Voltage-gated sodium (Nav) channels in cardiomyocytes are localized in specialized membrane domains that optimize their functions in propagating action potentials across cell junctions and in stimulating voltage-gated calcium channels located in T tubules. Mutation of the ankyrin-binding site of Nav1.5, the principal Nav channel in the heart, was previously known to cause cardiac arrhythmia and the retention of Nav1.5 in an intracellular compartment in cardiomyocytes. Conclusive evidence is now provided that direct interaction between Nav1.5 and ankyrin-G is necessary for the expression of Nav1.5 at the cardiomyocyte cell surface.
Eukaryotic, voltage-gated sodium (NaV) channels are large membrane proteins which underlie generation and propagation of rapid electrical signals in nerve, muscle and heart. Nine different NaV receptor sites, for natural ligands and/or drugs, have been identified, based on functional analyses and site-directed mutagenesis. In the marine ecosystem, numerous toxins have evolved to disrupt NaV channel function, either by inhibition of current flow through the channels, or by modifying the activation and inactivation gating processes by which the channels open and close. These toxins function in their native environment as offensive or defensive weapons in prey capture or deterrence of predators. In composition, they range from organic molecules of varying size and complexity to peptides consisting of ~10–70 amino acids. We review the variety of known NaV-targeted marine toxins, outlining, where known, their sites of interaction with the channel protein and their functional effects. In a number of cases, these natural ligands have the potential applications as drugs in clinical settings, or as models for drug development.
voltage-gated sodium channel; marine neurotoxins; sodium channel receptor sites
The cardiac voltage-gated sodium channel, Nav1.5, plays a central role in cardiac excitability and impulse propagation and associates with the dystrophin multiprotein complex at the lateral membrane of cardiomyocytes. It was previously shown that Nav1.5 protein content and the sodium current (lNa) were both decreased in cardiomyocytes of dystrophin-deficient mdx5cv mice. In this study, wild-type and mdx5cv mice were treated for 7 days with the proteasome inhibitor MG132 (10 μg/Kg/24 h) using implanted osmotic mini pumps. MG132 rescued both the total amount of Nav1.5 protein and lNa but, unlike in previous studies, de novo expression of dystrophin was not observed in skeletal or cardiac muscle. This study suggests that the reduced expression of Nav1.5 in dystrophin-deficient cells is dependent on proteasomal degradation.
sodium channels; dystrophin; proteasome; proteasome inhibitors; MG132; electrophysiology
The human genome encodes nine functional voltage-gated Na+ channels. Three of them, namely Nav1.5, Nav1.8, and Nav1.9, are resistant to nanomolar concentrations of tetrodotoxin (TTX; IC50 ≥ 1 μM). The other isoforms, which are predominantly expressed in the skeletal muscle and nervous system, are highly sensitive to TTX (IC50 ~ 10 nM). During the last two decades, it has become evident that in addition to the major cardiac isoform Nav1.5, several of those TTX sensitive isoforms are expressed in the mammalian heart. Whereas immunohistochemical and electrophysiological methods demonstrated functional expression in various heart regions, the physiological importance of those isoforms for cardiac excitation in higher mammals is still debated. This review summarizes our knowledge on the systemic cardiovascular effects of TTX in animals and humans, with a special focus on cardiac excitation and performance at lower concentrations of this marine drug. Altogether, these data strongly suggest that TTX sensitive Na+ channels, detected more recently in various heart tissues, are not involved in excitation phenomena in the healthy adult heart of higher mammals.
