Expression and distribution of voltage-gated sodium channels in the cerebellum

In order to understand the effects of sodium channels on synaptic signaling and response in the cerebellum, it is essential to know for each class of neuron what sodium channel isoforms are present, and the properties and distribution of each. Sodium channels are heteromultimeric membrane proteins, consisting of a large alpha subunit that forms the pore, and one or more beta subunits. Ten genes encode an alpha subunit in mammals, and of these, four are expressed in the cerebellum: Nav1.1, Nav1.2, Nav1.3 and Nav1.6. Three genes encode beta subunits (Nabeta1-3), and all three are expressed in the cerebellum. However, Nav1.3 and Nabeta3 have been found only in the developing cerebellum. All sodium channels recorded in the cerebellum are TTX-sensitive with similar kinetics, making it difficult to identify the isoforms electrically. Thus, most of the expression studies have relied on techniques that allow visualization of sodium channel subtypes at the level of mRNA and protein. In situ hybridization and immunolocalization studies demonstrated that granule cells predominantly express Nav1.2, Nav1.6, Nabeta1, and Nabeta2. Protein for Nav1.2 and Nav1.6 is localized primarily in granule cell parallel fibers. Purkinje cells express Nav1.1, Nav1.6, Nabeta1 and Nabeta2. The somato-dendritic localization of Nav1.1 and Nav1.6 in Purkinje cells suggests that these isoforms are involved in the integration of synaptic input. Deep cerebellar nuclei neurons expressed Nav1.1 and Nav1.6 as well as Nabeta1. Bergmann glia expressed Nav1.6, but not granule cell layer astrocytes. Some sodium channel isoforms that are not expressed normally in the adult cerebellum are expressed in animals with mutations or disease. Electrophysiological studies suggest that Nav1.6 is responsible for spontaneous firing and bursting features in Purkinje cells, but the specialized functions of the other subunits in the cerebellum remain unknown.     

Distribution of high-voltage-activated calcium channels in cultured gamma-aminobutyric acidergic neurons from mouse cerebral cortex.

The localization of voltage-gated calcium channel (VGCC) alpha(1) subunits in cultured GABAergic mouse cortical neurons was examined by immunocytochemical methods. Ca(v)1.2 and Ca(v)1.3 subunits of L-type VGCCs were found in cell bodies and dendrites of GABA-immunopositive neurons. Likewise, the Ca(v)2.3 subunit of R-type VGCCs was expressed in a somatodendritic pattern. Ca(v)2.2 subunits of N-type channels were found exclusively in small varicosities that were identified as presynaptic nerve terminals based on their expression of synaptic marker proteins. Two splice variants of the Ca(v)2.1 subunit of P/Q-type VGCCs showed widely differing expression patterns. The rbA isoform displayed a purely somatodendritic staining pattern, whereas the BI isoform was confined to axon-like fibers and nerve terminals. The nerve terminals of these cultured GABAergic neurons express Ca(v)2.2 either alone or in combination with Ca(v)2.1 (BI isoform) but never express Ca(v)2.1 alone. The functional association between VGCCs and the neurotransmitter release machinery was probed using the FM1-43 dye-labeling technique. N-type VGCCs were found to be tightly coupled to exocytosis in these cultured cortical neurons, and P-type VGCCs were also important in a fraction of the cells. The predominant role of N-type VGCCs in neurotransmitter release and the specific localization of the BI isoform of Ca(v)2.1 in the nerve terminals of these neurons distinguish them from previously studied central neurons. The complementary localization patterns observed for two different isoforms of the Ca(v)2.1 subunits provide direct evidence for alternative splicing as a means of generating functional diversity among neuronal calcium channels.     

Voltage-gated Na+ channel β1B: a secreted cell adhesion molecule involved in human epilepsy

