Use of the parmbsc0 force field and trajectory analysis to study the binding of netropsin to the DNA fragment (5'CCAATTGG)(2) in the presence of excess NaCl salt in aqueous solution

The parmbsc0 force field was applied to study in detail the binding of netropsin, at a salt concentration of 0.28M Na(+), to the minor groove of an 8-mer (5'CCAATTGG)(2) DNA duplex forming a netropsin·DNA complex which previously has been characterized by X-ray crystallography, albeit with the use of closely related DNA duplexes. The X-ray structure revealed that the terminal guanidinium and amidinium groups of netropsin interact with the extreme ends of the palindromic AATT sequence of the receptor DNA. The parmbsc0 parameters of B-DNA and AMBER v9 parameters of netropsin generated a stable 6ns molecular dynamics (MD) trajectory for a 1:1 class I binding motif of this complex. Trajectory analysis for the salt and hydration effects on the binding of netropsin to the 8-mer DNA duplex revealed that 18 water molecules and 2 Na(+) are displaced from the DNA upon netropsin binding. A hydration density map of the complex parallels the X-ray data showing that two structured water molecules are localized near the netropsin guanidinium and amidinium groups forming H-bond bridges between the receptor and the ligand.     

Novel isoforms of the sodium channels Nav1.8 and Nav1.5 are produced by a conserved mechanism in mouse and rat

The voltage-gated sodium channel Na(v)1.8 is only expressed in subsets of neurons in dorsal root ganglia (DRG) and trigeminal and nodose ganglia. We have isolated mouse partial length Na(v)1.8 cDNA clones spanning the exon 17 sequence, which have 17 nucleotide substitutions and 12 predicted amino acid differences from the published sequence. The absence of a mutually exclusive alternative exon 17 was confirmed by sequencing 4.1 kilobases of genomic DNA spanning exons 16-18 of Scn10a. A novel cDNA isoform was identified, designated Na(v)1.8c, which results from alternative 3'-splice site selection at a CAG/CAG motif to exclude the codon for glutamine 1031 within the interdomain cytoplasmic loop IDII/III. The ratio of Na(v)1.8c (CAG-skipped) to Na(v)1.8 (CAG-inclusive) mRNA in mouse is approximately 2:1 in adult DRG, trigeminal ganglion, and neonatal DRG. A Na(v)1.8c isoform also occurs in rat DRG, but is less common. Of the two other tetrodotoxin-resistant channels, no analogous alternative splicing of mouse Na(v)1.9 was detected, whereas rare alternative splicing of Na(v)1.5 at a CAG/CAG motif resulted in the introduction of a CAG trinucleotide. This isoform, designated Na(v)1.5c, is conserved in rat and encodes an additional glutamine residue that disrupts a putative CK2 phosphorylation site. In summary, novel isoforms of Na(v)1.8 and Na(v)1.5 are each generated by alternative splicing at CAG/CAG motifs, which result in the absence or presence of predicted glutamine residues within the interdomain cytoplasmic loop IDII/III. Mutations of sodium channels within this cytoplasmic loop have previously been demonstrated to alter electrophysiological properties and cause cardiac arrhythmias and epilepsy.     

Direct interaction with contactin targets voltage-gated sodium channel Na(v)1.9/NaN to the cell membrane

The mechanisms that target various sodium channels within different regions of the neuronal membrane, which they endow with different physiological properties, are not yet understood. To examine this issue we studied the voltage-gated sodium channel Na(v)1.9/NaN, which is preferentially expressed in small sensory neurons of dorsal root ganglia and trigeminal ganglia and the nonmyelinated axons that arise from them. Our results show that the cell adhesion molecule contactin binds directly to Na(v)1.9/NaN and recruits tenascin to the protein complex in vitro. Na(v)1.9/NaN and contactin co-immunoprecipitate from dorsal root ganglia and transfected Chinese hamster ovary cell line, and co-localize in the C-type neuron soma and along nonmyelinated C-fibers and at nerve endings in the skin. Co-transfection of Chinese hamster ovary cells with Na(v)1.9/NaN and contactin enhances the surface expression of the sodium channel over that of Na(v)1.9/NaN alone. Thus contactin binds directly to Na(v)1.9/NaN and participates in the surface localization of this channel along nonmyelinated axons.     

