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     

Two Nedd4-binding Motifs Underlie Modulation of Sodium Channel Nav1.6 by p38 MAPK

Sodium channel Na_v1.6 is essential for neuronal excitability in central and peripheral nervous systems. Loss-of-function mutations in Na_v1.6 underlie motor disorders, with homozygous-null mutations causing juvenile lethality. Phosphorylation of Na_v1.6 by the stress-induced p38 MAPK at a Pro-Gly-Ser⁵⁵³-Pro motif in its intracellular loop L1 reduces Na_v1.6 current density in a dorsal root ganglion-derived cell line, without changing its gating properties. Phosphorylated Pro-Gly-Ser⁵⁵³-Pro motif is a putative binding site to Nedd4 ubiquitin ligases, and we hypothesized that Nedd4-like ubiquitin ligases may contribute to channel ubiquitination and internalization. We report here that p38 activation in hippocampal neurons from wild-type mice, but not from Scn8a^medtg mice that lack Na_v1.6, reduces tetrodotoxin-S sodium currents, suggesting isoform-specific modulation of Na_v1.6 by p38 in these neurons. Pharmacological block of endocytosis completely abolishes p38-mediated Na_v1.6 current reduction, supporting our hypothesis that channel internalization underlies current reduction. We also report that the ubiquitin ligase Nedd4-2 interacts with Na_v1.6 via a Pro-Ser-Tyr¹⁹⁴⁵ motif in the C terminus of the channel and reduces Na_v1.6 current density, and we show that this regulation requires both the Pro-Gly-Ser-Pro motif in L1 and the Pro-Ser-Tyr motif in the C terminus. Similarly, both motifs are necessary for p38-mediated reduction of Na_v1.6 current, whereas abrogating binding of the ubiquitin ligase Nedd4-2 to the Pro-Ser-Tyr motif results in stress-mediated increase in Na_v1.6 current density. Thus, phosphorylation of the Pro-Gly-Ser-Pro motif within L1 of Na_v1.6 is necessary for stress-induced current modulation, with positive or negative regulation depending upon the availability of the C-terminal Pro-Ser-Tyr motif to bind Nedd4-2.     

Regulation of the voltage-gated K(+) channels KCNQ2/3 and KCNQ3/5 by ubiquitination. Novel role for Nedd4-2

The muscarine-sensitive K(+) current (M-current) stabilizes the resting membrane potential in neurons, thus limiting neuronal excitability. The M-current is mediated by heteromeric channels consisting of KCNQ3 subunits in association with either KCNQ2 or KCNQ5 subunits. The role of KCNQ2/3/5 in the regulation of neuronal excitability is well established; however, little is known about the mechanisms that regulate the cell surface expression of these channels. Ubiquitination by the Nedd4/Nedd4-2 ubiquitin ligases is known to regulate a number of membrane ion channels and transporters. In this study, we investigated whether Nedd4/Nedd4-2 could regulate KCNQ2/3/5 channels. We found that the amplitude of the K(+) currents mediated by KCNQ2/3 and KCNQ3/5 were reduced by Nedd4-2 (but not Nedd4) in a Xenopus oocyte expression system. Deletion experiments showed that the C-terminal region of the KCNQ3 subunit is required for the Nedd4-2-mediated regulation of the heteromeric channels. Glutathione S-transferase fusion pulldowns and co-immunoprecipitations demonstrated a direct interaction between KCNQ2/3 and Nedd4-2. Furthermore, Nedd4-2 could ubiquitinate KCNQ2/3 in transfected cells. Taken together, these data suggest that Nedd4-2 is potentially an important regulator of M-current activity in the nervous system.     

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.     