Scn1b-null mice have a severe neurological and cardiac phenotype. Human mutations in SCN1B result in epilepsy and cardiac arrhythmia. SCN1B is expressed as two developmentally regulated splice variants, β1 and β1B, that are each expressed in brain and heart in rodents and humans. Here, we studied the structure and function of β1B and investigated a novel human SCN1B epilepsy-related mutation (p.G257R) unique to β1B. We show that wild-type β1B is not a transmembrane protein, but a soluble protein expressed predominantly during embryonic development that promotes neurite outgrowth. Association of β1B with voltage-gated Na+ channels Na(v)1.1 or Na(v)1.3 is not detectable by immunoprecipitation and β1B does not affect Na(v)1.3 cell surface expression as measured by [(3)H]saxitoxin binding. However, β1B coexpression results in subtle alteration of Na(v)1.3 currents in transfected cells, suggesting that β1B may modulate Na+ current in brain. Similar to the previously characterized p.R125C mutation, p.G257R results in intracellular retention of β1B, generating a functional null allele. In contrast, two other SCN1B mutations associated with epilepsy, p.C121W and p.R85H, are expressed at the cell surface. We propose that β1B p.G257R may contribute to epilepsy through a mechanism that includes intracellular retention resulting in aberrant neuronal pathfinding.     

Contactin regulates the current density and axonal expression of tetrodotoxin-resistant but not tetrodotoxin-sensitive sodium channels in DRG neurons

Contactin, a glycosyl-phosphatidylinositol (GPI)-anchored predominantly neuronal cell surface glycoprotein, associates with sodium channels Nav1.2, Nav1.3 and Nav1.9, and enhances the density of these channels on the plasma membrane in mammalian expression systems. However, a detailed functional analysis of these interactions and of untested putative interactions with other sodium channel isoforms in mammalian neuronal cells has not been carried out. We examined the expression and function of sodium channels in small-diameter dorsal root ganglion (DRG) neurons from contactin-deficient (CNTN-/-) mice, compared to CNTN+/+ litter mates. Nav1.9 is preferentially expressed in isolectin B4 (IB4)-positive neurons and thus we used this marker to subdivide small-diameter DRG neurons. Using whole-cell patch-clamp recording, we observed a greater than two-fold reduction of tetrodotoxin-resistant (TTX-R) Nav1.8 and Nav1.9 current densities in IB4+ DRG neurons cultured from CNTN-/- vs. CNTN+/+ mice. Current densities for TTX-sensitive (TTX-S) sodium channels were unaffected. Contactin's effect was selective for IB4+ neurons as current densities for both TTX-R and TTX-S channels were not significantly different in IB4- DRG neurons from the two genotypes. Consistent with these results, we have demonstrated a reduction in Nav1.8 and Nav1.9 immunostaining on peripherin-positive unmyelinated axons in sciatic nerves from CNTN-/- mice but detected no changes in the expression for the two major TTX-S channels Nav1.6 and Nav1.7. These data provide evidence of a role for contactin in selectively regulating the cell surface expression and current densities of TTX-R but not TTX-S Na+ channel isoforms in nociceptive DRG neurons; this regulation could modulate the membrane properties and excitability of these neurons.     

Expression of tetrodotoxin-sensitive and resistant sodium channels by rat melanotrophs

Rat melanotrophs fire Na+ and Ca2(+)-dependent action potentials. Whereas the molecular identity of Ca2+ channels expressed by these cells is well documented, less is known about Na channels. We characterize the expression of seven sodium channel alpha-subunit and the beta1- and beta2-subunit mRNAs. The tetrodotoxin-resistant Nav1.8 and Nav1.9 alpha subunit mRNAs are detected in the newborn intermediate lobe and in cultured melanotrophs. Electrophysiological recordings further demonstrate the expression of both tetrodotoxin-sensitive and tetrodotoxin-resistant currents by dissociated melanotrophs. Moreover, activated sodium channels are able to elicit intracellular calcium waves, both in the absence or in the presence of tetrodotoxin. This work shows that rat melanotrophs express functional tetrodotoxin-resistant sodium channels, whose activation can lead to the generation of intracellular calcium waves.     

Non-uniform expression of alpha subunit isoforms of the Na+/K+ pump in rat dorsal root ganglia neurons

Tissue sections and antibodies selectively recognizing isoforms of the alpha subunit of the Na+/K+ pump were used to determine the expression of alpha1, alpha2 and alpha3 pump isoforms in the plasma membrane of adult rat dorsal root ganglia (DRG) neurons. There was no detectable membrane signal from DRG neurons that were probed with antibodies to the alpha2 isoform of the Na+/K+ pump. The alpha1 isoform of the Na+/K+ pump was found in most (77+/-4%) studied DRG neurons, regardless of cell size. Only 16+/-7% of the neurons expressed a detectable level of the alpha3 Na+/K+ pump and all were apparently from a subpopulation of large DRG neurons. Comparison of cell size distributions and a study of neurons identified in serial sections suggested that of the alpha3 positive DRG neurons about 75% coexpressed the alpha1 isoform of the Na+/K+ pump. These data show that the expression of the protein of the alpha subunit isoforms of the Na+/K+ pump is not uniform throughout the population of DRG neurons and that alpha1 is the predominant isoform in the plasma membrane of these neurons.     

