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Low voltage-activated calcium currents are mediated by T-type calcium channels CaV3.1, CaV3.2, and CaV3.3, which modulate a variety of physiological processes including sleep, cardiac pace-making, pain, and epilepsy. CaV3 isoforms’ biophysical properties, overlapping expression and lack of subtype-selective pharmacology hinder the determination of their specific physiological roles in health and disease. Notably, CaV3.3’s contribution to normal and pathophysiological function has remained largely unexplored. We have identified Pn3a as the first subtype-selective spider venom peptide inhibitor of CaV3.3, with >100-fold lower potency against the other T-type isoforms. Pn3a modifies CaV3.3 gating through a depolarizing shift in the voltage dependence of activation thus decreasing CaV3.3-mediated currents in the normal range of activation potentials. Paddle chimeras of KV1.7 channels bearing voltage sensor sequences from all four CaV3.3 domains revealed preferential binding of Pn3a to the S3-S4 region of domain II (CaV3.3DII). This novel T-type channel pharmacological site was explored through computational docking simulations of Pn3a into all T-type channel isoforms highlighting it as subtype-specific pharmacophore with therapeutic potential. This research expands our understanding of T-type calcium channel pharmacology and supports the suitability of Pn3a as a molecular tool in the study of the physiological roles of CaV3.3 channels.

Batrachotoxin (BTX) causes paralysis and death by activating “pseudosymmetric” eukaryotic sodium (Nav) channels. Finol-Urdaneta et al. investigate its action on the prokaryotic homotetrameric homologues NaChBac and NavSp1, revealing use-dependent facilitation of activation, and inhibition of deactivation, caused by BTX binding to the symmetric pore., Batrachotoxin (BTX), an alkaloid from skin secretions of dendrobatid frogs, causes paralysis and death by facilitating activation and inhibiting deactivation of eukaryotic voltage-gated sodium (Nav) channels, which underlie action potentials in nerve, muscle, and heart. A full understanding of the mechanism by which BTX modifies eukaryotic Nav gating awaits determination of high-resolution structures of functional toxin–channel complexes. Here, we investigate the action of BTX on the homotetrameric prokaryotic Nav channels NaChBac and NavSp1. By combining mutational analysis and whole-cell patch clamp with molecular and kinetic modeling, we show that BTX hinders deactivation and facilitates activation in a use-dependent fashion. Our molecular model shows the horseshoe-shaped BTX molecule bound within the open pore, forming hydrophobic H-bonds and cation-π contacts with the pore-lining helices, leaving space for partially dehydrated sodium ions to permeate through the hydrophilic inner surface of the horseshoe. We infer that bulky BTX, bound at the level of the gating-hinge residues, prevents the S6 rearrangements that are necessary for closure of the activation gate. Our results reveal general similarities to, and differences from, BTX actions on eukaryotic Nav channels, whose major subunit is a single polypeptide formed by four concatenated, homologous, nonidentical domains that form a pseudosymmetric pore. Our determination of the mechanism by which BTX activates homotetrameric voltage-gated channels reveals further similarities between eukaryotic and prokaryotic Nav channels and emphasizes the tractability of bacterial Nav channels as models of voltage-dependent ion channel gating. The results contribute toward a deeper, atomic-level understanding of use-dependent natural and synthetic Nav channel agonists and antagonists, despite their overlapping binding motifs on the channel proteins.