Are voltage-dependent ion channels involved in the endothelial cell control of vasomotor tone?

In the microcirculation, longitudinal conduction of vasomotor responses provides an essential means of coordinating flow distribution among vessels in a complex network. Spread of current along the vessel axis can display a regenerative component, which leads to propagation of vasomotor signals over many millimeters; the ionic basis for the regenerative response is unknown. We examined the responses to 10 s of focal electrical stimulation (30 Hz, 2 ms, 30 V) of mouse cremaster arterioles to test the hypothesis that voltage-dependent Na(+) (Na(v)) and Ca(2+) channels might be activated in long-distance signaling in microvessels. Electrical stimulation evoked a vasoconstriction at the site of stimulation and a spreading, nondecremental conducted dilation. Endothelial damage (air bubble) blocked conduction of the vasodilation, indicating an involvement of the endothelium. The Na(v) channel blocker bupivacaine also blocked conduction, and TTX attenuated it. The Na(v) channel activator veratridine induced an endothelium-dependent dilation. The Na(v) channel isoforms Na(v)1.2, Na(v)1.6, and Na(v)1.9 were detected in the endothelial cells of cremaster arterioles by immunocytochemistry. These findings are consistent with the involvement of Na(v) channels in the conducted response. BAPTA buffering of endothelial cell Ca(2+) delayed and reduced the conducted dilation, which was almost eliminated by Ni(2+), amiloride, or deletion of alpha(1H) T-type Ca(2+) (Ca(v)3.2) channels. Blockade of endothelial nitric oxide synthase or Ca(2+)-activated K(+) channels also inhibited the conducted vasodilation. Our findings indicate that an electrically induced signal can propagate along the vessel axis via the endothelium and can induce sequential activation of Na(v) and Ca(v)3.2 channels. The resultant Ca(2+) influx activates endothelial nitric oxide synthase and Ca(2+)-activated K(+) channels, triggering vasodilation.     

Nav1.4 deregulation in dystrophic skeletal muscle leads to Na+ overload and enhanced cell death

Duchenne muscular dystrophy (DMD) is a hereditary degenerative disease manifested by the absence of dystrophin, a structural, cytoskeletal protein, leading to muscle degeneration and early death through respiratory and cardiac muscle failure. Whereas the rise of cytosolic Ca(2+) concentrations in muscles of mdx mouse, an animal model of DMD, has been extensively documented, little is known about the mechanisms causing alterations in Na(+) concentrations. Here we show that the skeletal muscle isoform of the voltage-gated sodium channel, Na(v)1.4, which represents over 90% of voltage-gated sodium channels in muscle, plays an important role in development of abnormally high Na(+) concentrations found in muscle from mdx mice. The absence of dystrophin modifies the expression level and gating properties of Na(v)1.4, leading to an increased Na(+) concentration under the sarcolemma. Moreover, the distribution of Na(v)1.4 is altered in mdx muscle while maintaining the colocalization with one of the dystrophin-associated proteins, syntrophin alpha-1, thus suggesting that syntrophin is an important linker between dystrophin and Na(v)1.4. Additionally, we show that these modifications of Na(v)1.4 gating properties and increased Na(+) concentrations are strongly correlated with increased cell death in mdx fibers and that both cell death and Na(+) overload can be reversed by 3 nM tetrodotoxin, a specific Na(v)1.4 blocker.     

Importance of coulombic end effects on cation accumulation near oligoelectrolyte B-DNA: a demonstration using 23Na NMR.