Na+ channel; TTX sensitivity; cardiac conduction; TTX poisoning
Ankyrins are critical components of ion channel and transporter signaling complexes in the cardiovascular system. Over the past five years, ankyrin dysfunction has been linked with abnormal ion channel and transporter membrane organization and fatal human arrhythmias. Loss-of-function variants in the ankyrin-B gene (ANK2) cause “ankyrin-B syndrome” (previously called type 4 long QT syndrome), manifested by a complex cardiac phenotype including ventricular arrhythmias and sudden cardiac death. More recently, dysfunction in the ankyrin-B-based targeting pathway has been linked with a highly penetrant and severe form of human sinus node disease. Ankyrin-G (a second ankyrin gene product) is required for normal expression, membrane localization, and biophysical function of the primary cardiac voltage-gated sodium channel, Nav1.5. Loss of the ankyrin-G/Nav1.5 interaction is associated with human cardiac arrhythmia (Brugada syndrome). Finally, in the past year ankyrin dysfunction has been associated with more common arrhythmia and cardiovascular disease phenotypes. Specifically, large animal studies reveal striking remodeling of ankyrin-B and associated proteins following myocardial infarction. Additionally, the ANK2 locus has been linked with QTc interval variability in the general human population. Together, these findings identify a host of unanticipated and exciting roles for ankyrin polypeptides in cardiac function. More broadly, these findings illustrate the importance of local membrane organization for normal cardiac physiology.
Voltage-gated sodium channels (Navs) are glycoproteins composed of a pore-forming α-subunit and associated β-subunits that regulate Nav α-subunit plasma membrane density and biophysical properties. Glycosylation of the Nav α-subunit also directly affects Navs gating. β-subunits and glycosylation thus comodulate Nav α-subunit gating. We hypothesized that β-subunits could directly influence α-subunit glycosylation. Whole-cell patch clamp of HEK293 cells revealed that both β1- and β3-subunits coexpression shifted V½ of steady-state activation and inactivation and increased Nav1.7-mediated INa density. Biotinylation of cell surface proteins, combined with the use of deglycosydases, confirmed that Nav1.7 α-subunits exist in multiple glycosylated states. The α-subunit intracellular fraction was found in a core-glycosylated state, migrating at ~250 kDa. At the plasma membrane, in addition to the core-glycosylated form, a fully glycosylated form of Nav1.7 (~280 kDa) was observed. This higher band shifted to an intermediate band (~260 kDa) when β1-subunits were coexpressed, suggesting that the β1-subunit promotes an alternative glycosylated form of Nav1.7. Furthermore, the β1-subunit increased the expression of this alternative glycosylated form and the β3-subunit increased the expression of the core-glycosylated form of Nav1.7. This study describes a novel role for β1- and β3-subunits in the modulation of Nav1.7 α-subunit glycosylation and cell surface expression.
voltage-gated sodium channels (Navs); Navs β-subunits; glycosylation; biophysical properties; trafficking
Nav1.5 is the principal voltage-gated sodium channel expressed in heart, and is also expressed at lower abundance in embryonic dorsal root ganglia (DRG) with little or no expression reported postnatally. We report here the expression of Nav1.5 mRNA isoforms in adult mouse and rat DRG. The major isoform of mouse DRG is Nav1.5a, which encodes a protein with an IDII/III cytoplasmic loop reduced by 53 amino acids. Western blot analysis of adult mouse DRG membrane proteins confirmed the expression of Nav1.5 protein. The Na+ current produced by the Nav1.5a isoform has a voltage-dependent inactivation significantly shifted to more negative potentials (by ~5 mV) compared to the full-length Nav1.5 when expressed in the DRG neuroblastoma cell line ND7/23. These results imply that the alternatively spliced exon 18 of Nav1.5 plays a role in channel inactivation and that Nav1.5a is likely to make a significant contribution to adult DRG neuronal function.
Human voltage-activated sodium (Nav) channels are adept at rapidly transmitting electrical signals across long distances in various excitable tissues. As such, they are amongst the most widely targeted ion channels by drugs and animal toxins. Of the nine isoforms, Nav1.8 and Nav1.9 are preferentially expressed in DRG neurons where they are thought to play an important role in pain signaling. Although the functional properties of Nav1.8 have been relatively well characterized, difficulties with expressing Nav1.9 in established heterologous systems limit our understanding of the gating properties and toxin pharmacology of this particular isoform. This review summarizes our current knowledge of the role of Nav1.8 and Nav1.9 in pain perception and elaborates on the approaches used to identify molecules capable of influencing their function.