Molecular characterization of voltage-gated sodium channels in human gliomas

Voltage-sensitive sodium channels appear to be an electrophysiological hallmark of gliomas. However, the expression of channel subtypes is unclear in these tumors. In this study different gliomas were investigated for the expression of sodium channel subtypes Na(v)1.1, Na(v)1.2, Na(v)1.3, Na(v)1.4, Na(v)1.6, and Na(x)(Na(v)2.1) using RT-PCR. At least one subtype of channels could be detected in each tumor. High-grade gliomas expressed fewer sodium channel subtypes and these at weaker levels than low-grade tumors. Expression of Na(v)1.6, the most abundant isoform in the CNS, was almost absent in the gliomas except the pilocytic variant. Our study gives clear evidence for a differential expression of sodium channel subtypes in gliomas and indicates a predominant expression of channels related to malignancy grades.     

The alpha1I T-type calcium channel exhibits faster gating properties when overexpressed in neuroblastoma/glioma NG 108-15 cells

The recently cloned T-type calcium channel alpha1I (Cav3.3) displays atypically slow kinetics when compared to native T-channels. Possible explanations might involve alternative splicing of the alpha1I subunit, or the use of expression systems that do not provide a suitable environment (auxiliary subunit, phosphorylation, glycosylation...). In this study, two human alpha1I splice variants, the alpha1I-a and alpha1I-b isoforms that harbour distinct carboxy-terminal regions were studied using various expression systems. As the localization of the alpha1I subunit is primarily restricted to neuronal tissues, its functional expression was conducted in the neuroblastoma/glioma cell line NG 108-15, and the results compared to those obtained in HEK-293 cells and Xenopus oocytes. In Xenopus oocytes, both isoforms exhibited very slow current kinetics compared to those obtained in HEK-293 cells, but the alpha1I-b isoform generated faster currents than the alpha1I-a isoform. Both activation and inactivation kinetics of alpha1I currents were significantly faster in NG 108-15 cells, while deactivating tail currents were two times slower, compared to those obtained in HEK-293 cells. Moreover, the alpha1-b isoform showed significantly slower deactivation kinetics both in NG 1080-15 and in HEK-293 cells. Altogether, these data emphasize the advantage of combining several expression systems to reveal subtle differences in channel properties and further indicate that the major functional differences between both human alpha1I isoforms are related to current kinetics. More importantly, these data suggest that the expression of the alpha1I subunit in neuronal cells contributes to the "normalization" of current kinetics to the more classical, fast-gated T-type Ca2+ current.     

The roles of sodium channels in nociception: implications for mechanisms of neuropathic pain

Animal models have provided useful insights into the development and treatment of neuropathic pain. New genetic data from both human studies and transgenic mouse models suggest that specific voltage-gated sodium channel subtypes are associated with specific types of pain and, as such, may be useful analgesic drug targets for a variety of pain types including neuropathic pain. Global voltage-gated sodium channel blockers such as lidocaine have proven efficacy in treating pain but can be limited by adverse effects when administered systemically. Selective sodium channel blockers targeting channels at the periphery (Nav1.7, Nav1.8, and Nav1.9) could potentially reduce the side effect profile. Individual isoforms of voltage-gated sodium channels have been linked to particular types of pain. Nav1.7 is a useful target for ameliorating acute mechanical pain and inflammatory pain, and strong evidence also suggests that Nav1.9 could be targeted for treating inflammatory pain. Selective blockers of Nav1.8 could also have clinical benefit for visceral pain. Although there is no association between a single sodium channel isoform and neuropathic pain, combined blockade of peripherally expressed isoforms Nav1.7, Nav1.8, and Nav1.9 may prove useful.     