The local cation concentration at the surface of oligomeric or polymeric B-DNA is expected, on the basis of MC simulations (Olmsted, M. C., C. F. Anderson, and M. T. Record, Jr. 1989. Proc. Natl. Acad. Sci. USA. 86:7766-7770), to decrease sharply as either end of the molecule is approached. In this paper we report 23Na NMR measurements indicating the importance of this "coulombic" end effect on the average extent of association of Na+ with oligomeric duplex DNA. In solutions containing either 20-bp synthetic DNA or 160-bp mononucleosomal calf thymus DNA at phosphate monomer concentrations [P] of 4-10 mM, measurements were made over the range of ratios 1 < or = [Na]/[LP] < or = 20, corresponding to Na+ concentrations of 4-200 nM. The longitudinal 23Na NMR relaxation rates measured in these NaDNA solutions, Robs, are interpreted as population-weighted averages of contributions from "bound" (RB) and "free" (RF) 23Na relaxation rates. The observed enhancements of Robs indicate that RB significantly exceeds RF, which is approximately equal to the 23Na relaxation rate in an aqueous solution containing only NaCl. Under salt-fre-tconditions ([Na]/[P] = 1), where the enhancement in Robs is maximal, we find that Robs--RF in the solution containing 160-bp DNA is approximately 1.8 times that observed for the 20-bp DNA. For the 160-bp oligomer (which theoretical calculations predict to be effectively polyion-like), we find that a plot of Robs v. [P]/[Na] is linear, as observed previously for sonicated (approximately 700 bp) DNA samples. For the 20-bp oligonucleotide this plot exhibits a marked departure from linearity that can be fitted to a quadratic function of [P]/[Na]. Monte Carlo simulations based on a simplified model are capable of reproducing the qualitative trends in the 23Na NMR measurements analyzed here. In particular, the dependences of Robs--RF on DNA charge magnitude of Z(320 vs. 38 phosphates) and (for the 20-bp oligomer) on [Na]/[P] are well correlated with the calculated average surface concentration of Na+. Thus, effects of sodium concentration on RB appear to be of secondary importance. We conclude that 23Na NMR relaxation measurements are a sensitive probe of the effects of oligomer charge on the extent of ion accumulation near B-DNA oligonucleotides, as a function of [Na] and [P].     

A pore-blocking hydrophobic motif at the cytoplasmic aperture of the closed-state Nav1.7 channel is disrupted by the erythromelalgia-associated F1449V mutation

Sodium channel Na(v)1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Na(v)1.7 produce painful disorders, whereas loss-of-function Na(v)1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Na(v)1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Na(v)1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Na(v)1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Na(v)1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Na(v)1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Na(v)1.7.     

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.     

The cardiac sodium channel is post-translationally modified by arginine methylation

The α subunit of the cardiac sodium channel (Na(v)1.5) is an essential protein in the initial depolarization phase of the cardiomyocyte action potential. Post-translational modifications such as phosphorylation are known to regulate Na(v)1.5 function. Here, we used a proteomic approach for the study of the post-translational modifications of Na(v)1.5 using tsA201 cells as a model system. We generated a stable cell line expressing Na(v)1.5, purified the sodium channel, and analyzed Na(v)1.5 by MALDI-TOF and LC-MS/MS. We report the identification of arginine methylation as a novel post-translational modification of Na(v)1.5. R513, R526, and R680, located in the linker between domains I and II in Na(v)1.5, were found in mono- or dimethylated states. The functional relevance of arginine methylation in Na(v)1.5 is underscored by the fact that R526H and R680H are known Na(v)1.5 mutations causing Brugada and long QT type 3 syndromes, respectively. Our work describes for the first time arginine methylation in the voltage-gated ion channel superfamily.     

Structural and energetic determinants of apo calmodulin binding to the IQ motif of the Na(V)1.2 voltage-dependent sodium channel

The neuronal voltage-dependent sodium channel (Na(v)1.2), essential for generation and propagation of action potentials, is regulated by calmodulin (CaM) binding to the IQ motif in its α subunit. A peptide (Na(v)1.2(IQp), KRKQEEVSAIVIQRAYRRYLLKQKVKK) representing the IQ motif had higher affinity for apo CaM than (Ca(2+))(4)-CaM. Association was mediated solely by the C-domain of CaM. A solution structure (2KXW.pdb) of apo (13)C,(15)N-CaM C-domain bound to Na(v)1.2(IQp) was determined with NMR. The region of Na(v)1.2(IQp) bound to CaM was helical; R1902, an Na(v)1.2 residue implicated in familial autism, did not contact CaM. The apo C-domain of CaM in this complex shares features of the same domain bound to myosin V IQ motifs (2IX7) and bound to an SK channel peptide (1G4Y) that does not contain an IQ motif. Thermodynamic and structural studies of CaM-Na(v)1.2(IQp) interactions show that apo and (Ca(2+))(4)-CaM adopt distinct conformations that both permit tight association with Na(v)1.2(IQp) during gating.     