Nav1.8; Nav1.9; pain; animal toxins; voltage sensor; voltage-activated sodium channel
The number of voltage-gated sodium (NaV) channels available to generate action potentials in muscles and nerves is adjusted over seconds to minutes by prior electrical activity, a process called slow inactivation (SI). The basis for SI is uncertain. NaV channels have four domains (DI–DIV), each with a voltage sensor that moves in response to depolarizing stimulation over milliseconds to activate the channels. Here, SI of the skeletal muscle channel NaV1.4 is induced by repetitive stimulation and is studied by recording of sodium currents, gating currents, and changes in the fluorescence of probes on each voltage sensor to assess their movements. The magnitude, voltage dependence, and time course of the onset and recovery of SI are observed to correlate with voltage-sensor movements 10,000-fold slower than those associated with activation. The behavior of each voltage sensor is unique. Development of SI over 1–160 s correlates best with slow immobilization of the sensors in DI and DII; DIII tracks the onset of SI with less fidelity. Showing linkage to the sodium conduction pathway, pore block by tetrodotoxin affects both SI and immobilization of all the sensors, with DI and DII significantly suppressed. Recovery from SI correlates best with slow restoration of mobility of the sensor in DIII. The findings suggest that voltage-sensor movements determine SI and thereby mediate NaV channel availability.
Scorpion β toxins, peptides of ∼70 residues, specifically target voltage-gated sodium (NaV) channels to cause use-dependent subthreshold channel openings via a voltage–sensor trapping mechanism. This excitatory action is often overlaid by a not yet understood depressant mode in which NaV channel activity is inhibited. Here, we analyzed these two modes of gating modification by β-toxin Tz1 from Tityus zulianus on heterologously expressed NaV1.4 and NaV1.5 channels using the whole cell patch-clamp method. Tz1 facilitated the opening of NaV1.4 in a use-dependent manner and inhibited channel opening with a reversed use dependence. In contrast, the opening of NaV1.5 was exclusively inhibited without noticeable use dependence. Using chimeras of NaV1.4 and NaV1.5 channels, we demonstrated that gating modification by Tz1 depends on the specific structure of the voltage sensor in domain 2. Although residue G658 in NaV1.4 promotes the use-dependent transitions between Tz1 modification phenotypes, the equivalent residue in NaV1.5, N803, abolishes them. Gating charge neutralizations in the NaV1.4 domain 2 voltage sensor identified arginine residues at positions 663 and 669 as crucial for the outward and inward movement of this sensor, respectively. Our data support a model in which Tz1 can stabilize two conformations of the domain 2 voltage sensor: a preactivated outward position leading to NaV channels that open at subthreshold potentials, and a deactivated inward position preventing channels from opening. The results are best explained by a two-state voltage–sensor trapping model in that bound scorpion β toxin slows the activation as well as the deactivation kinetics of the voltage sensor in domain 2.
Voltage-gated sodium (Nav) channels are responsible for initiation and propagation of action potential in the neurons. To explore the mechanisms for chronic heart failure (CHF)-induced baroreflex dysfunction, we measured the expression and current density of Nav channel subunits (Nav1.7, Nav1.8, and Nav1.9) in the aortic baroreceptor neurons and investigated the role of Nav channels on aortic baroreceptor neuron excitability and baroreflex sensitivity in sham and CHF rats. CHF was induced by left coronary artery ligation. The development of CHF (6–8 weeks after the coronary ligation) was confirmed by hemodynamic and morphological characteristics. Immunofluorescent data indicated that Nav1.7 was expressed in A-type (myelinated) and C-type (unmyelinated) nodose neurons but Nav1.8 and Nav1.9 were expressed only in C-type nodose neurons. Real-time RT-PCR and western blot data showed that CHF reduced mRNA and protein expression levels of Nav channels in nodose neurons. In addition, using the whole cell patch-clamp technique, we found that Nav current density and cell excitability of the aortic baroreceptor neurons were lower in CHF rats than that in sham rats. Aortic baroreflex sensitivity was blunted in anesthetized CHF rats, compared with that in sham rats. Furthermore, Nav channel activator (rATX II, 100 nM) significantly enhanced Nav current density and cell excitability of aortic baroreceptor neurons and improved aortic baroreflex sensitivity in CHF rats. These results suggest that reduced expression and activation of the Nav channels is involved in the attenuation of baroreceptor neuron excitability, which subsequently contributes to the impairment of baroreflex in CHF state.