Functional imaging of the intracellular calcium concentration [Ca2+]i using fluorescent indicators is a powerful and frequently applied method for assessing various biological questions in vitro, including ion channel function and intracellular signaling in homeostasis and disease. In functional [Ca2+]i imaging experiments, the fluorescence intensity of single cells is typically recorded during application of a chemical stimulus, i.e. by exchange of modified extracellular media, exposure to drugs and/or ligands. The concomitant mechanical perturbation caused by the perfusion of different solution during experimentation severely hinders calcium imaging in non-adherent cells, including peripheral immune cells, as cells in suspension are dislocated by turbulent flow during chemical stimulation. The quantitative analysis, involving time-courses of intracellular fluorescence signal changes, necessitates cells to remain at the same position throughout the experiment. To prevent dislocation of cells during solution exchange, and to enable imaging as well as analysis of Ca2+ responses in immune cells, a gelatin-based method for immobilization of non-adherent cells was developed. Gelatin has been a long-serving material for cell immobilization, e.g. in 3D bio-printing of cells and has thus, also been employed in the context of this study. To demonstrate the applicability of the established method for functional Ca2+ imaging in gelatin-immobilized suspension cells, a proof-of-concept study was conducted using human peripheral blood model cell lines (Jurkat/T-lymphocytes and THP-1/monocytes), Ca2+ indicators (Fluo-4 and Fura-2) and two different fluorescence microscopy rigs. The data presented that the established methodology is applicable for studying Ca2+ signaling by in vitro high-content functional imaging of [Ca2+]i in suspension cells, including but not restricted to human immune cells.

Hille’s (1971) seminal study of organic cation selectivity of eukaryotic voltage-gated sodium channels showed a sharp size cut-off for ion permeation, such that no ion possessing a methyl group was permeant. Using the prokaryotic channel, NaChBac, we found some similarity and two peculiar differences in the selectivity profiles for small polyatomic cations. First, we identified a diverse group of minimally permeant cations for wildtype NaChBac, ranging in sizes from ammonium to guanidinium and tetramethylammonium; and second, for both ammonium and hydrazinium, the charge-conserving selectivity filter mutation (E191D) yielded substantial increases in relative permeability (PX/PNa). The relative permeabilities varied inversely with relative Kd calculated from 1D Potential of Mean Force profiles (PMFs) for the single cations traversing the channel. Several of the cations bound more strongly than Na+, and hence appear to act as blockers, as well as charge carriers. Consistent with experimental observations, the E191D mutation had little impact on Na+ binding to the selectivity filter, but disrupted the binding of ammonium and hydrazinium, consequently facilitating ion permeation across the NaChBac-like filter. We concluded that for prokaryotic sodium channels, a fine balance among filter size, binding affinity, occupancy, and flexibility seems to contribute to observed functional differences.

&micro, Conotoxin PIIIA, in the sub-picomolar, range inhibits the archetypal bacterial sodium channel NaChBac (NavBh) in a voltage- and use-dependent manner. Peptide &micro, conotoxins were first recognized as potent components of the venoms of fish-hunting cone snails that selectively inhibit voltage-gated skeletal muscle sodium channels, thus preventing muscle contraction. Intriguingly, computer simulations predicted that PIIIA binds to prokaryotic channel NavAb with much higher affinity than to fish (and other vertebrates) skeletal muscle sodium channel (Nav 1.4). Here, using whole-cell voltage clamp, we demonstrate that PIIIA inhibits NavBac mediated currents even more potently than predicted. From concentration-response data, with [PIIIA] varying more than 6 orders of magnitude (10&minus, 12 to 10&minus, 5 M), we estimated an IC50 = ~5 pM, maximal block of 0.95 and a Hill coefficient of 0.81 for the inhibition of peak currents. Inhibition was stronger at depolarized holding potentials and was modulated by the frequency and duration of the stimulation pulses. An important feature of the PIIIA action was acceleration of macroscopic inactivation. Docking of PIIIA in a NaChBac (NavBh) model revealed two interconvertible binding modes. In one mode, PIIIA sterically and electrostatically blocks the permeation pathway. In a second mode, apparent stabilization of the inactivated state was achieved by PIIIA binding between P2 helices and trans-membrane S5s from adjacent channel subunits, partially occluding the outer pore. Together, our experimental and computational results suggest that, besides blocking the channel-mediated currents by directly occluding the conducting pathway, PIIIA may also change the relative populations of conducting (activated) and non-conducting (inactivated) states.

