules. The S4 segments are the primary voltage sensors while the intracellular activation gate is located near the COOH-terminal end of S6, although the coupling mechanism between them remains unknown. In the present study, we found that two short, complementary sequences in voltage-gated K+ channels are essential for coupling the voltage sensors to the intracellular activation gate. One sequence is the so called S4-S5 linker distal to the voltage-sensing S4, while the other is around the COOH-terminal end of S6, a region containing the actual gate-forming residues.     

alpha-helical structural elements within the voltage-sensing domains of a K(+) channel

Voltage-gated K(+) channels are tetramers with each subunit containing six (S1-S6) putative membrane spanning segments. The fifth through sixth transmembrane segments (S5-S6) from each of four subunits assemble to form a central pore domain. A growing body of evidence suggests that the first four segments (S1-S4) comprise a domain-like voltage-sensing structure. While the topology of this region is reasonably well defined, the secondary and tertiary structures of these transmembrane segments are not. To explore the secondary structure of the voltage-sensing domains, we used alanine-scanning mutagenesis through the region encompassing the first four transmembrane segments in the drk1 voltage-gated K(+) channel. We examined the mutation-induced perturbation in gating free energy for periodicity characteristic of alpha-helices. Our results are consistent with at least portions of S1, S2, S3, and S4 adopting alpha-helical secondary structure. In addition, both the S1-S2 and S3-S4 linkers exhibited substantial helical character. The distribution of gating perturbations for S1 and S2 suggest that these two helices interact primarily with two environments. In contrast, the distribution of perturbations for S3 and S4 were more complex, suggesting that the latter two helices make more extensive protein contacts, possibly interfacing directly with the shell of the pore domain.     

Sp-tetraKCNG: A novel cyclic nucleotide gated K(+) channel

The sequence of a novel cGMP-regulated, tetrameric, K(+) selective channel (Sp-tetraKCNG) was discovered in the sea urchin Strongylocentrotus purpuratus. The Sp-tetraKCNG is a single polypeptide made of four KCNG domains similar to voltage-dependent Na(+) and Ca(2+) channels. Each KCNG domain has six transmembrane segments (S1-S6), the ion pore having the K(+) selectivity signature GYGD and a cyclic nucleotide-binding domain (CNBD). This novel channel is evolutionary located between K(+)-selective and voltage-dependent EAG channels and voltage-independent cationic CNG channels. Bilayer reconstitutions demonstrate such a cGMP-regulated K(+) selective channel in sea urchin spermatozoa.     

Batrachotoxin-resistant Na+ channels derived from point mutations in transmembrane segment D4-S6.

Local anesthetics (LAs) block voltage-gated Na+ channels in excitable cells, whereas batrachotoxin (BTX) keeps these channels open persistently. Previous work delimited the LA receptor within the D4-S6 segment of the Na+ channel alpha-subunit, whereas the putative BTX receptor was found within the D1-S6. We mutated residues at D4-S6 critical for LA binding to determine whether such mutations modulate the BTX phenotype in rat skeletal muscle Na+ channels (mu1/rSkm1). We show that mu1-F1579K and mu1-N1584K channels become completely resistant to 5 microM BTX. In contrast, mu1-Y1586K channels remain BTX-sensitive; their fast and slow inactivation is eliminated by BTX after repetitive depolarization. Furthermore, we demonstrate that cocaine elicits a profound time-dependent block after channel activation, consistent with preferential LA binding to BTX-modified open channels. We propose that channel opening promotes better exposure of receptor sites for binding with BTX and LAs, possibly by widening the bordering area around D1-S6, D4-S6, and the pore region. The BTX receptor is probably located at the interface of D1-S6 and D4-S6 segments adjacent to the LA receptor. These two S6 segments may appose too closely to bind BTX and LAs simultaneously when the channel is in its resting closed state.     

Residues in Na(+) channel D3-S6 segment modulate both batrachotoxin and local anesthetic affinities

Batrachotoxin (BTX) alters the gating of voltage-gated Na(+) channels and causes these channels to open persistently, whereas local anesthetics (LAs) block Na(+) conductance. The BTX and LA receptors have been mapped to several common residues in D1-S6 and D4-S6 segments of the Na(+) channel alpha-subunit. We substituted individual residues with lysine in homologous segment D3-S6 of the rat muscle mu1 Na(+) channel from F1274 to N1281 to determine whether additional residues are involved in BTX and LA binding. Two mutant channels, mu1-S1276K and mu1-L1280K, when expressed in mammalian cells, become completely resistant to 5 microM BTX during repetitive pulses. The activation and/or fast inactivation gating of these mutants is substantially different from that of wild type. These mutants also display approximately 10-20-fold reduction in bupivacaine affinity toward their inactivated state but show only approximately twofold affinity changes toward their resting state. These results demonstrate that residues mu1-S1276 and mu1-L1280 in D3-S6 are critical for both BTX and LA binding interactions. We propose that LAs interact readily with these residues from D3-S6 along with those from D1-S6 and D4-S6 in close proximity when the Na(+) channel is in its inactivated state. Implications of this state-dependent binding model for the S6 alignment are discussed.     

