Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines: A possible basis for modulation of excitability in vivo

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Sensitivity of cloned muscle,heart and neuronal voltage-gated sodium channels to block by polyamines

Spermidine and spermine, are endogenous polyamines (PAs) that regulate cell growth and modulate the activity of numerous ion channel proteins. In particular, intracellular PAs are potent blockers of many different cation channels and are responsible for strong suppression of outward K⁺ current, a phenomenon known as inward rectification characteristic of a major class of K_IR K⁺ channels. We previously described block of heterologously expressed voltage-gated Na⁺ channels (Na_V) of rat muscle by intracellular PAs and PAs have recently been found to modulate excitability of brain neocortical neurons by blocking neuronal Na_V channels. In this study, we compared the sensitivity of four different cloned mammalian Na_V isoforms to PAs to investigate whether PA block is a common feature of Na_V channel pharmacology. We find that outward Na⁺ current of muscle (Na_V1.4), heart (Na_V1.5), and neuronal (Na_V1.2, Na_V1.7) Na_V isoforms is blocked by PAs, suggesting that PA metabolism may be linked to modulation of action potential firing in numerous excitable tissues. Interestingly, the cardiac Na_V1.5 channel is more sensitive to PA block than other isoforms. Our results also indicate that rapid binding of PAs to blocking sites in the Na_V1.4 channel is restricted to access from the cytoplasmic side of the channel, but plasma membrane transport pathways for PA uptake may contribute to long-term Na_V channel modulation. PAs may also play a role in drug interactions since spermine attenuates the use-dependent effect of the lidocaine, a typical local anesthetic and anti-arrhythmic drug.     

Structural basis of cytoplasmic NaV1.5 and NaV1.4 regulation

Voltage-gated sodium channels (Na_Vs) are membrane proteins responsible for the rapid upstroke of the action potential in excitable cells. There are nine human voltage-sensitive Na_V1 isoforms that, in addition to their sequence differences, differ in tissue distribution and specific function. This review focuses on isoforms Na_V1.4 and Na_V1.5, which are primarily expressed in skeletal and cardiac muscle cells, respectively. The determination of the structures of several eukaryotic Na_Vs by single-particle cryo-electron microscopy (cryo-EM) has brought new perspective to the study of the channels. Alignment of the cryo-EM structure of the transmembrane channel pore with x-ray crystallographic structures of the cytoplasmic domains illustrates the complementary nature of the techniques and highlights the intricate cellular mechanisms that modulate these channels. Here, we review structural insights into the cytoplasmic C-terminal regulation of Na_V1.4 and Na_V1.5 with special attention to Ca²⁺ sensing by calmodulin, implications for disease, and putative channel dimerization.     

Development of high-affinity nanobodies specific for NaV1.4 and NaV1.5 voltage-gated sodium channel isoforms

Voltage-gated sodium channels, Na_Vs, are responsible for the rapid rise of action potentials in excitable tissues. Na_V channel mutations have been implicated in several human genetic diseases, such as hypokalemic periodic paralysis, myotonia, and long-QT and Brugada syndromes. Here, we generated high-affinity anti-Na_V nanobodies (Nbs), Nb17 and Nb82, that recognize the Na_V1.4 (skeletal muscle) and Na_V1.5 (cardiac muscle) channel isoforms. These Nbs were raised in llama (Lama glama) and selected from a phage display library for high affinity to the C-terminal (CT) region of Na_V1.4. The Nbs were expressed in Escherichia coli, purified, and biophysically characterized. Development of high-affinity Nbs specifically targeting a given human Na_V isoform has been challenging because they usually show undesired crossreactivity for different Na_V isoforms. Our results show, however, that Nb17 and Nb82 recognize the CTNa_V1.4 or CTNa_V1.5 over other CTNav isoforms. Kinetic experiments by biolayer interferometry determined that Nb17 and Nb82 bind to the CTNa_V1.4 and CTNa_V1.5 with high affinity (K_D ∼ 40–60 nM). In addition, as proof of concept, we show that Nb82 could detect Na_V1.4 and Na_V1.5 channels in mammalian cells and tissues by Western blot. Furthermore, human embryonic kidney cells expressing holo Na_V1.5 channels demonstrated a robust FRET-binding efficiency for Nb17 and Nb82. Our work lays the foundation for developing Nbs as anti-Na_V reagents to capture Na_Vs from cell lysates and as molecular visualization agents for Na_Vs.     