The vast complexity of native heteromeric K+ channels is largely unexplored. Defining the composition and subunit arrangement of individual subunits in native heteromeric K+ channels and establishing their physiological roles is experimentally challenging. Here we systematically explored this “zone of ignorance” in molecular neuroscience. Venom components, such as peptide toxins, appear to have evolved to modulate physiologically relevant targets by discriminating among closely related native ion channel complexes. We provide proof-of-principle for this assertion by demonstrating that κM-conotoxin RIIIJ (κM-RIIIJ) from Conus radiatus precisely targets “asymmetric” Kv channels composed of three Kv1.2 subunits and one Kv1.1 or Kv1.6 subunit with 100-fold higher apparent affinity compared with homomeric Kv1.2 channels. Our study shows that dorsal root ganglion (DRG) neurons contain at least two major functional Kv1.2 channel complexes: a heteromer, for which κM-RIIIJ has high affinity, and a putative Kv1.2 homomer, toward which κM-RIIIJ is less potent. This conclusion was reached by (i) covalent linkage of members of the mammalian Shaker-related Kv1 family to Kv1.2 and systematic assessment of the potency of κM-RIIIJ block of heteromeric K+ channel-mediated currents in heterologous expression systems; (ii) molecular dynamics simulations of asymmetric Kv1 channels providing insights into the molecular basis of κM-RIIIJ selectivity and potency toward its targets; and (iii) evaluation of calcium responses of a defined population of DRG neurons to κM-RIIIJ. Our study demonstrates that bioactive molecules present in venoms provide essential pharmacological tools that systematically target specific heteromeric Kv channel complexes that operate in native tissues.

Voltage-gated sodium channels (Navs) play crucial roles in excitable cells. Although vertebrate Nav function has been extensively studied, the detailed structural basis for voltage-dependent gating mechanisms remain obscure. We have assessed the structural changes of the Nav voltage sensor domain using lanthanide-based resonance energy transfer (LRET) between the rat skeletal muscle voltage-gated sodium channel (Nav1.4) and fluorescently labeled Nav1.4-targeting toxins. We generated donor constructs with genetically encoded lanthanide-binding tags (LBTs) inserted at the extracellular end of the S4 segment of each domain (with a single LBT per construct). Three different Bodipy-labeled, Nav1.4-targeting toxins were synthesized as acceptors: β-scorpion toxin (Ts1)-Bodipy, KIIIA-Bodipy, and GIIIA-Bodipy analogs. Functional Nav-LBT channels expressed in Xenopus oocytes were voltage-clamped, and distinct LRET signals were obtained in the resting and slow inactivated states. Intramolecular distances computed from the LRET signals define a geometrical map of Nav1.4 with the bound toxins, and reveal voltage-dependent structural changes related to channel gating.

The molecular strategy for alkali cation selectivity by a bacterial sodium channel resembles those of eukaryotic calcium and potassium channels, rather than those of eukaryotic sodium channels., Striking structural differences between voltage-gated sodium (Nav) channels from prokaryotes (homotetramers) and eukaryotes (asymmetric, four-domain proteins) suggest the likelihood of different molecular mechanisms for common functions. For these two channel families, our data show similar selectivity sequences among alkali cations (relative permeability, Pion/PNa) and asymmetric, bi-ionic reversal potentials when the Na/K gradient is reversed. We performed coordinated experimental and computational studies, respectively, on the prokaryotic Nav channels NaChBac and NavAb. NaChBac shows an “anomalous,” nonmonotonic mole-fraction dependence in the presence of certain sodium–potassium mixtures; to our knowledge, no comparable observation has been reported for eukaryotic Nav channels. NaChBac’s preferential selectivity for sodium is reduced either by partial titration of its highly charged selectivity filter, when extracellular pH is lowered from 7.4 to 5.8, or by perturbation—likely steric—associated with a nominally electro-neutral substitution in the selectivity filter (E191D). Although no single molecular feature or energetic parameter appears to dominate, our atomistic simulations, based on the published NavAb crystal structure, revealed factors that may contribute to the normally observed selectivity for Na over K. These include: (a) a thermodynamic penalty to exchange one K+ for one Na+ in the wild-type (WT) channel, increasing the relative likelihood of Na+ occupying the binding site; (b) a small tendency toward weaker ion binding to the selectivity filter in Na–K mixtures, consistent with the higher conductance observed with both sodium and potassium present; and (c) integrated 1-D potentials of mean force for sodium or potassium movement that show less separation for the less selective E/D mutant than for WT. Overall, tight binding of a single favored ion to the selectivity filter, together with crucial inter-ion interactions within the pore, suggests that prokaryotic Nav channels use a selective strategy more akin to those of eukaryotic calcium and potassium channels than that of eukaryotic Nav channels.