Regulation of the Human Ether-a-go-go-related Gene (hERG) Channel by Rab4 Protein through Neural Precursor Cell-expressed Developmentally Down-regulated Protein 4-2 (Nedd4-2)

The human ether-a-go-go-related gene (hERG) encodes the pore-forming α-subunit of the rapidly activating delayed rectifier K⁺ channel in the heart, which plays a critical role in cardiac action potential repolarization. Dysfunction of I_Kr causes long QT syndrome, a cardiac electrical disorder that predisposes affected individuals to fatal arrhythmias and sudden death. The homeostasis of hERG channels in the plasma membrane depends on a balance between protein synthesis and degradation. Our recent data indicate that hERG channels undergo enhanced endocytic degradation under low potassium (hypokalemia) conditions. The GTPase Rab4 is known to mediate rapid recycling of various internalized proteins to the plasma membrane. In the present study, we investigated the effect of Rab4 on the expression level of hERG channels. Our data revealed that overexpression of Rab4 decreases the expression level of hERG in the plasma membrane. Rab4 does not affect the expression level of the Kv1.5 or EAG K⁺ channels. Mechanistically, our data demonstrate that overexpression of Rab4 increases the expression level of endogenous Nedd4-2, a ubiquitin ligase that targets hERG but not Kv1.5 or EAG channels for ubiquitination and degradation. Nedd4-2 undergoes self- ubiquitination and degradation. Rab4 interferes with Nedd4-2 degradation, resulting in an increased expression level of Nedd4-2, which targets hERG. In summary, the present study demonstrates a novel pathway for hERG regulation; Rab4 decreases the hERG density at the plasma membrane by increasing the endogenous Nedd4-2 expression.     

Regulation of neuronal voltage-gated sodium channels by the ubiquitin-protein ligases Nedd4 and Nedd4-2

Nedd4 and Nedd4-2 are ubiquitin-protein ligases known to regulate a number of membrane proteins including receptors and ion transporters. Regulation of the epithelial Na(+) channel by Nedd4 and Nedd4-2 is mediated via interactions between the PY motifs of the epithelial sodium channel subunits and the Nedd4/Nedd4-2 WW domains. This example serves as a model for the regulation of other PY motif-containing ion channels by Nedd4 and Nedd4-2. We found that the carboxyl termini of the six voltage-gated Na(+) (Na(v)) channels contain typical PY motifs (PPXY), and a further Na(v) contains a PY motif variant (LPXY). Not only did we demonstrate by Far-Western analysis that Nedd4 and Nedd4-2 interact with the PY motif-containing Na(v) channels, but we also showed that these channels have conserved WW domain binding specificity. We further showed that the carboxyl termini fusion proteins of one central nervous system and one peripheral nervous system-derived Na(+) channel (Na(v)1.2 and Na(v)1.7, respectively) are readily ubiquitinated by Nedd4-2. In Xenopus oocytes, Nedd4-2 strongly inhibited the activities of all three Na(v)s (Na(v)1.2, Na(v)1.7, and Na(v)1.8) tested. Interestingly, Nedd4 suppressed the activity of Na(v)1.2 and Na(v)1.7 but was a poor inhibitor of Na(v)1.8. Our results provide evidence that Nedd4 and Nedd4-2 are likely to be key regulators of specific neuronal Na(v) channels in vivo.     

Time-Dependent Molecular Memory in Single Voltage-Gated Sodium Channel

Excitability in neurons is associated with firing of action potentials and requires the opening of voltage-gated sodium channels with membrane depolarization. Sustained membrane depolarization, as seen in pathophysiological conditions like epilepsy, can have profound implications on the biophysical properties of voltage-gated ion channels. Therefore, we sought to characterize the effect of sustained membrane depolarization on single voltage-gated Na⁺ channels. Single-channel activity was recorded in the cell-attached patch-clamp mode from the rNa_v1.2α channels expressed in CHO cells. Classical statistical analysis revealed complex nonlinear changes in channel dwell times and unitary conductance of single Na⁺ channels as a function of conditioning membrane depolarization. Signal processing tools like weighted wavelet Z (WWZ) and discrete Fourier transform analyses attributed a “pseudo-oscillatory” nature to the observed nonlinear variation in the kinetic parameters. Modeling studies using the hidden Markov model (HMM) illustrated significant changes in kinetic states and underlying state transition rate constants upon conditioning depolarization. Our results suggest that sustained membrane depolarization induces novel nonlinear properties in voltage-gated Na⁺ channels. Prolonged membrane depolarization also induced a “molecular memory” phenomenon, characterized by clusters of dwell time events and strong autocorrelation in the dwell time series similar to that reported recently for single enzyme molecules. The persistence of such molecular memory was found to be dependent on the duration of depolarization. Voltage-gated Na⁺ channel with the observed time-dependent nonlinear properties and the molecular memory phenomenon may determine the functional state of the channel and, in turn, the excitability of a neuron.     