The unique pharmacology of the scorpion alpha-like toxin Lqh3 is associated with its flexible C-tail

The affinity of scorpion alpha-toxins for various voltage-gated sodium channels (Na(v)s) differs considerably despite similar structures and activities. It has been proposed that key bioactive residues of the five-residue-turn (residues 8-12) and the C-tail form the NC domain, whose topology is dictated by a cis or trans peptide-bond conformation between residues 9 and 10, which correlates with the potency on insect or mammalian Na(v)s. We examined this hypothesis using Lqh3, an alpha-like toxin from Leiurus quinquestriatus hebraeus that is highly active in insects and mammalian brain. Lqh3 exhibits slower association kinetics to Na(v)s compared with other alpha-toxins and its binding to insect Na(v)s is pH-dependent. Mutagenesis of Lqh3 revealed a bi-partite bioactive surface, composed of the Core and NC domains, as found in other alpha-toxins. Yet, substitutions at the five-residue turn and stabilization of the 9-10 bond in the cis conformation did not affect the activity. However, substitution of hydrogen-bond donors/acceptors at the NC domain reduced the pH-dependency of toxin binding, while retaining its high potency at Drosophila Na(v)s expressed in Xenopus oocytes. Based on these results and the conformational flexibility and rearrangement of intramolecular hydrogen-bonds at the NC domain, evident from the known solution structure, we suggest that acidic pH or specific mutations at the NC domain favor toxin conformations with high affinity for the receptor by stabilizing the bound toxin-receptor complex. Moreover, the C-tail flexibility may account for the slower association rates and suggests a novel mechanism of dynamic conformer selection during toxin binding, enabling alpha-like toxins to affect a broad range of Na(v)s.     

Compact myelin dictates the differential targeting of two sodium channel isoforms in the same axon

Voltage-dependent sodium channels are uniformly distributed along unmyelinated axons, but are highly concentrated at nodes of Ranvier in myelinated axons. Here, we show that this pattern is associated with differential localization of distinct sodium channel alpha subunits to the unmyelinated and myelinated zones of the same retinal ganglion cell axons. In adult axons, Na(v)1.2 is localized to the unmyelinated zone, whereas Na(v)1.6 is specifically targeted to nodes. During development, Na(v)1.2 is expressed first and becomes clustered at immature nodes of Ranvier, but as myelination proceeds, Na(v)1.6 replaces Na(v)1.2 at nodes. In Shiverer mice, which lack compact myelin, Na(v)1.2 is found throughout adult axons, whereas little Na(v)1.6 is detected. Together, these data show that sodium channel isoforms are differentially targeted to distinct domains of the same axon in a process associated with formation of compact myelin.     

Solution NMR analysis of the binding mechanism of DIVS6 model peptides of voltage-gated sodium channels and the lipid soluble alkaloid veratridine

Voltage-gated sodium channels (VGSCs) are responsible for generating action potentials in nervous systems. Veratridine (VTD), a lipid soluble alkaloid isolated from sabadilla lily seed, is believed to bind to segment 6 of VGSCs and act as a partial agonist. However, high resolution structural interaction mechanism between VGSCs and VTD is difficult to elucidate because of the large size and membrane localization of VGSCs. Here, the authors designed model peptides corresponding to domain IV segment 6 (DIVS6) of rat skeletal muscle Na(v)1.4 and analyzed the complex of the model peptides and VTD by solution NMR analysis to obtain structural information of the interaction. The model peptides successfully formed an α-helices, which is the suspected native conformation of DIVS6, in aqueous 2,2,2-trifluoroethanol, a membrane-mimicking solvent. The VTD binding residues of the model peptide were identified using the NMR titration experiments with VTD, including a newly discovered VTD binding residue Leu14 (μ1-L1580 in Na(v)1.4), which has not been reported by point mutation studies. Mapping of VTD binding residues on the model peptide revealed the hydrophobic interaction surface. NMR titration experiments with a non-toxic analog of VTD, veracevine, also indicated that the steroidal backbone of VTD interacts with the hydrophobic interaction surface of DIVS6 and that the 3-acyl group of VTD possibly causes neurotoxicity by interacting with domain I segment 6 and/or domain IV segment 4.     