Voltage-gated sodium channels (Nav) play an essential role in the generation and propagation of action potentials. Mammalian Nav alpha subunits are large single polypeptide chains organized in four different domains (DI-DIV). Each domain is composed of 6 transmembrane segments (S1-S6) in which S1-S4 constitute the voltage sensing domain (VSD) and S5 and S6 form the pore. Although Nav function has been studied comprehensively, the precise structural basis for the gating mechanisms has not been fully elucidated. Recently, the crystal structures of several prokaryotic sodium channels have been solved, but they are homo-tetrameric proteins in contrast to the mammalian Navs and these studies only provide a single snapshot of the channels in one of many possible conformational states. Therefore, techniques that provide dynamic structural information are needed. In this work we have used Lanthanide-based resonance energy transfer (LRET) to obtain structural information and conformational dynamics of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4). We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which binds a lanthanide (Tb3+) ion with high affinity, inserted in several strategic places at the top of the S4 segment in each domain. Two new conotoxin analogs were synthesized to function as LRET acceptors: KIIIA-Bodipy and GIIIA-Bodipy. We calculated multiple distances from each one of the domains to the pore region where the labeled toxin binds in both resting and slow inactivated states in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs, which remained functionally active. The results give a geometrical map of the S4 positions of each domain in the mammalian Nav channels and provide evidence for voltage-dependent structural changes. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.

The actions of alcohols and anesthetics on ion channels are poorly understood. Controversy continues about whether bilayer restructuring is relevant to the modulatory effects of these surface active agents (SAAs). Some voltage-gated K channels (Kv), but not KvAP, have putative low affinity alcohol-binding sites, and because KvAP structures have been determined in bilayers, KvAP could offer insights into the contribution of bilayer mechanics to SAA actions. We monitored KvAP unitary conductance and macroscopic activation and inactivation kinetics in PE:PG/decane bilayers with and without exposure to classic SAAs (short-chain 1-alkanols, cholesterol, and selected anesthetics: halothane, isoflurane, chloroform). At levels that did not measurably alter membrane specific capacitance, alkanols caused functional changes in KvAP behavior including lowered unitary conductance, modified kinetics, and shifted voltage dependence for activation. A simple explanation is that the site of SAA action on KvAP is its entire lateral interface with the PE:PG/decane bilayer, with SAA-induced changes in surface tension and bilayer packing order combining to modulate the shape and stability of various conformations. The KvAP structural adjustment to diverse bilayer pressure profiles has implications for understanding desirable and undesirable actions of SAA-like drugs and, broadly, predicts that channel gating, conductance and pharmacology may differ when membrane packing order differs, as in raft versus nonraft domains.

