Voltage-gated Nav channel targeting in the heart requires an ankyrin-G dependent cellular pathway

Voltage-gated Na_v channels are required for normal electrical activity in neurons, skeletal muscle, and cardiomyocytes. In the heart, Na_v1.5 is the predominant Na_v channel, and Na_v1.5-dependent activity regulates rapid upstroke of the cardiac action potential. Na_v1.5 activity requires precise localization at specialized cardiomyocyte membrane domains. However, the molecular mechanisms underlying Na_v channel trafficking in the heart are unknown. In this paper, we demonstrate that ankyrin-G is required for Na_v1.5 targeting in the heart. Cardiomyocytes with reduced ankyrin-G display reduced Na_v1.5 expression, abnormal Na_v1.5 membrane targeting, and reduced Na⁺ channel current density. We define the structural requirements on ankyrin-G for Na_v1.5 interactions and demonstrate that loss of Na_v1.5 targeting is caused by the loss of direct Na_v1.5–ankyrin-G interaction. These data are the first report of a cellular pathway required for Na_v channel trafficking in the heart and suggest that ankyrin-G is critical for cardiac depolarization and Na_v channel organization in multiple excitable tissues.     

Caveolae, ion channels and cardiac arrhythmias

Caveolae are specialized membrane microdomains enriched in cholesterol and sphingolipids which are present in multiple cell types including cardiomyocytes. Along with the essential scaffolding protein caveolin-3, a number of different ion channels and transporters have been localized to caveolae in cardiac myocytes including L-type Ca²⁺ channels (Ca_v1.2), Na⁺ channels (Na_v1.5), pacemaker channels (HCN4), Na⁺/Ca²⁺ exchanger (NCX1) and others. Closely associated with these channels are specific macromolecular signaling complexes that provide highly localized regulation of the channels. Mutations in the caveolin-3 gene (CAV3) have been linked with the congenital long QT syndrome (LQT9), and mutations in caveolar-localized ion channels may contribute to other inherited arrhythmias. Changes in the caveolar microdomain in acquired heart disease may also lead to dysregulation and dysfunction of ion channels, altering the risk of arrhythmias in conditions such as heart failure. This review highlights the existing evidence identifying and characterizing ion channels localized to caveolae in cardiomyocytes and their role in arrhythmogenesis.     

Involvement of voltage-gated K+ and Na+ channels in gastric epithelial cell migration

Epithelial cell migration plays an important role in gastrointestinal mucosal repair. We previously reported that multiple functional ion channels, including a Ba²⁺-sensitive K⁺ inward rectifier K_ir1.2, 4-aminopyridine (4-AP)-sensitive voltage-gated K⁺ channels K_v1.1, K_v1.6 and K_v2.1, and a nifedipine-sensitive, tetrodotoxin (TTX)-insensitive voltage-gated Na⁺ channel Na_v1.5 were expressed in a non-transformed rat gastric epithelial cell line (RGM-1). In the present study, we further investigated whether these ion channels are involved in the modulation of gastric epithelial cell migration. Cell migration was determined by monolayer wound healing assay. Results showed that blockade of K_v with 4-AP or Na_v1.5 with nifedipine inhibited RGM-1 cell migration in the absence or presence of epidermal growth factor (EGF), which effectively stimulated RGM-1 cell migration. Moreover, high concentration of TTX mimicked the action of nifedipine, suggesting that the action of nifedipine was mediated through specific blockade of Na_v1.5. In contrast, inhibition of K_ir1.2 with Ba²⁺, either in basal or EGF-stimulated condition, had no effect on RGM-1 cell migration. In conclusion, the present study demonstrates for the first time that voltage-gated K⁺ and Na⁺ channels are involved in the modulation of gastric epithelial cell migration.     