Modulation of Nav1.5 channel function by an alternatively spliced sequence in the DII/DIII linker region

In the present study, we identified a novel splice variant of the human cardiac Na(+) channel Na(v)1.5 (Na(v)1.5d), in which a 40-amino acid sequence of the DII/DIII intracellular linker is missing due to a partial deletion of exon 17. Expression of Na(v)1.5d occurred in embryonic and adult hearts of either sex, indicating that the respective alternative splicing is neither age-dependent nor gender-specific. In contrast, Na(v)1.5d was not detected in the mouse heart, indicating that alternative splicing of Na(v)1.5 is species-dependent. In HEK293 cells, splice variant Na(v)1.5d generated voltage-dependent Na(+) currents that were markedly reduced compared with wild-type Na(v)1.5. Experiments with mexiletine and 8-bromo-cyclic AMP suggested that the trafficking of Na(v)1.5d channels was not impaired. However, single-channel recordings showed that the whole-cell current reduction was largely due to a significantly reduced open probability. Additionally, steady-state activation and inactivation were shifted to depolarized potentials by 15.9 and 5.1 mV, respectively. Systematic mutagenesis analysis of the spliced region provided evidence that a short amphiphilic region in the DII/DIII linker resembling an S4 voltage sensor of voltage-gated ion channels is an important determinant of Na(v)1.5 channel gating. Moreover, the present study identified novel short sequence motifs within this amphiphilic region that specifically affect the voltage dependence of steady-state activation and inactivation and current amplitude of human Na(v)1.5.     

Discovery of potent furan piperazine sodium channel blockers for treatment of neuropathic pain

The synthesis and pharmacological characterization of a novel furan-based class of voltage-gated sodium channel blockers is reported. Compounds were evaluated for their ability to block the tetrodotoxin-resistant sodium channel Na(v)1.8 (PN3) as well as the Na(v)1.2 and Na(v)1.5 subtypes. Benchmark compounds from this series possessed enhanced potency, oral bioavailability, and robust efficacy in a rodent model of neuropathic pain, together with improved CNS and cardiovascular safety profiles compared to the clinically used sodium channel blockers mexiletine and lamotrigine.     

Molecular basis of isoform-specific micro-conotoxin block of cardiac,skeletal muscle,and brain Na+ channels

mu-Conotoxins (mu-CTXs) block skeletal muscle Na(+) channels with an affinity 1-2 orders of magnitude higher than cardiac and brain Na(+) channels. Although a number of conserved pore residues are recognized as critical determinants of mu-CTX block, the molecular basis of isoform-specific toxin sensitivity remains unresolved. Sequence comparison of the domain II (DII) S5-S6 loops of rat skeletal muscle (mu1, Na(v)1.4), human heart (hh1, Na(v)1.5), and rat brain (rb1, Na(v)1.1) Na(+) channels reveals substantial divergence in their N-terminal S5-P linkers even though the P-S6 and C-terminal P segments are almost identical. We used Na(v)1.4 as the backbone and systematically converted these DII S5-P isoform variants to the corresponding residues in Na(v)1.1 and Na(v)1.5. The Na(v)1.4-->Na(v)1.5 variant substitutions V724R, C725S, A728S, D730S, and C731S (Na(v)1.4 numbering) reduced block of Na(v)1.4 by 4-, 86-, 12-, 185-, and 55-fold respectively, rendering the skeletal muscle isoform more "cardiac-like." Conversely, an Na(v)1.5--> Na(v)1.4 chimeric construct in which the Na(v)1.4 DII S5-P linker replaces the analogous segment in Na(v)1.5 showed enhanced mu-CTX block. However, these variant determinants are conserved between Na(v)1.1 and Na(v)1.4 and thus cannot explain their different sensitivities to mu-CTX. Comparison of their sequences reveals two variants at Na(v)1.4 positions 729 and 732: Ser and Asn in Na(v)1.4 compared with Thr and Lys in Na(v)1.1, respectively. The double mutation S729T/N732K rendered Na(v)1.4 more "brain-like" (30-fold downward arrow in block), and the converse mutation T925S/K928N in Na(v)1.1 reproduced the high affinity blocking phenotype of Na(v)1.4. We conclude that the DII S5-P linker, although lying outside the conventional ion-conducting pore, plays a prominent role in mu-CTX binding, thus shaping isoform-specific toxin sensitivity.     