Ion channels are membrane-spanning proteins that allow ions to permeate at high rates. The kinetic characteristics of the channels present in a cell determine the cell signaling profile and therefore cell function in many different physiological processes. We found that Kv1.7 channels from mouse heart muscle have two putative translation initiation start sites that generate two channel isoforms with different functional characteristics, mKv1.7L (489 aa) and a shorter mKv1.7S (457 aa). The electrophysiological analysis of mKv1.7L and mKv1.7S channels revealed that the two channel isoforms have different inactivation kinetics. The channel resulting from the longer protein (L) inactivates faster than the shorter channels (S). Our data supports the hypothesis that mKv1.7L channels inactivate predominantly due to an N-type related mechanism, which is impaired in the mKv1.7S form. Furthermore, only the longer version mKv1.7L is regulated by the cell redox state, whereas the shorter form mKv1.7S is not. Thus, expression starting at each translation initiation site results in significant functional divergence. Our data suggest that the redox modulation of mKv1.7L may occur through a site in the cytoplasmic N-terminal domain that seems to encompass a metal coordination motif resembling those found in many redox-sensitive proteins. The mRNA expression profile and redox modulation of mKv1.7 kinetics identify these channels as molecular entities of potential importance in cellular redox-stress states such as hypoxia.

Batrachotoxin (BTX) is an excitatory component in the skin secretions of dendrobatid frogs, which advertise their armament with the gaudy colors of their lethal skin. BTX is a hydrophobic alkaloid, which crosses cell membranes and activates voltage-gated sodium (Nav) channels of muscle, nerve and heart. BTX binding causes a shift of the activation curve toward more negative voltages, and suppresses fast and slow inactivation resulting in a massive increase in excitability of the tissue and ultimately causing paralysis. BTX is thought to act by binding in the cytoplasmic cavity of the ion-conducting pore, of pseudo-symmetric eukaryotic Nav channels. This binding site is near, or overlapping, receptor sites for amphiphylic inhibitor drugs including local anesthetics, anticonvulsants and anti-arrhythmics.The four homologous domains of eukaryotic Navs parallel the homo-tetrameric arrangement of BacNav channels, which have yielded high-resolution crystal structures. Despite the absence of a “ball-and chain” or “hinged-lid” fast inactivation mechanism, BacNavs display a “pore based” inactivation, that in some cases is quite rapid. Thus, they offer an opportunity to explore BTX actions on a molecular system of gating that is amenable to structural verification.The BacNav channels NaChBac and NavSp1 both show dramatic modification by BTX, with negative shifts of activation and inhibition of inactivation. These actions are enhanced by phenylalanine substitutions of pore domain (S6) residues, at positions which align with Phe residues that are important for BTX activation in eukaryotic Nav channels.

Hille's (1971) seminal study of the organic cation selectivity of voltage-gated sodium channels, showed a striking, sharp size cut-off for ion permeation. No ion species with an aliphatic moiety having at least the diameter of a methyl group was measurably permeant. Here, we show that, despite similar alkali cation permeability sequences for pro- and eu-karyotic channels (Finol-Urdaneta et al 2014 J Gen Physiol), relative permeabilities among organic ions (ammonium, hydrazinium, guanidinium and tetramethylammonium) are different. Using molecular dynamics simulations, we have asked to what extent 1d-pmfs based on the NavAb crystal structure might suggest a basis for NavBac selectivity among organic ions. Unlike eukaryotic channels, NavBac permeabilities do not show a size cut-off based on minimal cross-section, but are correlated with apparent binding energies in 1-dimensional potentials of mean force from simulations of single ions moving through the channel. In contrast, earlier analyses for eukaryotic Nav1s, suggest that differences among ion entry barriers are sufficient to account for relative permeabilities (Hille 1975 J Gen Physiol). We surmise that multi-ion occupancy and structural flexibility of Bac-Navs may also contribute to the observed and simulated functional differences. Meanwhile, we examined the consistency of our PMFs based on Zhu and Hummer's paper (Zhu & Hummer, 2011 J Comput Chem). The results indicate that it needs at least 10ns for particular windows to get accurate PMFs even for small molecules like ammonium.