Aortic baroreceptor neuron; Baroreflex; Heart failure; Sodium channel
Ranolazine is an antianginal agent that targets a number of ion channels in the heart, including cardiac voltage-gated Na+ channels. However, ranolazine block of muscle and neuronal Na+ channel isoforms has not been examined. We compared the state- and use-dependent ranolazine block of Na+ currents carried by muscle Nav1.4, cardiac Nav1.5, and neuronal Nav1.7 isoforms expressed in human embryonic kidney 293T cells. Resting and inactivated block of Na+ channels by ranolazine were generally weak, with a 50% inhibitory concentration (IC50) ≥ 60 μM. Use-dependent block of Na+ channel isoforms by ranolazine during repetitive pulses (+50 mV/10 ms at 5 Hz) was strong at 100 μM, up to 77% peak current reduction for Nav1.4, 67% for Nav1.5, and 83% for Nav1.7. In addition, we found conspicuous time-dependent block of inactivation-deficient Nav1.4, Nav1.5, and Nav1.7 Na+ currents by ranolazine with estimated IC50 values of 2.4, 6.2, and 1.7 μM, respectively. On- and off-rates of ranolazine were 8.2 μM−1 s−1 and 22 s−1, respectively, for Nav1.4 open channels and 7.1 μM−1 s−1 and 14 s−1, respectively, for Nav1.7 counterparts. A F1579K mutation at the local anesthetic receptor of inactivation-deficient Nav1.4 Na+ channels reduced the potency of ranolazine ~17-fold. We conclude that: 1) both muscle and neuronal Na+ channels are as sensitive to ranolazine block as their cardiac counterparts; 2) at its therapeutic plasma concentrations, ranolazine interacts predominantly with the open but not resting or inactivated Na+ channels; and 3) ranolazine block of open Na+ channels is via the conserved local anesthetic receptor albeit with a relatively slow on-rate.
Skeletal muscle sodium channel (Nav1.4) expression in border zone myocardium increases action potential upstroke velocity in depolarized isolated tissue. Because resting membrane potential in the 1 week canine infarct is reduced, we hypothesized that conduction velocity (CV) is greater in Nav1.4 dogs compared to control dogs.
To measure CV in the infarct border zone border in dogs with and without Nav1.4 expression.
Adenovirus was injected in the infarct border zone in 34 dogs. The adenovirus incorporated the Nav1.4- and a green fluorescent protein (GFP) gene (Nav1.4 group, n=16) or only GFP (n=18). After 1 week, upstroke velocity and CV were measured by sequential microelectrode recordings at 4 and 7 mM [K+] in superfused epicardial slabs. High density in vivo epicardial activation mapping was performed in a subgroup (8 Nav1.4, 6 GFP) at 3–4 locations in the border zone. Microscopy and antibody staining confirmed GFP or Nav1.4 expression.
Infarct sizes were similar between groups (30.6+/−3 % of LV mass, mean+/−SEM). Longitudinal CV was greater in Nav1.4- than in GFP- sites (58.5+/−1.8 vs 53.3+/−1.2 cm/s, 20 and 15 sites, respectively, p<0.05). Transverse CV was not different between the groups. In tissue slabs dV/dtmax was higher and CV was greater in Nav1.4 than in control at 7 mM [K+] (P<0.05). Immunohistochemical Nav1.4 staining was seen at the longitudinal ends of the myocytes.