KcsA crystal structure as framework for a molecular model of the Na(+) channel pore

The crystal structure of the pore-forming part of the KcsA bacterial K(+)-selective channel suggests a possible motif for related voltage-gated channels. We examined the hypothesis that the spacial orientation of the KcsA M1 and M2 alpha-helices also predicts the backbone location of S5 and S6 helices of the voltage-gated Na(+) channel. That channel's P region structure is expected to be different because selectivity is determined by side-chain interactions rather than by main-chain carbonyls, and its outer vestibule accommodates relatively large toxin molecules, tetrodotoxin (TTX) and saxitoxin (STX), which interact with selectivity ring residues. The Na(+) channel P loop was well-modeled by the alpha-helix-turn-beta-strand motif, which preserves the relationships for toxin interaction with the Na(+) channel found experimentally. This outer vestibule was docked into the extracellular part of the inverted teepee structure formed by the S5 and S6 helices that were spacially located by coordinates of the KcsA M1 and M2 helix main chains [Doyle et al. (1998) Science 280, 69-74], but populated with side chains of the respective S5 and S6 structures. van der Waals contacts were optimized with minimal adjustment of the S5, S6, and P loop structures, forming a densely packed pore structure. Nonregular external S5-P and P-S6 segments were not modeled here, except the P-S6 segment of domain II. The resulting selectivity region structure is consistent with Na(+) channel permeation properties, offering suggestions for the molecular processes involved in selectivity. The ability to construct a Na(+) channel pore model consistent with most of the available biophysical and mutational information suggests that the KcsA structural framework may be conserved in voltage-gated channels.     

Evolutionary genomics reveals the premetazoan origin of opposite gating polarity in animal-type voltage-gated ion channels

Electrical signaling in animals ensures the rapid and accurate transmission of information, often carried by voltage-gated Na(+), Ca(2+) and K(+) channels that are activated by membrane depolarization. In heart and neurons, a distinct type of ion channel called the hyperpolarization-activated, cyclic nucleotide-regulated (HCN) channel is activated by membrane hyperpolarization. Recent genomic studies have revealed that animal-type voltage-gated Na(+) channels (Liebeskind BJ, et al. 2011. Proc Natl Acad Sci U S A. 108:9154) had evolved in choanoflagellates, one of the unicellular relatives of animals. To date, HCN channels have been considered to be animal-specific. Here, we demonstrate the presence of an HCN channel homolog (SroHCN) in the choanoflagellate protist Salpingoeca rosetta. SroHCN contains highly conserved functional domains and sequence motifs that are correlated with the unique biophysical activities of HCN channels. These findings provide novel genomic insights into the evolution of complex electrical signaling before the emergence of multicellular animals.     

Helical structure of the COOH terminus of S3 and its contribution to the gating modifier toxin receptor in voltage-gated ion channels

The voltage-sensing domains in voltage-gated K(+) channels each contain four transmembrane (TM) segments, termed S1 to S4. Previous scanning mutagenesis studies suggest that S1 and S2 are amphipathic membrane spanning alpha-helices that interface directly with the lipid membrane. In contrast, the secondary structure of and/or the environments surrounding S3 and S4 are more complex. For S3, although the NH(2)-terminal part displays significant helical character in both tryptophan- and alanine-scanning mutagenesis studies, the structure of the COOH-terminal portion of this TM is less clear. The COOH terminus of S3 is particularly interesting because this is where gating modifier toxins like Hanatoxin interact with different voltage-gated ion channels. To further examine the secondary structure of the COOH terminus of S3, we lysine-scanned this region in the drk1 K(+) channel and examined the mutation-induced changes in channel gating and Hanatoxin binding affinity, looking for periodicity characteristic of an alpha-helix. Both the mutation-induced perturbation in the toxin-channel interaction and in gating support the presence of an alpha-helix of at least 10 residues in length in the COOH terminus of S3. Together with previous scanning mutagenesis studies, these results suggest that, in voltage-gated K(+) channels, the entire S3 segment is helical, but that it can be divided into two parts. The NH(2)-terminal part of S3 interfaces with both lipid and protein, whereas the COOH-terminal part interfaces with water (where Hanatoxin binds) and possibly protein. A conserved proline residue is located near the boundary between the two parts of S3, arguing for the presence of a kink in this region. Several lines of evidence suggest that these structural features of S3 probably exist in all voltage-gated ion channels.     