We have examined the interactions of protons and an inhibitory, poly-cationic conotoxin with the human voltage-gated potassium channel, hKv1.7. This channel differs from some members of the Kv1 sub family by having a titratable histidine residue near the N-terminal end of the putative pore-supporting P-helix, suggesting that intrinsic channel functions and pharmacology may depend on the pH of the external solution. Channels were expressed in HEK-293 cells and studied by whole-cell patch clamp. The voltage dependence of channel activation was evaluated using a tail-current protocol in which the voltage was stepped to -40 mV following a variable activating pre-pulse. Lowering of the pH of the external solution from 7.4 to 5.0 produced a positive shift in the half-activation of 36±3 mV (n=4). The lowering of pH also dramatically decreased the ability of the conotoxin to inhibit currents through the channels. Thus, following the largest depolarization, at pH5.0 very little inhibition was observed at a toxin concentration near IC50 for pH7.4. The tail current in the presence of the conotoxin was near the value seen in the absence of the toxin for external pH of 7.4. Our observations are consistent with a two-fold action of the conotoxin. First, they suggest that the positively charged toxin binds close enough to the S4 segment of the voltage sensor to inhibit activation following a depolarizing voltage step. Second, the observed decrease in maximal conductance at pH 7.4 following addition of the toxin is consistent with a block of current through the open channels. Toxin binding appears to be inhibited by protonation of a residue on the external surface of the channel, perhaps the histidine residue near the N-terminal end of the pore helix.

“Use-” or “state-” dependent block of voltage-gated sodium channels is characteristic of therapeutic agents used to moderate electrical activity in pathologically hyperactive tissues and combat certain forms of epilepsy, cardiac arrhythmia, myotonia, and pain. Externally acting Na channel pore blockers are not commonly used as therapeutic drugs, but intraperitoneal μ-conotoxin (μCTX) KIIIA can act as an effective analgesic against inflammatory pain in mice, at concentrations that do not cause obvious systemic side effects (Zhang et al 2007 J Biol Chem 42:30699). This action appears to result from highly specific targeting to neuronal Na channels, without essential reliance on the use-dependent potency that contributes to the efficacy of clinical local anesthetics. μCTXs, although highly selective among closely related eukaryotic Na channels, have recently been found to block bacterial Nav channels with high affinity (Chen & Chung 2012 Biophys J 102:483; Finol-Urdaneta et al 2013 Biophys J 104:136a). In addition, the block was potentiated at more depolarized holding potentials. Here, we explore the dependence of μCTX PIIIA block of NaChBac and NavSp1 on varying patterns of conditioning depolarization. Briefly, in the low picomolar range, PIIIA application was associated with faster inactivation decay during a test depolarization, and block of the peak current. The speeding of inactivation precedes the block of current following PIIIA application, and this kinetic effect was obvious at more negative holding potentials than was the decrease in peak current. In NaChBac, both activation and inactivation were shifted in the hyperpolarizing direction by ∼25-30 mV in the presence of 5pM PIIIA. The data suggest that PIIIA binding is conformation dependent, with high affinity for non-conducting, depolarization-induced states. At present, the relative importance of “pre-activated” and “in-activated” states in potentiating inhibition by PIIIA is unclear.