Nav1.4 channels in myocardium surviving 1 week infarction increases longitudinal but not transverse CV, consistent with the increased dV/dtmax and with the cellular localization of Nav1.4.
conduction; arrhythmias; gene therapy; skeletal muscle; sodium channel; myocardial infarction
▶ The β3 subunit masks the ER retention signal of NaV1.8 and release the channel from the ER. ▶ p11 directly binds to NaV1.8 and help its translocation to the plasma membrane. ▶ PDZD2 is responsible for the functional expression of NaV1.8 on the plasma membrane. ▶ Contactin KO mice exhibit a reduction of NaV1.8 along unmyelinated axons in the sciatic nerve. ▶ PKA activation increases the NaV1.8 density on the membrane through direct phosphorylation.
The α-subunit of tetrodotoxin-resistant voltage-gated sodium channel NaV1.8 is selectively expressed in sensory neurons. It has been reported that NaV1.8 is involved in the transmission of nociceptive information from sensory neurons to the central nervous system in nociceptive  and neuropathic  pain conditions. Thus NaV1.8 has been a promising target to treat chronic pain. Here we discuss the recent advances in the study of trafficking mechanism of NaV1.8. These pieces of information are particularly important as such trafficking machinery could be new targets for painkillers.
Sodium Channel; Sensory Neuron; Pain; Trafficking
Inherited mutations in voltage-gated sodium channels (VGSCs; or Nav) cause many
disorders of excitability, including epilepsy, chronic pain, myotonia, and cardiac
arrhythmias. Understanding the functional consequences of the disease-causing
mutations is likely to provide invaluable insight into the roles that VGSCs play in
normal and abnormal excitability. Here, we sought to test the hypothesis that
disease-causing mutations lead to increased resurgent currents, unusual sodium
currents that have not previously been implicated in disorders of excitability. We
demonstrated that a paroxysmal extreme pain disorder (PEPD) mutation in the human
peripheral neuronal sodium channel Nav1.7, a paramyotonia congenita (PMC) mutation in
the human skeletal muscle sodium channel Nav1.4, and a long-QT3/SIDS mutation in the
human cardiac sodium channel Nav1.5 all substantially increased the amplitude of
resurgent sodium currents in an optimized adult rat–derived dorsal root
ganglion neuronal expression system. Computer simulations indicated that resurgent
currents associated with the Nav1.7 mutation could induce high-frequency action
potential firing in nociceptive neurons and that resurgent currents associated with
the Nav1.5 mutation could broaden the action potential in cardiac myocytes. These
effects are consistent with the pathophysiology associated with the respective
channelopathies. Our results indicate that resurgent currents are associated with
multiple channelopathies and are likely to be important contributors to neuronal and
muscle disorders of excitability.
Despite increasing evidence for the presence of voltage-gated Na+ channels (Nav) isoforms and measurements of Nav channel currents with the patch-clamp technique in arterial myocytes, no information is available to date as to whether or not Nav channels play a functional role in arteries. The aim of the present work was to look for a physiological role of Nav channels in the control of rat aortic contraction.
Nav channels were detected in the aortic media by Western blot analysis and double immunofluorescence labeling for Nav channels and smooth muscle α-actin using specific antibodies. In parallel, using real time RT-PCR, we identified three Nav transcripts: Nav1.2, Nav1.3, and Nav1.5. Only the Nav1.2 isoform was found in the intact media and in freshly isolated myocytes excluding contamination by other cell types. Using the specific Nav channel agonist veratridine and antagonist tetrodotoxin (TTX), we unmasked a contribution of these channels in the response to the depolarizing agent KCl on rat aortic isometric tension recorded from endothelium-denuded aortic rings. Experimental conditions excluded a contribution of Nav channels from the perivascular sympathetic nerve terminals. Addition of low concentrations of KCl (2–10 mM), which induced moderate membrane depolarization (e.g., from −55.9±1.4 mV to −45.9±1.2 mV at 10 mmol/L as measured with microelectrodes), triggered a contraction potentiated by veratridine (100 µM) and blocked by TTX (1 µM). KB-R7943, an inhibitor of the reverse mode of the Na+/Ca2+ exchanger, mimicked the effect of TTX and had no additive effect in presence of TTX.