Current view on regulation of voltage‐gated sodium channels by calcium and auxiliary proteins

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage‐gated Na⁺ channels (Na_V channels) triggers action potentials, a process that is regulated via the interactions of the channels’ intercellular C‐termini with auxiliary proteins and/or Ca²⁺. The molecular and structural details for how Ca²⁺ and/or auxiliary proteins modulate Na_V channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca²⁺ acts directly upon the Na_V channel or through interacting proteins, such as the Ca²⁺ binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of Na_V intracellular C‐termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca²⁺ affects Na_V function, and how altered Ca²⁺‐dependent or FHF‐mediated regulation of Na_V channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.     

Proton Sensors in the Pore Domain of the Cardiac Voltage-gated Sodium Channel

Protons impart isoform-specific modulation of inactivation in neuronal, skeletal muscle, and cardiac voltage-gated sodium (Na_V) channels. Although the structural basis of proton block in Na_V channels has been well described, the amino acid residues responsible for the changes in Na_V kinetics during extracellular acidosis are as yet unknown. We expressed wild-type (WT) and two pore mutant constructs (H880Q and C373F) of the human cardiac Na_V channel, Na_V1.5, in Xenopus oocytes. C373F and H880Q both attenuated proton block, abolished proton modulation of use-dependent inactivation, and altered pH modulation of the steady-state and kinetic parameters of slow inactivation. Additionally, C373F significantly reduced the maximum probability of use-dependent inactivation and slow inactivation, relative to WT. H880Q also significantly reduced the maximum probability of slow inactivation and shifted the voltage dependence of activation and fast inactivation to more positive potentials, relative to WT. These data suggest that Cys-373 and His-880 in Na_V1.5 are proton sensors for use-dependent and slow inactivation and have implications in isoform-specific modulation of Na_V channels.     

Tuning voltage-gated channel activity and cellular excitability with a sphingomyelinase

Voltage-gated ion channels generate action potentials in excitable cells and help set the resting membrane potential in nonexcitable cells like lymphocytes. It has been difficult to investigate what kinds of phospholipids interact with these membrane proteins in their native environments and what functional impacts such interactions create. This problem might be circumvented if we could modify specific lipid types in situ. Using certain voltage-gated K⁺ (K_V) channels heterologously expressed in Xenopus laevis oocytes as a model, our group has shown previously that sphingomyelinase (SMase) D may serve this purpose. SMase D is known to remove the choline group from sphingomyelin, a phospholipid primarily present in the outer leaflet of plasma membranes. This SMase D action lowers the energy required for voltage sensors of a K_V channel to enter the activated state, causing a hyperpolarizing shift of the Q-V and G-V curves and thus activating them at more hyperpolarized potentials. Here, we find that this SMase D effect vanishes after removing most of the voltage-sensor paddle sequence, a finding supporting the notion that SMase D modification of sphingomyelin molecules alters these lipids’ interactions with voltage sensors. Then, using SMase D to probe lipid–channel interactions, we find that SMase D not only similarly stimulates voltage-gated Na⁺ (Na_V) and Ca²⁺ channels but also markedly slows Na_V channel inactivation. However, the latter effect is not observed in tested mammalian cells, an observation highlighting the profound impact of the membrane environment on channel function. Finally, we directly demonstrate that SMase D stimulates both native K_V1.3 in nonexcitable human T lymphocytes at their typical resting membrane potential and native Na_V channels in excitable cells, such that it shifts the action potential threshold in the hyperpolarized direction. These proof-of-concept studies illustrate that the voltage-gated channel activity in both excitable and nonexcitable cells can be tuned by enzymatically modifying lipid head groups.     

Multiscale modeling shows that dielectric differences make NaV channels faster than KV channels

The generation of action potentials in excitable cells requires different activation kinetics of voltage-gated Na (Na_V) and K (K_V) channels. Na_V channels activate much faster and allow the initial Na⁺ influx that generates the depolarizing phase and propagates the signal. Recent experimental results suggest that the molecular basis for this kinetic difference is an amino acid side chain located in the gating pore of the voltage sensor domain, which is a highly conserved isoleucine in K_V channels but an equally highly conserved threonine in Na_V channels. Mutagenesis suggests that the hydrophobicity of this side chain in Shaker K_V channels regulates the energetic barrier that gating charges cross as they move through the gating pore and control the rate of channel opening. We use a multiscale modeling approach to test this hypothesis. We use high-resolution molecular dynamics to study the effect of the mutation on polarization charge within the gating pore. We then incorporate these results in a lower-resolution model of voltage gating to predict the effect of the mutation on the movement of gating charges. The predictions of our hierarchical model are fully consistent with the tested hypothesis, thus suggesting that the faster activation kinetics of Na_V channels comes from a stronger dielectric polarization by threonine (Na_V channel) produced as the first gating charge enters the gating pore compared with isoleucine (K_V channel).     

Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels Na<Subscript>V</Subscript>1.1, Na<Subscript>V</Subscript>1.4 and Na<Subscript>V</Subscript>1.7

Human voltage-gated sodium (Na_V) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane β-subunits. Na_V pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all Na_V isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of Na_V1.2, Na_V1.5, and Na_V1.6 showed that CaM binding was mediated by the C-domain of CaM (CaM_C), while the N-domain (CaM_N) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other Na_V isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of Na_V1.1, Na_V1.4, and Na_V1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaM_C; resonances of CaM_N were identical to free CaM. Thus, CaM_C residues contribute to the interface with the IQ motif, while CaM_N is available to interact elsewhere on the channel.     

Electrophysiological characterization of Tityus obscurus β toxin 1 (To1) on Na+-channel isoforms

To1, previously named Tc49b, is a peptide neurotoxin isolated from venom of the scorpion Tityus obscurus that is responsible for lethal human poisoning cases in the Brazilian Amazonian region. Previously, To1 was shown to be lethal to mice and to change Na⁺ permeation in cerebellum granular neurons from rat brain. In addition, To1 did not affect Shaker B K⁺ channels. Based on sequence similarities, To1 was described as a β-toxin. In the present work, To1 was purified from T. obscurus venom and submitted to an electrophysiological characterization in human and invertebrate Na_V channels. The analysis of the electrophysiological experiments reveal that To1 enhances the open probability at more negative potentials of human Na_V 1.3 and 1.6, of the insect channel BgNa_V1 and of arachnid VdNa_V1 channel. In addition, To1 reduces the peak of Na⁺ currents in some of the Na_Vs tested. These results support the classification of the To1 as a β-toxin. A structure and functional comparison to other β-toxins that share sequence similarity to To1 is also presented.     

Models of the structure and gating mechanisms of the pore domain of the NaChBac ion channel

The NaChBac prokaryotic sodium channel appears to be a descendent of an evolutionary link between voltage-gated K_V and Ca_V channels. Like K_V channels, four identical six-transmembrane subunits comprise the NaChBac channel, but its selectivity filter possesses a signature sequence of eukaryotic Ca_V channels. We developed structural models of the NaChBac channel in closed and open conformations, using K⁺-channel crystal structures as initial templates. Our models were also consistent with numerous experimental results and modeling criteria. This study concerns the pore domain. The major differences between our models and K⁺ crystal structures involve the latter portion of the selectivity filter and the bend region in S6 of the open conformation. These NaChBac models may serve as a stepping stone between K⁺ channels of known structure and Na_V, Ca_V, and TRP channels of unknown structure.     

Protein arginine methyl transferases-3 and -5 increase cell surface expression of cardiac sodium channel

The α-subunit of the cardiac voltage-gated sodium channel (Na_V1.5) plays a central role in cardiomyocyte excitability. We have recently reported that Na_V1.5 is post-translationally modified by arginine methylation. Here, we aimed to identify the enzymes that methylate Na_V1.5, and to describe the role of arginine methylation on Na_V1.5 function. Our results show that protein arginine methyl transferase (PRMT)-3 and -5 methylate Na_V1.5 in vitro, interact with Na_V1.5 in human embryonic kidney (HEK) cells, and increase Na_V1.5 current density by enhancing Na_V1.5 cell surface expression. Our observations are the first evidence of regulation of a voltage-gated ion channel, including calcium, potassium, sodium and TRP channels, by arginine methylation.     

Discovery of novel sodium channel inhibitors—A gene family-based approach

Voltage-gated sodium (Na_V) channel inhibitors are an important class of drugs that are used to treat a number of CNS indications including pain, local anaesthesia, epilepsy and bipolar disorder. These drugs all have their origins in traditional “empirical” pharmacology, and it was only some time after discovery that they were found to inhibit Na_V channels. The basis for therapeutic selectivity of these drugs within different disease indications is currently unknown. However, the subsequent discovery of a multi-gene family of Na_V channels suggests a possible mechanism and has opened the way for more targeted approaches to finding improved therapeutic inhibitors. This article describes some ongoing approaches to systematically clone, express and characterise the entire family of Na_V subtypes in order to better understand their properties and define their individual physiological and pathophysiological roles. As well as providing specific disease validation for individual subtypes, this also provides a panel of reagents for comprehensively exploring the efficacy, selectivity and potency relationships of existing Na_V-blocking drugs. In this way, a gene family-based approach to Na_V channels has enabled a “drug-to-target” approach, reversing the more usual “gene-to-target-to-drug” paradigm. Together with recent advances in assay technology, gene family-based approaches are increasing the tractability of these targets and are re-invigorating Na_V drug discovery within the pharmaceutical industry.     