The sodium channel Nav1.5a is the predominant isoform expressed in adult mouse dorsal root ganglia and exhibits distinct inactivation properties from the full-length Nav1.5 channel

Nav1.5 is the principal voltage-gated sodium channel expressed in heart, and is also expressed at lower abundance in embryonic dorsal root ganglia (DRG) with little or no expression reported postnatally. We report here the expression of Nav1.5 mRNA isoforms in adult mouse and rat DRG. The major isoform of mouse DRG is Nav1.5a, which encodes a protein with an IDII/III cytoplasmic loop reduced by 53 amino acids. Western blot analysis of adult mouse DRG membrane proteins confirmed the expression of Nav1.5 protein. The Na+ current produced by the Nav1.5a isoform has a voltage-dependent inactivation significantly shifted to more negative potentials (by approximately 5 mV) compared to the full-length Nav1.5 when expressed in the DRG neuroblastoma cell line ND7/23. These results imply that the alternatively spliced exon 18 of Nav1.5 plays a role in channel inactivation and that Nav1.5a is likely to make a significant contribution to adult DRG neuronal function.     

Expression of calcium transporters in the retina of the tiger salamander (Ambystoma tigrinum)

Changes in intracellular calcium concentration, [Ca2+]i, modulate the flow of visual signals across all stages of processing in the retina, yet the identities of Ca2+ transporters responsible for these changes are still largely unknown. In the current study, the distribution of plasma membrane and intracellular Ca2+ transporters in the retina of tiger salamander, a model system for physiological studies of retinal function, was determined. Plasma membrane calcium ATPases (PMCAs), responsible for high-affinity Ca2+ extrusion, were highly expressed in the salamander retina. PMCA isoforms 1, 2, and 4 were localized to photoreceptors, whereas the inner retina expressed all four isoforms. PMCA3 was expressed in a sparse population of amacrine and ganglion neurons, whereas PMCA2 was expressed in most amacrine and ganglion cells. Na+/Ca2+ exchangers, a high-capacity Ca2+ extrusion system, were expressed in the outer plexiform layer and in a subset of inner nuclear and ganglion layer cells. Intracellular Ca2+ store transporters were also represented prominently. SERCA2a, a splice variant of the sarcoplasmic-endoplasmic Ca2+ ATPase, was found mostly in photoreceptors, whereas SERCA2b was found in the majority of retinal neurons and in glial cells. The predominant endoplasmic reticulum (ER) Ca2+ channels in the salamander retina are represented by the isoform 2 of the IP3 receptor family and the isoform 2 of the ryanodine receptor family. These results indicate that Ca2+ transporters in the salamander retina are expressed in a cell type-specific manner.     

Retinal lesions induce differential changes in the expression of flip and flop isoforms of the glutamate receptor subunit GluR1 in the chick optic tectum

A sensitive RNase protection assay was employed to determine the levels of mRNA encoding the GluR1 subunit flip and flop isoforms in the chick optic tectum and forebrain. We found that the flip GluR1 mRNA predominates in the forebrain, whereas the flop variant is more strongly expressed in the optic tectum. A temporal analysis of GluR1 variants in the embryonic and adult chick brain revealed that the flip isoform is more highly expressed at E12 than at P15-21, whereas mRNA levels of the flop isoform are higher at P15-21 than at E12. To study the effect of deafferentation on GluR1 expression, unilateral retinal lesions were performed. Two days later the mRNA levels of GluR1 flip and flop variants were decreased in the deafferented tectum, especially for the flop isoform. However, 7 days after the lesion, the mRNA levels of both GluR1 isoforms were increased, especially for the flip isoform. These results reveal an important control of the retinal input upon the expression of the different GluR1 isoforms. Furthermore, they indicate a differential spatial and temporal regulation of the flip and flop splice variants, suggesting the existence of a mechanism regulating differential splicing or possibly differential RNA stability.     

Na+, K+ ATPase alpha-subunit isoform distribution and abundance in guinea-pig longitudinal muscle/myenteric plexus after exposure to morphine

Previous work in the myenteric plexus has shown that the resting membrane potential of morphine-tolerant guinea-pig myenteric S neurons is significantly depolarized relative to placebo-implanted controls, and that this depolarization is associated with reduced electrogenic Na+, K+ pumping. Identification of the subunits of the sodium pump which are in the myenteric plexus was undertaken in order to facilitate direct qualitative and quantitative measurements of the abundance of sodium pump isoforms after morphine exposure, thereby confirming and extending the electrophysiological data to the molecular level. Seven days prior to the experiments, tolerance was induced by subcutaneous implantation of morphine pellets (one pellet, 75 mg/100 g body weight) while control guinea pigs received placebo pellets. Using immunohistochemistry and confocal microscopy, the distribution of the alpha subunit isoforms of the Na+/K+ -ATPase in placebo and morphine-tolerant guinea-pig ileum was determined. Only the alpha1 and alpha3 subunit isoforms were in sufficient abundance to be observed. The alpha1 subunit isoform was most highly concentrated in the mucosa and in neurons. In contrast, the alpha3 subunit isoform was uniquely localized to neurons. Western and slot blot analyses of longitudinal muscle/myenteric plexus homogenates identified a significant reduction of the alpha3 but not the alpha1 subunit isoform in tolerant preparations. It is concluded that the reduced electrogenic pumping in the S neurons after morphine exposure is associated with a reduction in the alpha3 subunit isoform.     