These results define a new role for Nav channels in arterial physiology, and suggest that the TTX-sensitive Nav1.2 isoform, together with the Na+/Ca2+ exchanger, contributes to the contractile response of aortic myocytes at physiological range of membrane depolarization.
The voltage-gated sodium channel Nav1.6 plays unique roles in the nervous system, but its functional properties and neuromodulation are not as well established as for NaV1.2 channels. We found no significant differences in voltage-dependent activation or fast inactivation between NaV1.6 and NaV1.2 channels expressed in non-excitable cells. In contrast, the voltage dependence of slow inactivation was more positive for Nav1.6 channels, they conducted substantially larger persistent sodium currents than Nav1.2 channels, and they were much less sensitive to inhibtion by phosphorylation by cAMP-dependent protein kinase and protein kinase C. Resurgent sodium current, a hallmark of Nav1.6 channels in neurons, was not observed for NaV1.6 expressed alone or with the auxiliary β4 subunit. The unique properties of NaV1.6 channels, together with the resurgent currents that they conduct in neurons, make these channels well-suited to provide the driving force for sustained repetitive firing, a crucial property of neurons.
The axon initial segment is an excitable membrane highly enriched in voltage-gated sodium channels that integrates neuronal inputs and initiates action potentials. This study identifies Nav1.6 as the voltage-gated sodium channel isoform at mature Purkinje neuron initial segments and reports an essential role for ankyrin-G in coordinating the physiological assembly of Nav1.6, βIV spectrin, and the L1 cell adhesion molecules (L1 CAMs) neurofascin and NrCAM at initial segments of cerebellar Purkinje neurons. Ankyrin-G and βIV spectrin appear at axon initial segments by postnatal day 2, whereas L1 CAMs and Nav1.6 are not fully assembled at continuous high density along axon initial segments until postnatal day 9. L1 CAMs and Nav1.6 therefore do not initiate protein assembly at initial segments. βIV spectrin, Nav1.6, and L1 CAMs are not clustered in adult Purkinje neuron initial segments of mice lacking cerebellar ankyrin-G. These results support the conclusion that ankyrin-G coordinates the physiological assembly of a protein complex containing transmembrane adhesion molecules, voltage-gated sodium channels, and the spectrin membrane skeleton at axon initial segments.
βIV spectrin; sodium channel Nav1.6; neurofascin; NrCAM; axon hillock
NaV1.5 is a mechanosensitive voltage-gated Na+ channel encoded by the gene SCN5A, expressed in cardiac myocytes and required for phase 0 of the cardiac action potential (AP). In the cardiomyocyte, ranolazine inhibits depolarizing Na+ current and delayed rectifier (IKr) currents. Recently, ranolazine was also shown to be an inhibitor of NaV1.5 mechanosensitivity. Stretch also accelerates the firing frequency of the SA node, and fluid shear stress increases the beating rate of cultured cardiomyocytes in vitro. However, no cultured cell platform exists currently for examination of spontaneous electrical activity in response to mechanical stimulation. In the present study, flow of solution over atrial myocyte-derived HL-1 cultured cells was used to study shear stress mechanosensitivity of Na+ current and spontaneous, endogenous rhythmic action potentials. In voltage-clamped HL-1 cells, bath flow increased peak Na+ current by 14 ± 5%. In current-clamped cells, bath flow increased the frequency and decay rate of AP by 27 ± 12% and 18 ± 4%, respectively. Ranolazine blocked both responses to shear stress. This study suggests that cultured HL-1 cells are a viable in vitro model for detailed study of the effects of mechanical stimulation on spontaneous cardiac action potentials. Inhibition of the frequency and decay rate of action potentials in HL-1 cells are potential mechanisms behind the antiarrhythmic effect of ranolazine.