μ-Conotoxins (μCTXs) are potent blockers of eukaryotic sodium channels, and individual members of the μCTX family are highly specific for particular Nav1 isoforms. We have begun to explore μCTX interactions with NaChBac, a prokaryotic Nav channel closely related to NavAb, whose crystal structure was recently determined (Payandeh et al, 2011, Nature 475:353). Our data reveal actions on both ion conduction and gating, consistent with polymodal actions on the pore domain.Under voltage clamp, whole-cell currents from NaChBac expressed in tsA-201 cells were reduced, up to nearly complete block, by concentrations in the pM to μM range, for wildtype toxins and derivatives of μCTX PIIIA and KIIIA. For wildtype PIIIA, dose-response data (0.001-10,000nM) yielded the following: IC50=0.005nM; maximal fraction of current blocked = 0.95; Hill coefficient = 0.7. Chen & Chung (2012, Biophys. J. 102:483) predicted an IC50 of 0.1nM for PIIIA when the NavAb channel is occupied by 2 sodium ions.Even at very low [PIIIA] (1pM), the unblocked currents showed increasing rates of inactivation as the peptides were washed in, suggesting a gating modulation, that was not tightly associated with pore block. For μCTX PIIIA (0.1nM), or KIIIA (30nM), inactivation accelerated by ∼10-fold. Substitution of key basic residues (PIIIA-R14A and KIIIA-K7A) reduced blocking potency and decreased the speeding of inactivation to less than 2-fold.Given the remarkably high selectivity and affinity that μCTXs show for certain eukaryotic Nav channels with highly asymmetric pores, it may seem surprising that they bind to symmetric bacterial Nav channels with such high affinity. However, in both cases, there is the potential for multiple electrostatic interactions to sum and yield potent binding. Our data suggest that pore-block, and acceleration of inactivation, result from distinct molecular actions of the toxin.

NaChBac, from Bacillus halodurans, is a homotetrameric Nav channel, with each monomer having 6 putative transmembrane helices. Its selectivity filter appears to be lined by a ring of 4 glutamate residues (E191 from each monomer). We expressed NaChBac channels in HEK293 cells and studied their selectivity to monovalent organic and alkali cations. Reversal potential shifts were determined from whole-cell currents, following substitution of a test cation for extracellular Na+, and relative permeabilities (PX/PNa) were calculated using the Goldman-Hodgkin-Katz equation. Reversal potentials (Erev) were routinely measured from plots of peak INa vs V, but for ions showing small inward currents, values were checked against tail currents, measured immediately after a maximally activating prepulse. Among the alkali cations, only Na+, Li+ and K+ were measurably permeant. Of 14 organic cations, at pH 7.4, only hydrazinium (HZ+) was measurably permeant (PX/PNa, ≈ 0.8, 0.3, 0.7 respectively, for X= Li, K, HZ

Voltage-gated sodium channels (Nav) are fundamental for the generation and the propagation of action potentials. Mammalian Nav alpha subunits are single macromolecules organized in four different domains (DI-DIV). Each is composed of 6 transmembrane segments (S1-S6) from which S1-S4 constitute the voltage sensing domain (VSD) and with S5 and S6 constituting the pore. While Nav function has been studied extensively, the exact structural mechanisms of gating are not fully understood. Recently, the crystal structure of the prokaryotic sodium channel, NavAb, has been solved, but NavAb is a homotetrameric protein in contract to the mammalian Navs. Thus many questions were not answered by the prokaryotic channel structures. To resolve the voltage dependent conformational changes of Nav, we tracked conformational changes of the VSD from each domain of the rat skeletal muscle sodium channel (Nav1.4) using Lanthanide-based Resonance Energy Transfer (LRET), a FRET technique that allows for precise measurement of intermolecular distances by taking advantage of the special properties of lanthanide as an energy donor. We prepared Nav1.4 constructs with a genetically encoded lanthanide binding tag (LBT), which holds a lanthanide (Tb3+) ion with high affinity, inserted at the top of the S4 segment in each domain. Also, we synthesized two toxins conjugated to dyes to function as acceptors: the pore-blocking small molecule tetrodotoxin conjugated with a HiLyte fluor488 (TTX-F), and the peptide β scorpion toxin Ts1, from the Brazilian scorpion Tityus serrulatus, conjugated with Alexa488 (Ts1-Alexa488). Having several donor positions (Tb3+ ions in LBT's) and two different acceptor positions (TTX-F and Ts1-Alexa488), we calculated multiple distances in voltage-clamped Xenopus laevis oocytes expressing our Nav1.4 constructs that remained functionally active. The results provide new insight to structure-function information in mammalian Nav channels. Support: 13POST14800031 (AHA), MOP-10053 (CIHR), GM68044-07, U54GM087519 and GM030376.