Small GTPases SAR1A and SAR1B regulate the trafficking of the cardiac sodium channel Nav1.5

Background

The cardiac sodium channel Na_v1.5 is essential for the physiological function of the heart and causes cardiac arrhythmias and sudden death when mutated. Many disease-causing mutations in Na_v1.5 cause defects in protein trafficking, a cellular process critical to the targeting of Na_v1.5 to cell surface. However, the molecular mechanisms underlying the trafficking of Na_v1.5, in particular, the exit from the endoplasmic reticulum (ER) for cell surface trafficking, remain poorly understood.

Comparison of Gating Properties and Use-Dependent Block of Nav1.5 and Nav1.7 Channels by Anti-Arrhythmics Mexiletine and Lidocaine

Mexiletine and lidocaine are widely used class IB anti-arrhythmic drugs that are considered to act by blocking voltage-gated open sodium currents for treatment of ventricular arrhythmias and relief of pain. To gain mechanistic insights into action of anti-arrhythmics, we characterized biophysical properties of Na_v1.5 and Na_v1.7 channels stably expressed in HEK293 cells and compared their use-dependent block in response to mexiletine and lidocaine using whole-cell patch clamp recordings. While the voltage-dependent activation of Na_v1.5 or Na_v1.7 was not affected by mexiletine and lidocaine, the steady-state fast and slow inactivation of Na_v1.5 and Na_v1.7 were significantly shifted to hyperpolarized direction by either mexiletine or lidocaine in dose-dependent manner. Both mexiletine and lidocaine enhanced the slow component of closed-state inactivation, with mexiletine exerting stronger inhibition on either Na_v1.5 or Na_v1.7. The recovery from inactivation of Na_v1.5 or Na_v1.7 was significantly prolonged by mexiletine compared to lidocaine. Furthermore, mexiletine displayed a pronounced and prominent use-dependent inhibition of Na_v1.5 than lidocaine, but not Na_v1.7 channels. Taken together, our findings demonstrate differential responses to blockade by mexiletine and lidocaine that preferentially affect the gating of Na_v1.5, as compared to Na_v1.7; and mexiletine exhibits stronger use-dependent block of Na_v1.5. The differential gating properties of Na_v1.5 and Na_v1.7 in response to mexiletine and lidocaine may help explain the drug effectiveness and advance in new designs of safe and specific sodium channel blockers for treatment of cardiac arrhythmia or pain.     

Differential regulation of Na(v)beta subunits during myogenesis

Voltage-gated sodium channels (Na_v) consist of a pore-forming α subunit (Na_vα) associated with β regulatory subunits (Na_vβ). Adult skeletal myocytes primarily express Na_v1.4 channels. We found, however, using neonatal L6E9 myocytes, that myofibers acquire a Na_v1.5-cardiac-like phenotype efficiently. Differentiated myotubes elicited faster Na_v1.5 currents than those recorded from myoblasts. Unlike myoblasts, I_Na recorded in myotubes exhibited an accumulation of inactivation after the application of trains of pulses, due to a slower recovery from inactivation. Since Na_vβ subunits modulate channel gating and pharmacology, the goal of the present work was to study Na_vβ subunits during myogenesis. All four Na_vβ (Na_vβ1-4) isoforms were present in L6E9 myocytes. While Na_vβ1-3 subunits were up-regulated by myogenesis, Na_vβ4 subunits were not. These results show that Na_vβ genes are strongly regulated during muscle differentiation and further support a physiological role for voltage-gated Na⁺ channels during development and myotube formation.     

Crystal Structure and Molecular Imaging of the Nav Channel β3 Subunit Indicates a Trimeric Assembly

The vertebrate sodium (Na_v) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Na_v channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Na_v 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Na_v channel cross-linking and provide new structural insights into some pathological Na_v channel mutations.     