Solution NMR structure of Apo-calmodulin in complex with the IQ motif of human cardiac sodium channel NaV1.5

The function of the human voltage-gated sodium channel Na_V1.5 is regulated in part by intracellular calcium signals. The ubiquitous calcium sensor protein calmodulin (CaM) is an important part of the complex calcium-sensing apparatus in Na_V1.5. CaM interacts with an IQ (isoleucine-glutamine) motif in the large intracellular C-terminal domain of the channel. Using co-expression and co-purification, we have been able to isolate a CaM-IQ motif complex and to determine its high-resolution structure in absence of calcium using multi-dimensional solution NMR. Under these conditions, the Na_V1.5 IQ motif interacts with the C-terminal domain (C-lobe) of CaM, with the N-terminal domain remaining free in solution. The structure reveals that the C-lobe adopts a semi-open conformation with the IQ motif bound in a narrow hydrophobic groove. Sequence similarities between voltage-gated sodium channels and voltage-gated calcium channels suggest that the structure of the CaM-Na_V1.5 IQ motif complex can serve as a general model for the interaction between CaM and ion channel IQ motifs under low-calcium conditions. The structure also provides insight into the biochemical basis for disease-associated mutations that map to the IQ motif in Na_V1.5.     

Of cilia,titin, and neurosteroids

Missense mutations at arginine residues in the S4 voltage-sensor domains of Na_V1.4 are an established cause of hypokalemic periodic paralysis, an inherited disorder of skeletal muscle involving recurrent episodes of weakness in conjunction with low serum K⁺. Expression studies in oocytes have revealed anomalous, hyperpolarization-activated gating pore currents in mutant channels. This aberrant gating pore conductance creates a small inward current at the resting potential that is thought to contribute to susceptibility to depolarization in low K⁺ during attacks of weakness. A critical component of this hypothesis is the magnitude of the gating pore conductance relative to other conductances that are active at the resting potential in mammalian muscle: large enough to favor episodes of paradoxical depolarization in low K⁺, yet not so large as to permanently depolarize the fiber. To improve the estimate of the specific conductance for the gating pore in affected muscle, we sequentially measured Na⁺ current through the channel pore, gating pore current, and gating charge displacement in oocytes expressing R669H, R672G, or wild-type Na_V1.4 channels. The relative conductance of the gating pore to that of the pore domain pathway for Na⁺ was 0.03%, which implies a specific conductance in muscle from heterozygous patients of ∼10 µS/cm² or 1% of the total resting conductance.Unexpectedly, our data also revealed a substantial decoupling between gating charge displacement and peak Na⁺ current for both R669H and R672G mutant channels. This decoupling predicts a reduced Na⁺ current density in affected muscle, consistent with the observations that the maximal dV/dt and peak amplitude of the action potential are reduced in fibers from patients with R672G and in a knock-in mouse model of R669H. The defective coupling between gating charge displacement and channel activation identifies a previously unappreciated mechanism that contributes to the reduced excitability of affected fibers seen with these mutations and possibly with other R/X mutations of S4 of Na_V, Ca_V, and K_V channels associated with human disease.     

Sigma-1 Receptor Agonists Directly Inhibit NaV1.2/1.4 Channels

(+)-SKF 10047 (N-allyl-normetazocine) is a prototypic and specific sigma-1 receptor agonist that has been used extensively to study the function of sigma-1 receptors. (+)-SKF 10047 inhibits K⁺, Na⁺ and Ca2+ channels via sigma-1 receptor activation. We found that (+)-SKF 10047 inhibited Na_V1.2 and Na_V1.4 channels independently of sigma-1 receptor activation. (+)-SKF 10047 equally inhibited Na_V1.2/1.4 channel currents in HEK293T cells with abundant sigma-1 receptor expression and in COS-7 cells, which barely express sigma-1 receptors. The sigma-1 receptor antagonists BD 1063,BD 1047 and NE-100 did not block the inhibitory effects of (+)-SKF-10047. Blocking of the PKA, PKC and G-protein pathways did not affect (+)-SKF 10047 inhibition of Na_V1.2 channel currents. The sigma-1 receptor agonists Dextromethorphan (DM) and1,3-di-o-tolyl-guanidine (DTG) also inhibited Na_V1.2 currents through a sigma-1 receptor-independent pathway. The (+)-SKF 10047 inhibition of Na_V1.2 currents was use- and frequency-dependent. Point mutations demonstrated the importance of Phe¹⁷⁶⁴ and Tyr¹⁷⁷¹ in the IV-segment 6 domain of the Na_V1.2 channel and Phe¹⁵⁷⁹ in the Na_V1.4 channel for (+)-SKF 10047 inhibition. In conclusion, our results suggest that sigma-1 receptor agonists directly inhibit Na_V1.2/1.4 channels and that these interactions should be given special attention for future sigma-1 receptor function studies.     