Using mutagenesis to study potassium channel mechanisms

The voltage-activated K⁺ channels are members of an ion channel family that includes the voltage-activated Na⁺ and Ca²⁺ channels. These ion channels mediate the transmembrane ionic currents that are responsible for the electrical signals produced by cells. The recent cloning of numerous voltage-activated K⁺ channels has made it possible to combine molecular-genetic and biophysical methods to study K⁺ channel mechanisms. These mutagenesis-function studies are beginning to provide new information about the architecture of K⁺ channel proteins and how they form a voltage-gated, K⁺-selective pore.     

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.     

Ubiquitination and Endocytosis of Plasma Membrane Proteins: Role of Nedd4/Rsp5p Family of Ubiquitin-Protein Ligases

In addition to its well-known role in recognition by the proteasome, ubiquitin-conjugation is also involved in downregulation of membrane receptors, transporters and channels. In most cases, ubiquitination of these plasma membrane proteins leads to their internalization followed by targeting to the lysosome/vacuole for degradation. A crucial role in ubiquitination of many plasma membrane proteins appears to be played by ubiquitin-protein ligases of the Nedd4/Rsp5p family. All family members carry an N-terminal Ca²⁺-dependent lipid/protein binding (C2) domain, two to four WW domains and a C-terminal catalytic Hect-domain. Nedd4 is involved in downregulation of the epithelial Na⁺ channel, by binding of its WW domains to specific PY motifs of the channel. Rsp5p, the unique family member in S. cerevisiae, is involved in ubiquitin-dependent endocytosis of a great number of yeast plasma membrane proteins. These proteins lack apparent PY motifs, but carry acidic sequences, and/or phosphorylated-based sequences that might be important, directly or indirectly, for their recognition by Rsp5p. In contrast to polyubiquitination leading to proteasomal recognition, a number of Rsp5p targets carry few ubiquitins per protein, and moreover with a different ubiquitin linkage. Accumulating evidence suggests that, at least in yeast, ubiquitin itself may constitute an internalization signal, recognized by a hypothetical receptor. Recent data also suggest that Nedd4/Rsp5p might play a role in the endocytic process possibly involving its C2 domain, in addition to its role in ubiquitinating endocytosed proteins. Recieved: 19 January 2000/Revised: 6 April 2000     

Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4‐2 using proteome arrays

Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2–4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4‐1) and Nedd4L (Nedd4‐2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4‐1 and Nedd4‐2, and rat‐Nedd4‐1, using protein microarrays spotted with ～8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4‐1 and Nedd4‐2, others were specific to only one, with several Tyr kinases preferred by Nedd4‐1 and some ion channels by Nedd4‐2; this was subsequently validated in vivo. Accordingly, Nedd4‐1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4‐1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases.     

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.     

Unique Regulation of Human Na+/H+ Exchanger 3 (NHE3) by Nedd4-2 Ligase That Differs from Non-primate NHE3s

Na⁺/H⁺ exchanger NHE3 expressed in the intestine and kidney plays a major role in NaCl and HCO₃⁻ absorption that is closely linked to fluid absorption and blood pressure regulation. The Nedd4 family of E3 ubiquitin ligases interacts with a number of transporters and channels via PY motifs. A comparison of NHE3 sequences revealed the presence of PY motifs in NHE3s from human and several non-human primates but not in non-primate NHE3s. In this study we evaluated the differences between human and non-primate NHE3s in ubiquitination and interaction with Nedd4-2. We found that Nedd4-2 ubiquitinated human NHE3 (hNHE3) and altered its expression and activity. Surprisingly, rat NHE3 co-immunoprecipitated Nedd4-2, but its expression and activity were not altered by silencing of Nedd4-2. Ubiquitination by Nedd4-2 rendered hNHE3 to undergo internalization at a significantly greater rate than non-primate NHE3s without altering protein stability. Insertion of a PY motif in rabbit NHE3 recapitulated the interaction with Nedd4-2 and enhanced internalization. Thus, we propose a new model where disruption of Nedd4-2 interaction elevates hNHE3 expression and activity.     