Voltage-dependent activation of Rac1 by Nav1.5 channels promotes cell migration

Ion channels can regulate the plasma membrane potential (V_m) and cell migration as a result of altered ion flux. However, the mechanism by which V_m regulates motility remains unclear. Here, we show that the Na_v1.5 sodium channel carries persistent inward Na⁺ current which depolarizes the resting V_m at the timescale of minutes. This Na_v1.5-dependent V_m depolarization increases Rac1 colocalization with phosphatidylserine, to which it is anchored at the leading edge of migrating cells, promoting Rac1 activation. A genetically encoded FRET biosensor of Rac1 activation shows that depolarization-induced Rac1 activation results in acquisition of a motile phenotype. By identifying Na_v1.5-mediated V_m depolarization as a regulator of Rac1 activation, we link ionic and electrical signaling at the plasma membrane to small GTPase-dependent cytoskeletal reorganization and cellular migration. We uncover a novel and unexpected mechanism for Rac1 activation, which fine tunes cell migration in response to ionic and/or electric field changes in the local microenvironment.     

Importance of Gradients in Membrane Properties and Electrical Coupling in Sinoatrial Node Pacing

The sinoatrial node (SAN) is heterogeneous in terms of cell size, ion channels, current densities, connexins and electrical coupling. For example, Na_v1.5 (responsible for I _Na) and Cx43 (responsible for electrical coupling) are absent from the centre of the SAN (normally the leading pacemaker site), but present in the periphery (at SAN-atrial muscle junction). To test whether the heterogeneity is important for the functioning of the SAN, one- and two-dimensional models of the SAN and surrounding atrial muscle were created. Normal functioning of the SAN (in terms of cycle length, position of leading pacemaker site, conduction times, activation and repolarization sequences and space constants) was observed when, from the centre to the periphery, (i) cell characteristics (cell size and ionic current densities) were changed in a gradient fashion from a central-type (lacking I _Na) to a peripheral-type (possessing I _Na) and (ii) coupling conductance was increased in a gradient fashion. We conclude that the heterogeneous nature of the node is important for its normal functioning. The presence of Na_v1.5 and Cx43 in the periphery may be essential for the node to be able to drive the atrial muscle: Na_v1.5 provides the necessary depolarizing current and Cx43 delivers it to the atrial muscle.     

Preferential Targeting of Nav1.6 Voltage-Gated Na+ Channels to the Axon Initial Segment during Development

During axonal maturation, voltage-gated sodium (Na_v) channels accumulate at the axon initial segment (AIS) at high concentrations. This localization is necessary for the efficient initiation of action potentials. The mechanisms underlying channel trafficking to the AIS during axonal development have remained elusive due to a lack of Na_v reagents suitable for high resolution imaging of channels located specifically on the cell surface. Using an optical pulse-chase approach in combination with a novel Na_v1.6 construct containing an extracellular biotinylation domain we demonstrate that Na_v1.6 channels are preferentially inserted into the AIS membrane during neuronal development via direct vesicular trafficking. Single-molecule tracking illustrates that axonal channels are immediately immobilized following delivery, while channels delivered to the soma are often mobile. Neither a Na_v1.6 channel lacking the ankyrin-binding motif nor a chimeric K_v2.1 channel containing the Na_v ankyrinG-binding domain show preferential AIS insertion. Together these data support a model where ankyrinG-binding is required for preferential Na_v1.6 insertion into the AIS plasma membrane. In contrast, ankyrinG-binding alone does not confer the preferential delivery of proteins to the AIS.     

The Mechanism of Na+/K+ Selectivity in Mammalian Voltage-Gated Sodium Channels Based on Molecular Dynamics Simulation

Voltage-gated sodium (Na_v) channels and their Na⁺/K⁺ selectivity are of great importance in the mammalian neuronal signaling. According to mutational analysis, the Na⁺/K⁺ selectivity in mammalian Na_v channels is mainly determined by the Lys and Asp/Glu residues located at the constriction site within the selectivity filter. Despite successful molecular dynamics simulations conducted on the prokaryotic Na_v channels, the lack of Lys at the constriction site of prokaryotic Na_v channels limits how much can be learned about the Na⁺/K⁺ selectivity in mammalian Na_v channels. In this work, we modeled the mammalian Na_v channel by mutating the key residues at the constriction site in a prokaryotic Na_v channel (Na_vRh) to its mammalian counterpart. By simulating the mutant structure, we found that the Na⁺ preference in mammalian Na_v channels is collaboratively achieved by the deselection from Lys and the selection from Asp/Glu within the constriction site.     