A "minimal" sodium channel construct consisting of ligated S5-P-S6 segments forms a toxin-activatable ionophore

The large size (six membrane-spanning repeats in each of four domains) and asymmetric architecture of the voltage-dependent Na+ channel has hindered determination of its structure. With the goal of determining the minimum structure of the Na+ channel permeation pathway, we created two stable cell lines expressing the voltage-dependent rat skeletal muscle Na+ channel (micro1) with a polyhistidine tag on the C terminus (muHis) and pore-only micro1 (muPore) channels with S1-S4 in all domains removed. Both constructs were recognized by a Na+ channel-specific antibody on a Western blot. muHis channels exhibited the same functional properties as wild-type micro1. In contrast, muPore channels did not conduct Na+ currents nor did they bind [3H]saxitoxin. Veratridine caused 40 and 54% cell death in muHis- and muPore-expressing cells, respectively. However, veratridine-induced cell death could only be blocked by tetrodotoxin in cells expressing muHis, but not muPore. Furthermore, using a fluorescent Na+ indicator, we measured changes in intracellular Na+ induced by veratridine and a brevotoxin analogue, pumiliotoxin. When calibrated to the maximum signal after addition of gramicidin, the maximal percent increases in fluorescence (deltaF) were 35 and 31% in cells expressing muHis and muPore, respectively. Moreover, in the presence of 1 microm tetrodotoxin, deltaF decreased significantly to 10% in muHis- but not in muPore-expressing cells (43%). In conclusion, S5-P-S6 segments of micro1 channels form a toxin-activable ionophore but do not reconstitute the Na+ channel permeation pathway with full fidelity.     

Convergent evolution of sodium ion selectivity in metazoan neuronal signaling

Ion selectivity of metazoan voltage-gated Na(+) channels is critical for neuronal signaling and has long been attributed to a ring of four conserved amino acids that constitute the ion selectivity filter (SF) at the channel pore. Yet, in addition to channels with a preference for Ca(2+) ions, the expression and characterization of Na(+) channel homologs from the sea anemone Nematostella vectensis, a member of the early-branching metazoan phylum Cnidaria, revealed a sodium-selective channel bearing a noncanonical SF. Mutagenesis and physiological assays suggest that pore elements additional to the SF determine the preference for Na(+) in this channel. Phylogenetic analysis assigns the Nematostella Na(+)-selective channel to a channel group unique to Cnidaria, which diverged >540 million years ago from Ca(2+)-conducting Na(+) channel homologs. The identification of Cnidarian Na(+)-selective ion channels distinct from the channels of bilaterian animals indicates that selectivity for Na(+) in neuronal signaling emerged independently in these two animal lineages.     

Membrane topology of NAADP-sensitive two-pore channels and their regulation by N-linked glycosylation

Two-pore channels (TPCs) localize to the endolysosomal system and have recently emerged as targets for the Ca(2+)-mobilizing messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). However, their membrane topology is unknown. Using fluorescence protease protection assays, we show that human TPC1 and TPC2 possess cytosolic N and C termini and therefore an even number of transmembrane regions. Fluorophores placed at position 225 or 347 in TPC1, or 339 in TPC2 were also cytosolic, whereas a fluorophore at position 628 in TPC1 was luminal. These data together with sequence similarity to voltage-gated Ca(2+) and Na(+) channels, and unbiased in silico predictions are consistent with a topology in which two homologous domains are present, each comprising 6 transmembrane regions and a re-entrant pore loop. Immunocytochemical analysis of selectively permeabilized cells using antipeptide antibodies confirmed that the C-terminal tails of recombinant TPCs are cytosolic and that residues 240-254 of TPC2 prior to putative pore 1 are luminal. Both TPC1 and TPC2 are N-glycosylated with residues 599, 611, and 616 contributing to glycosylation of TPC1. This confirms the luminal position of these residues, which immediately precede the putative pore loop of the second domain. Mutation of all three glycosylation sites in TPC1 enhances NAADP-evoked cytosolic Ca(2+) signals. Our data establish essential features of the topology of two-pore channels.     