electrophysiology; mechanosensitivity; ion channels; drugs; myocytes
In skeletal muscle, slow inactivation (SI) of NaV1.4 voltage-gated sodium channels prevents spontaneous depolarization and fatigue. Inherited mutations in NaV1.4 that impair SI disrupt activity-induced regulation of channel availability and predispose patients to hyperkalemic periodic paralysis. In our companion paper in this issue (Silva and Goldstein. 2013. J. Gen. Physiol. http://dx.doi.org/10.1085/jgp.201210909), the four voltage sensors in NaV1.4 responsible for activation of channels over microseconds are shown to slowly immobilize over 1–160 s as SI develops and to regain mobility on recovery from SI. Individual sensor movements assessed via attached fluorescent probes are nonidentical in their voltage dependence, time course, and magnitude: DI and DII track SI onset, and DIII appears to reflect SI recovery. A causal link was inferred by tetrodotoxin (TTX) suppression of both SI onset and immobilization of DI and DII sensors. Here, the association of slow sensor immobilization and SI is verified by study of NaV1.4 channels with a hyperkalemic periodic paralysis mutation; L689I produces complex changes in SI, and these are found to manifest directly in altered sensor movements. L689I removes a component of SI with an intermediate time constant (∼10 s); the mutation also impedes immobilization of the DI and DII sensors over the same time domain in support of direct mechanistic linkage. A model that recapitulates SI attributes responsibility for intermediate SI to DI and DII (10 s) and a slow component to DIII (100 s), which accounts for residual SI, not impeded by L689I or TTX.
Bisphenol A (BPA) has attracted considerable public attention as it leaches from plastic used in food containers, is detectable in human fluids and recent epidemiologic studies link BPA exposure with diseases including cardiovascular disorders. As heart-toxicity may derive from modified cardiac electrophysiology, we investigated the interaction between BPA and hNav1.5, the predominant voltage-gated sodium channel subtype expressed in the human heart. Electrophysiology studies of heterologously-expressed hNav1.5 determined that BPA blocks the channel with a Kd of 25.4±1.3 µM. By comparing the effects of BPA and the local anesthetic mexiletine on wild type hNav1.5 and the F1760A mutant, we demonstrate that both compounds share an overlapping binding site. With a key binding determinant thus identified, an homology model of hNav1.5 was generated based on the recently-reported crystal structure of the bacterial voltage-gated sodium channel NavAb. Docking predictions position both ligands in a cavity delimited by F1760 and contiguous with the DIII–IV pore fenestration. Steered molecular dynamics simulations used to assess routes of ligand ingress indicate that the DIII–IV pore fenestration is a viable access pathway. Therefore BPA block of the human heart sodium channel involves the local anesthetic receptor and both BPA and mexiletine may enter the closed-state pore via membrane-located side fenestrations.
Tetrodotoxin (TTX)-resistant voltage-gated Na (NaV) channels have been implicated in nociception. In particular, NaV1.9 contributes to expression of persistent Na current in small diameter, nociceptive sensory neurons in dorsal root ganglia and is required for inflammatory pain sensation. Using ND7/23 cells stably expressing human NaV1.9, we elucidated the biophysical mechanisms responsible for potentiation of channel activity by G-protein signaling to better understand the response to inflammatory mediators. Heterologous NaV1.9 expression evoked TTX-resistant Na current with peak activation at −40 mV with extensive overlap in voltage dependence of activation and inactivation. Inactivation kinetics were slow and incomplete, giving rise to large persistent Na currents. Single-channel recording demonstrated long openings and correspondingly high open probability (Po) accounting for the large persistent current amplitude. Channels exposed to intracellular GTPγS, a proxy for G-protein signaling, exhibited twofold greater current density, slowing of inactivation, and a depolarizing shift in voltage dependence of inactivation but no change in activation voltage dependence. At the single-channel level, intracellular GTPγS had no effect on single-channel amplitude but caused an increased mean open time and greater Po compared with recordings made in the absence of GTPγS. We conclude that G-protein activation potentiates human NaV1.9 activity by increasing channel open probability and mean open time, causing the larger peak and persistent current, respectively. Our results advance our understanding about the mechanism of NaV1.9 potentiation by G-protein signaling during inflammation and provide a cellular platform useful for the discovery of NaV1.9 modulators with potential utility in treating inflammatory pain.