Sodium Channel Carboxyl-terminal Residue Regulates Fast Inactivation

The Na_v1.2 and Na_v1.3 voltage-gated sodium channel isoforms demonstrate distinct differences in their kinetics and voltage dependence of fast inactivation when expressed in Xenopus oocytes. Co-expression of the auxiliary β1 subunit accelerated inactivation of both the Na_v1.2 and Na_v1.3 isoforms, but it did not eliminate the differences, demonstrating that this property is inherent in the α subunit. By constructing chimeric channels between Na_v1.2 and Na_v1.3, we demonstrate that the carboxyl terminus is responsible for the differences. The Na_v1.2 carboxyl terminus caused faster inactivation in the Na_v1.3 backbone, and the Na_v1.3 carboxyl terminus caused slower inactivation in the Na_v1.2 channel. Through analysis of truncated channels, we identified a homologous 60-amino acid region within the carboxyl terminus of the Na_v1.2 and the Na_v1.3 channels that is responsible for this modulation of fast inactivation. Site-directed replacement of Na_v1.3 lysine 1826 in this region to its Na_v1.2 analogue glutamic acid 1880 (K1826E) shifted the voltage dependence of inactivation toward that of Na_v1.2. The K1826E mutation also accelerated the inactivation kinetics to a level comparable with that of Na_v1.2. The reverse Na_v1.2 E1880K mutation exhibited much slower inactivation kinetics and depolarized inactivation voltage dependence. A complementary mutation located within the inactivation linker of Na_v1.3 (K1453E) caused inactivation changes mirroring those caused by the K1826E mutation in Na_v1.3. Therefore, we have identified a homologous carboxyl-terminal residue that regulates the kinetics and voltage dependence of fast inactivation in sodium channels, possibly via a charge-dependent interaction with the inactivation linker.     

Action potentials in primary osteoblasts and in the MG-63 osteoblast-like cell line

Whole-cell patch-clamp analysis revealed a resting membrane potential of −60 mV in primary osteoblasts and in the MG-63 osteoblast-like cells. Depolarization-induced action potentials were characterized by duration of 60 ms, a minimal peak-to-peak distance of 180 ms, a threshold value of −20 mV and a repolarization between the spikes to −45 mV. Expressed channels were characterized by application of voltage pulses between −150 mV and 90 mV in 10 mV steps, from a holding potential of −40 mV. Voltages below −60 mV induced an inward current. Depolarizing voltages above −30 mV evoked two currents: (a) a fast activated and inactivated inward current at voltages between −30 and 30 mV, and (b) a delayed-activated outward current that was induced by voltages above −30 mV. Electrophysiological and pharmacological parameters indicated that hyperpolarization activated strongly rectifying K⁺ (K_ir) channels, whereas depolarization activated tetrodotoxin sensitive voltage gated Na⁺ (Na_v) channels as well as delayed, slowly activated, non-inactivating, and tetraethylammonium sensitive voltage gated K⁺ (K_v) channels. In addition, RT-PCR showed expression of Na_v1.3, Na_v1.4, Na_v1.5, Na_v1.6, Na_v1.7, and K_ir2.1, K_ir2.3, and K_ir2.4 as well as K_v2.1. We conclude that osteoblasts express channels that allow firing of action potentials.     