Tethered blockers as molecular 'tape measures' for a voltage-gated K+ channel

The propagation of electrical signals in excitable cells is orchestrated by a molecular family of voltage-dependent ion channel proteins. These K+, Na+, and Ca++ channels are all composed of four identical or similar units, each containing six transmembrane segments (S1-S6) in a roughly four-fold symmetric structure. The S5-S6 sequences fold into a central pore unit, which is surrounded by a voltage-gating module composed of S1-S4. The recent structure of KcsA, a two-transmembrane bacterial K+ channel, illuminates the physical character of the pore unit, but little is known about the arrangement of the surrounding S1-S4 sequences. To locate regions of this gating module in space, we synthesized a series of compounds of varying length that function as molecular 'tape measures': quaternary ammonium (QA) pore blockers that can be tethered to specific test residues. We show that in a Shaker K+ channel, the extracellular ends of S1 and S3 are approximately 30 ? from the tetraethylammonium (TEA) blocking site at the external opening of the pore. A portion of the S3-S4 loop is, at 17-18 ?, considerably closer.     

Role of glycine residues highly conserved in the S2-S3 linkers of domains I and II of voltage-gated calcium channel α1 subunits

The pore-forming component of voltage-gated calcium channels, α₁ subunit, contains four structurally conserved domains (I-IV), each of which contains six transmembrane segments (S1-S6). We have shown previously that a Gly residue in the S2-S3 linker of domain III is completely conserved from yeasts to humans and important for channel activity. The Gly residues in the S2-S3 linkers of domains I and II, which correspond positionally to the Gly in the S2-S3 linker of domain III, are also highly conserved. Here, we investigated the role of the Gly residues in the S2-S3 linkers of domains I and II of Ca_v1.2. Each of the Gly residues was replaced with Glu or Gln to produce mutant Ca_v1.2s; G182E, G182Q, G579E, G579Q, and the resulting mutants were transfected into BHK6 cells. Whole-cell patch-clamp recordings showed that current-voltage relationships of the four mutants were the same as those of wild-type Ca_v1.2. However, G182E and G182Q showed significantly smaller current densities because of mislocalization of the mutant proteins, suggesting that Gly¹⁸² in domain I is involved in the membrane trafficking or surface expression of α₁ subunit. On the other hand, G579E showed a slower voltage-dependent current inactivation (VDI) compared to Ca_v1.2, although G579Q showed a normal VDI, implying that Gly⁵⁷⁹ in domain II is involved in the regulation of VDI and that the incorporation of a negative charge alters the VDI kinetics. Our findings indicate that the two conserved Gly residues are important for α₁ subunit to become functional.     

Role of amino-terminal half of the S4-S5 linker in type 1 ryanodine receptor (RyR1) channel gating

The type 1 ryanodine receptor (RyR1) is a Ca(2+) release channel found in the sarcoplasmic reticulum of skeletal muscle and plays a pivotal role in excitation-contraction coupling. The RyR1 channel is activated by a conformational change of the dihydropyridine receptor upon depolarization of the transverse tubule, or by Ca(2+) itself, i.e. Ca(2+)-induced Ca(2+) release (CICR). The molecular events transmitting such signals to the ion gate of the channel are unknown. The S4-S5 linker, a cytosolic loop connecting the S4 and S5 transmembrane segments in six-transmembrane type channels, forms an α-helical structure and mediates signal transmission in a wide variety of channels. To address the role of the S4-S5 linker in RyR1 channel gating, we performed alanine substitution scan of N-terminal half of the putative S4-S5 linker (Thr(4825)-Ser(4829)) that exhibits high helix probability. The mutant RyR1 was expressed in HEK cells, and CICR activity was investigated by caffeine-induced Ca(2+) release, single-channel current recordings, and [(3)H]ryanodine binding. Four mutants (T4825A, I4826A, S4828A, and S4829A) had reduced CICR activity without changing Ca(2+) sensitivity, whereas the L4827A mutant formed a constitutive active channel. T4825I, a disease-associated mutation for malignant hyperthermia, exhibited enhanced CICR activity. An α-helical wheel representation of the N-terminal S4-S5 linker provides a rational explanation to the observed activities of the mutants. These results suggest that N-terminal half of the S4-S5 linker may form an α-helical structure and play an important role in RyR1 channel gating.     

NAADP-evoked Ca2+ signals through two-pore channel-1 require arginine residues in the first S4-S5 linker

Two-pore channels (TPCs) are two-domain members of the voltage-gated ion channel superfamily that localize to acidic organelles. Their mechanism of activation (ligands such as NAADP/PI(3,5)P₂ versus voltage) and ion selectivity (Ca²⁺ versus Na⁺) is debated. Here we report that a cluster of arginine residues in the first domain required for selective voltage-gating of TPC1 map not to the voltage-sensing fourth transmembrane region (S4) but to a cytosolic downstream region (S4-S5 linker). These residues are conserved between TPC isoforms suggesting a generic role in TPC activation. Accordingly, mutation of residues in TPC1 but not the analogous region in the second domain prevents Ca²⁺ release by NAADP in intact cells. Our data affirm the role of TPCs in NAADP-mediated Ca²⁺ signalling and unite differing models of channel activation through identification of common domain-specific residues.     