Photocontrol of Voltage-Gated Ion Channel Activity by Azobenzene Trimethylammonium Bromide in Neonatal Rat Cardiomyocytes

The ability of azobenzene trimethylammonium bromide (azoTAB) to sensitize cardiac tissue excitability to light was recently reported. The dark, thermally relaxed trans- isomer of azoTAB suppressed spontaneous activity and excitation propagation speed, whereas the cis- isomer had no detectable effect on the electrical properties of cardiomyocyte monolayers. As the membrane potential of cardiac cells is mainly controlled by activity of voltage-gated ion channels, this study examined whether the sensitization effect of azoTAB was exerted primarily via the modulation of voltage-gated ion channel activity. The effects of trans- and cis- isomers of azoTAB on voltage-dependent sodium (INav), calcium (ICav), and potassium (IKv) currents in isolated neonatal rat cardiomyocytes were investigated using the whole-cell patch-clamp technique. The experiments showed that azoTAB modulated ion currents, causing suppression of sodium (Na⁺) and calcium (Ca²⁺) currents and potentiation of net potassium (K⁺) currents. This finding confirms that azoTAB-effect on cardiac tissue excitability do indeed result from modulation of voltage-gated ion channels responsible for action potential.     

K+ Block Is the Mechanism of Functional Asymmetry in Bacterial Nav Channels

Crystal structures of several bacterial Na_v channels have been recently published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of eukaryotic channels. Bacterial Na_v channels have been characterized as functionally asymmetric, and the mechanism of this asymmetry has not been clearly understood. To address this question, we combined non-equilibrium simulation data with two-dimensional equilibrium unperturbed landscapes generated by umbrella sampling and Weighted Histogram Analysis Methods for multiple ions traversing the selectivity filter of bacterial Na_vAb channel. This approach provided new insight into the mechanism of selective ion permeation in bacterial Na_v channels. The non-equilibrium simulations indicate that two or three extracellular K⁺ ions can block the entrance to the selectivity filter of Na_vAb in the presence of applied forces in the inward direction, but not in the outward direction. The block state occurs in an unstable local minimum of the equilibrium unperturbed free-energy landscape of two K⁺ ions that can be ‘locked’ in place by modest applied forces. In contrast to K⁺, three Na⁺ ions move favorably through the selectivity filter together as a unit in a loose “knock-on” mechanism of permeation in both inward and outward directions, and there is no similar local minimum in the two-dimensional free-energy landscape of two Na⁺ ions for a block state. The useful work predicted by the non-equilibrium simulations that is required to break the K⁺ block is equivalent to large applied potentials experimentally measured for two bacterial Na_v channels to induce inward currents of K⁺ ions. These results illustrate how inclusion of non-equilibrium factors in the simulations can provide detailed information about mechanisms of ion selectivity that is missing from mechanisms derived from either crystal structures or equilibrium unperturbed free-energy landscapes.     

Analysis of the selectivity filter of the voltage-gated sodium channel NavRh

NaChBac is a bacterial voltage-gated sodium (Na_v) channel that shows sequence similarity to voltage-gated calcium channels. To understand the ion-permeation mechanism of Na_v channels, we combined molecular dynamics simulation, structural biology and electrophysiological approaches to investigate the recently determined structure of Na_vRh, a marine bacterial NaChBac ortholog. Two Na⁺ binding sites are identified in the selectivity filter (SF) in our simulations: The extracellular Na⁺ ion first approaches site 1 constituted by the side groups of Ser181 and Glu183, and then spontaneously arrives at the energetically more favorable site 2 formed by the carbonyl oxygens of Leu179 and Thr178. In contrast, Ca²⁺ ions are prone to being trapped by Glu183 at site 1, which then blocks the entrance of both Na⁺ and Ca²⁺ to the vestibule of the SF. In addition, Na⁺ permeates through the selective filter in an asymmetrical manner, a feature that resembles that of the mammalian Na_v orthologs. The study reported here provides insights into the mechanism of ion selectivity on Na⁺ over Ca²⁺ in mammalian Na_v channels.     

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.