Molecular determinants of voltage-gated sodium channel regulation by the Nedd4/Nedd4-like proteins

The voltage-gated Na⁺ channels (Na_v) form a family composed of 10 genes. The COOH termini of Na_v contain a cluster of amino acids that are nearly identical among 7 of the 10 members. This COOH-terminal sequence, PPSYDSV, is a PY motif known to bind to WW domains of E3 protein-ubiquitin ligases of the Nedd4 family. We recently reported that cardiac Na_v1.5 is regulated by Nedd4-2. In this study, we further investigated the molecular determinants of regulation of Na_v proteins. When expressed in HEK-293 cells and studied using whole cell voltage clamping, the neuronal Na_v1.2 and Na_v1.3 were also downregulated by Nedd4-2. Pull-down experiments using fusion proteins bearing the PY motif of Na_v1.2, Na_v1.3, and Na_v1.5 indicated that mouse brain Nedd4-2 binds to the Na_v PY motif. Using intrinsic tryptophan fluorescence imaging of WW domains, we found that Na_v1.5 PY motif binds preferentially to the fourth WW domain of Nedd4-2 with a K_d of 55 µM. We tested the binding properties and the ability to ubiquitinate and downregulate Na_v1.5 of three Nedd4-like E3s: Nedd4-1, Nedd4-2, and WWP2. Despite the fact that along with Nedd4-2, Nedd4-1 and WWP2 bind to Na_v1.5 PY motif, only Nedd4-2 robustly ubiquitinated and downregulated Na_v1.5. Interestingly, coexpression of WWP2 competed with the effect of Nedd4-2. Finally, using brefeldin A, we found that Nedd4-2 accelerated internalization of Na_v1.5 stably expressed in HEK-293 cells. This study shows that Nedd4-dependent ubiquitination of Na_v channels may represent a general mechanism regulating the excitability of neurons and myocytes via modulation of channel density at the plasma membrane. ubiquitin; Nedd4-2; PY motif; Na_v1.5; human ether-à-go-go-related gene     

The β1-Subunit of Nav1.5 Cardiac Sodium Channel Is Required for a Dominant Negative Effect through α-α Interaction

Brugada syndrome (BrS) is an inherited autosomal dominant cardiac channelopathy. Several mutations on the cardiac sodium channel Na_v1.5 which are responsible for BrS lead to misfolded proteins that do not traffic properly to the plasma membrane. In order to mimic patient heterozygosity, a trafficking defective mutant, R1432G was co-expressed with Wild Type (WT) Na_v1.5 channels in HEK293T cells. This mutant significantly decreased the membrane Na current density when it was co-transfected with the WT channel. This dominant negative effect did not result in altered biophysical properties of Na_v1.5 channels. Luminometric experiments revealed that the expression of mutant proteins induced a significant reduction in membrane expression of WT channels. Interestingly, we have found that the auxiliary Na channel β₁-subunit was essential for this dominant negative effect. Indeed, the absence of the β₁-subunit prevented the decrease in WT sodium current density and surface proteins associated with the dominant negative effect. Co-immunoprecipitation experiments demonstrated a physical interaction between Na channel α-subunits. This interaction occurred only when the β₁-subunit was present. Our findings reveal a new role for β₁-subunits in cardiac voltage-gated sodium channels by promoting α-α subunit interaction which can lead to a dominant negative effect when one of the α-subunits shows a trafficking defective mutation.     

Ts17, a Tityus serrulatus β-toxin structurally related to α-scorpion toxins

Ts17 was purified from the venom of the scorpion Tityus serrulatus, the most dangerous scorpion species in Brazil. The activity on Na_v1.1-Na_v1.7 channels was electrophysiologically characterized by patch-clamp technique. Ts17 amino acid sequence indicated high similarity to alpha-scorpion toxins; however, it presented beta-toxin activity, altering the kinetics of the Na⁺-channels. The most affected subtypes during activation (with and without prepulse) and inactivation phases were Na_v1.2 and Na_v1.5, respectively. For recovery from inactivation, the most affected voltage-gated sodium channel was Na_v1.5. Circular dichroism spectra showed that Ts17 presents mainly β-sheet and unordered structures at all analyzed pHs, and the maximum value of α-helix was found at pH 4.0 (13.3 %). Based on the results, Ts17 might be used as a template to develop a new cardiac drug.Key contributionPurification of Ts17 from Tityus serrulatus, electrophysiological characterization of Ts17 on voltage-gated sodium channel subtypes, β-toxin classification.