Colocalization of voltage-gated Na+ channels with the Na+/Ca2+ exchanger in rabbit cardiomyocytes during development

Reverse-mode activity of the Na(+)/Ca(2+) exchanger (NCX) has been previously shown to play a prominent role in excitation-contraction coupling in the neonatal rabbit heart, where we have proposed that a restricted subsarcolemmal domain allows a Na(+) current to cause an elevation in the Na(+) concentration sufficiently large to bring Ca(2+) into the myocyte through reverse-mode NCX. In the present study, we tested the hypothesis that there is an overlapping expression and distribution of voltage-gated Na(+) (Na(v)) channel isoforms and the NCX in the neonatal heart. For this purpose, Western blot analysis, immunocytochemistry, confocal microscopy, and image analyses were used. Here, we report the robust expression of skeletal Na(v)1.4 and cardiac Na(v)1.5 in neonatal myocytes. Both isoforms colocalized with the NCX, and Na(v)1.5-NCX colocalization was not statistically different from Na(v)1.4-NCX colocalization in the neonatal group. Western blot analysis also showed that Na(v)1.4 expression decreased by sixfold in the adult (P < 0.01) and Na(v)1.1 expression decreased by ninefold (P < 0.01), whereas Na(v)1.5 expression did not change. Although Na(v)1.4 underwent large changes in expression levels, the Na(v)1.4-NCX colocalization relationship did not change with age. In contrast, Na(v)1.5-NCX colocalization decreased ～50% with development. Distance analysis indicated that the decrease in Na(v)1.5-NCX colocalization occurs due to a statistically significant increase in separation distances between Na(v)1.5 and NCX objects. Taken together, the robust expression of both Na(v)1.4 and Na(v)1.5 isoforms and their colocalization with the NCX in the neonatal heart provides structural support for Na(+) current-induced Ca(2+) entry through reverse-mode NCX. In contrast, this mechanism is likely less efficient in the adult heart because the expression of Na(v)1.4 and NCX is lower and the separation distance between Na(v)1.5 and NCX is larger.     

BACE1 regulates voltage-gated sodium channels and neuronal activity

BACE1 activity is significantly increased in the brains of Alzheimer's disease patients, potentially contributing to neurodegeneration. The voltage-gated sodium channel (Na(v)1) beta2-subunit (beta2), a type I membrane protein that covalently binds to Na(v)1 alpha-subunits, is a substrate for BACE1 and gamma-secretase. Here, we find that BACE1-gamma-secretase cleavages release the intracellular domain of beta2, which increases mRNA and protein levels of the pore-forming Na(v)1.1 alpha-subunit in neuroblastoma cells. Similarly, endogenous beta2 processing and Na(v)1.1 protein levels are elevated in brains of BACE1-transgenic mice and Alzheimer's disease patients with high BACE1 levels. However, Na(v)1.1 is retained inside the cells and cell surface expression of the Na(v)1 alpha-subunits and sodium current densities are markedly reduced in both neuroblastoma cells and adult hippocampal neurons from BACE1-transgenic mice. BACE1, by cleaving beta2, thus regulates Na(v)1 alpha-subunit levels and controls cell-surface sodium current densities. BACE1 inhibitors may normalize membrane excitability in Alzheimer's disease patients with elevated BACE1 activity.