Solution structure of omega-grammotoxin SIA,a gating modifier of P/Q and N-type Ca(2+) channel

omega-Grammotoxin SIA (GrTx) is a 36 amino acid residue protein toxin from spider venom that inhibits P/Q and N-type voltage-gated Ca(2+) channels by modifying voltage-dependent gating. We determined the three-dimensional structure of GrTx using NMR spectroscopy. The toxin adopts an "inhibitor cystine knot" motif composed of two beta-strands (Leu19-Cys21 and Cys30-Trp32) and a beta-bulge (Trp6, Gly7-Cys30) with a +2x, -1 topology, which are connected by four chain reversals. Although GrTx was originally identified as an inhibitor of voltage-gated Ca(2+) channel, it also binds to K(+) channels with lower affinity. A similar cross-reaction was observed for Hanatoxin1 (HaTx), which binds to the voltage-sensing domains of K(+) and Ca(2+) channels with different affinities. A detailed comparison of the GrTx and HaTx structures identifies a conserved face containing a large hydrophobic patch surrounded by positively charged residues. The slight differences in the surface shape, which result from the orientation of the surface aromatic residues and/or the distribution of the charged residues, may explain the differences in the binding affinity of these gating modifiers with different voltage-gated ion channels.     

A voltage-gated calcium-selective channel encoded by a sodium channel-like gene

BSC1, which was originally identified by its sequence similarity to voltage-gated Na(+) channels, encodes a functional voltage-gated cation channel whose properties differ significantly from Na(+) channels. BSC1 has slower kinetics of activation and inactivation than Na(+) channels, it is more selective for Ba(2+) than for Na(+), it is blocked by Cd(2+), and Na(+) currents through BSC1 are blocked by low concentrations of Ca(2+). All of these properties are more similar to voltage-gated Ca(2+) channels than to voltage-gated Na(+) channels. The selectivity for Ba(2+) is partially due to the presence of a glutamate in the pore-forming region of domain III, since replacing that residue with lysine (normally present in voltage-gated Na(+) channels) makes the channel more selective for Na(+). BSC1 appears to be the prototype of a novel family of invertebrate voltage-dependent cation channels with a close structural and evolutionary relationship to voltage-gated Na(+) and Ca(2+) channels.     

Design of a specific activator for skeletal muscle sodium channels uncovers channel architecture

Gating modifiers of voltage-gated sodium channels (Na(v)s) are important tools in neuroscience research and may have therapeutic potential in medicinal disorders. Analysis of the bioactive surface of the scorpion beta-toxin Css4 (from Centruroides suffusus suffusus) toward rat brain (rNa(v)1.2a) and skeletal muscle (rNa(v)1.4) channels using binding studies revealed commonality but also substantial differences, which were used to design a specific activator, Css4(F14A/E15A/E28R), of rNa(v)1.4 expressed in Xenopus oocytes. The therapeutic potential of Css4(F14A/E15A/E28R) was tested using an rNa(v)1.4 mutant carrying the same mutation present in the genetic disorder hypokalemic periodic paralysis. The activator restored the impaired gating properties of the mutant channel expressed in oocytes, thus offering a tentative new means for treatment of neuromuscular disorders with reduced muscle excitability. Mutant double cycle analysis employing toxin residues involved in the construction of Css4(F14A/E15A/E28R) and residues whose equivalents in the rat brain channel rNa(v)1.2a were shown to affect Css4 binding revealed significant coupling energy (>1.3 kcal/mol) between F14A and E592A at Domain-2/voltage sensor segments 1-2 (D2/S1-S2), R27Q and E1251N at D3/SS2-S6, and E28R with both E650A at D2/S3-S4 and E1251N at D3/SS2-S6. These results show that despite the differences in interactions with the rat brain and skeletal muscle Na(v)s, Css4 recognizes a similar region on both channel subtypes. Moreover, our data indicate that the S3-S4 loop of the voltage sensor module in Domain-2 is in very close proximity to the SS2-S6 segment of the pore module of Domain-3 in rNa(v)1.4. This is the first experimental evidence that the inter-domain spatial organization of mammalian Na(v)s resembles that of voltage-gated potassium channels.