Sodium channels play an essential role in generating the action potential in eukaryotic cells, and their transcripts, especially those in insects, undergo extensive A-to-I RNA editing. The functional consequences of RNA editing of sodium channel transcripts, however, have yet to be determined. We characterized 20 splice variants of the German cockroach sodium channel gene BgNav. Functional analysis revealed that these variants exhibited a broad range of voltage-dependent activation and inactivation. Further analysis of two variants, BgNav1-1 and BgNav1-2, which activate at more depolarizing membrane potentials than other variants, showed that RNA editing events were responsible for variant-specific gating properties. Two U-to-C editing sites identified in BgNav1-1 resulted in a Leu to Pro change in segment 1 of domain III (IIIS1) and a Val to Ala change in IVS4. The Leu to Pro change shifted both the voltage dependence of activation and steady-state inactivation in the depolarizing direction. Two A-to-I editing events in BgNav1-2 resulted in a Lys to Arg change in IS2 and an Ile to Met change in IVS3. The Lys to Arg change shifted the voltage dependence of activation in the depolarizing direction. Moreover, these RNA editing events occurred in a tissue-specific and development-specific manner. Our findings provide direct evidence that RNA editing is an important mechanism generating tissue-/cell type-specific functional variants of sodium channels.
The lipid bilayer is important for maintaining the integrity of cellular compartments and plays a vital role in providing the hydrophobic and charged interactions necessary for membrane protein structure, conformational flexibility and function. To directly assess the lipid dependence of activity for voltage-gated sodium channels, we compared the activity of three bacterial sodium channel homologues (NaChBac, NavMs, and NavSp) by cumulative 22Na+ uptake into proteoliposomes containing a 3∶1 ratio of 1-palmitoyl 2-oleoyl phosphatidylethanolamine and different “guest” glycerophospholipids. We observed a unique lipid profile for each channel tested. NavMs and NavSp showed strong preference for different negatively-charged lipids (phosphatidylinositol and phosphatidylglycerol, respectively), whilst NaChBac exhibited a more modest variation with lipid type. To investigate the molecular bases of these differences we used synchrotron radiation circular dichroism spectroscopy to compare structures in liposomes of different composition, and molecular modeling and electrostatics calculations to rationalize the functional differences seen. We then examined pore-only constructs (with voltage sensor subdomains removed) and found that in these channels the lipid specificity was drastically reduced, suggesting that the specific lipid influences on voltage-gated sodium channels arise primarily from their abilities to interact with the voltage-sensing subdomains.
Cardiac sodium channel Nav1.5 plays a critical role in heart excitability and conduction. The molecular mechanism that underlies the expression of Nav1.5 at the cell membrane is poorly understood. Previous studies demonstrated that cytoskeleton proteins can be involved in the regulation of cell surface expression and localization of several ion channels. We performed a yeast two-hybrid screen to identify Nav1.5-associated proteins that may be involved in channel function and expression. We identified α-actinin-2 as an interacting partner of the cytoplasmic loop connecting domains III and IV of Nav1.5 (Nav1.5/LIII–IV). Co-immunoprecipitation and His6 pull-down assays confirmed the physical association between Nav1.5 and α-actinin-2 and showed that the spectrin-like repeat domain is essential for binding of α-actinin-2 to Nav1.5. Patch-clamp studies revealed that the interaction with α-actinin-2 increases sodium channel density without changing their gating properties. Consistent with these findings, coexpression of α-actinin-2 and Nav1.5 in tsA201 cells led to an increase in the level of expression of Nav1.5 at the cell membrane as determined by cell surface biotinylation. Lastly, immunostaining experiments showed that α-actinin-2 was colocalized with Nav1.5 along the Z-lines and in the plasma membrane. Our data suggest that α-actinin-2, which is known to regulate the functional expression of the potassium channels, may play a role in anchoring Nav1.5 to the membrane by connecting the channel to the actin cytoskeleton network.