Ancient Origin of Four-Domain Voltage-gated Na<Superscript>+</Superscript> Channels Predates the Divergence of Animals and Fungi

The four-domain voltage-gated Na⁺ channels are believed to have arisen in multicellular animals, possibly during the evolution of the nervous system. Recent genomic studies reveal that many ion channels, including Na⁺ channels and Ca²⁺ channels previously thought to be restricted to animals, can be traced back to one of the unicellular ancestors of animals, Monosiga brevicollis. The eukaryotic supergroup Opisthokonta contains animals, fungi, and a diverse group of their unicellular relatives including M. brevicollis. Here, we demonstrate the presence of a putative voltage-gated Na⁺ channel homolog (TtrNa_V) in the apusozoan protist Thecamonas trahens, which belongs to the unicellular sister group to Opisthokonta. TtrNa_V displays a unique selectivity motif distinct from most animal voltage-gated Na⁺ channels. The identification of TtrNa_V suggests that voltage-gated Na⁺ channels might have evolved before the divergence of animals and fungi. Furthermore, our analyses reveal that Na_V channels have been lost independently in the amoeboid holozoan Capsaspora owczarzaki of the animal lineage and in several basal fungi. These findings provide novel insights into the evolution of four-domain voltage-gated ion channels, ion selectivity, and membrane excitability in the Opisthokonta lineage.     

Mechanism of tetrodotoxin block and resistance in sodium channels

Tetrodotoxin (TTX) has been used for many decades to characterize the structure and function of biological ion channels. Yet, the precise mechanism by which TTX blocks voltage-gated sodium (Na_V) channels is not fully understood. Here molecular dynamics simulations are used to elucidate how TTX blocks mammalian voltage-gated sodium (Nav) channels and why it fails to be effective for the bacterial sodium channel, Na_VAb. We find that, in Na_VAb, a sodium ion competes with TTX for the binding site at the extracellular end of the filter, thus reducing the blocking efficacy of TTX. Using a model of the skeletal muscle channel, Na_V1.4, we show that the conduction properties of the channel observed experimentally are faithfully reproduced. We find that TTX occludes the entrance of Na_V1.4 by forming a network of hydrogen-bonds at the outer lumen of the selectivity filter. The guanidine group of TTX adopts a lateral orientation, rather than pointing into the filter as proposed previously. The acidic residues just above the selectivity filter are important in stabilizing the hydrogen-bond network between TTX and Na_V1.4. The effect of two single mutations of a critical tyrosine residue in the filter of Na_V1.4 on TTX binding observed experimentally is reproduced using computational mutagenesis.     

Targeted Expression of Channelrhodopsin-2 to the Axon Initial Segment Alters the Temporal Firing Properties of Retinal Ganglion Cells

The axon initial segment (AIS) is essential for initiating action potentials and maintaining neuronal excitability in axon-bearing neurons in the CNS. There is increasing interest in the targeting of optogenetic tools to subcellular compartments, including the AIS, to gain precise control of neuronal activity for basic research and clinical applications. In particular, targeted expression of optogenetic tools in retinal ganglion cells (RGCs) has been explored as an approach for restoring vision after photoreceptor degeneration. Thus, understanding the effects of such targeting on spiking abilities and/or patterns is important. Here, we examined the effects of recombinant adeno-associated virus (rAAV)-mediated targeted expression of channelrhodopsin-2 (ChR2)-GFP with a Na_V channel motif in mouse RGCs. We found that this targeted expression disrupted Na_V channel clustering at the AIS and converted the spike firing patterns of RGCs from sustained to transient. Our results suggest that the clustering of membrane channels, including Na_V channels, at the AIS is important for the ability of RGCs to generate sustained spike firing. Additionally, the targeting of optogenetic tools to the AIS with the Na_V channel motif may offer a way to create transient light responses in RGCs for vision restoration.