Tetrodotoxin-resistant Na+ channels in human neuroblastoma cells are encoded by new variants of Nav1.5/SCN5A

Both tetrodotoxin-sensitive (TTX-S) and TTX-resistant (TTX-R) voltage-dependent Na+ channels are expressed in the human neuroblastoma cell line NB-1, but a gene encoding the TTX-R Na+ channel has not been identified. In this study, we have cloned cDNA encoding the alpha subunit of the TTX-R Na+ channel in NB-1 cells and designated it hNbR1. The longest open reading frame of hNbR1 (accession no. AB158469) encodes 2016 amino acid residues. Sequence analysis has indicated that hNbR1 is highly homologous with human cardiac Nav1.5/SCN5A with > 99% amino acid identity. The presence of a cysteine residue (Cys373) in the pore-loop region of domain I is consistent with the supposition that hNbR1 is resistant to TTX. Analysis of the genomic sequence of SCN5A revealed a new exon encoding S3 and S4 of domain I (exon 6A). In addition, an alternative splicing variant, lacking exon 18, that encodes 54 amino acids in the intracellular loop between domains II and III was found (hNbR1-2; accession no. AB158470). Na+ currents in human embryonic kidney cells (HEK293) transfected with hNbR1 or hNbR1-2 showed electrophysiological properties similar to those for TTX-R I(Na) in NB-1 cells. The IC50 for the TTX block was approximately 8 microM in both variants. These results suggest that SCN5A has a newly identified exon for alternative splicing and is more widely expressed than previously thought.     

Expression, activity and distribution of Na,K-ATPase subunits during in vitro neuronal induction

The expression pattern of the alpha and beta isoforms and the gamma subunit of the Na,K-ATPase was investigated during in vitro induction of pluripotent murine embryonic stem (ES) cells into neuronal cells. alpha1 protein was expressed in undifferentiated ES (UES) cells and throughout all stages studied. In contrast, alpha3 protein was prominent only when neuronal cells have reached full differentiation. In this model, neuron-depleted cultures did not express the alpha3 isoform, indicating its specificity for mature neuronal cells. UES possessed Na,K-ATPase activity consistent with a single isoform (alpha1), whereas in fully mature neuronal cells a ouabain-sensitive isoform (alpha3) accounted for 27+/-4% of the activity, and a ouabain-resistant isoform (alpha1) 66+/-3%. Immunocytochemistry of mature neuronal cells for alpha1 and alpha3 proteins showed a similar distribution, including cell soma and processes, without evidence of polarization. beta1 protein was expressed in uninduced ES, embryonic bodies (EB) and neuronal cells. While proteins of the beta2 and beta3 isoforms were not detected by immunoblots (except for beta2 in UES), their mRNAs were detected in UES and EB (beta2 and beta3), and in immature and fully differentiated neuronal cells (beta3). Message for the beta2 isoform, however, was not present in neuronal cells. gamma subunit mRNA and protein were undetectable at any stage. These results provide further characterization of neuron-like cells obtained by induction of ES cells in vitro, and establish a model for the expression of isoforms of the Na,K-ATPase during neuronal differentiation. The relation to other aspects of neuronal cell development and relevance to a specialised function for the alpha3 subunit in neurons are discussed.     

Identification of residues within GABA(A) receptor alpha subunits that mediate specific assembly with receptor beta subunits.

GABA(A) receptors can be constructed from a range of differing subunit isoforms: alpha, beta, gamma, delta, and epsilon. Expression studies have revealed that production of GABA-gated channels is achieved after coexpression of alpha and beta subunits. The expression of a gamma subunit isoform is essential to confer benzodiazepine sensitivity on the expressed receptor. However, how the specificity of subunit interactions is controlled during receptor assembly remains unknown. Here we demonstrate that residues 58-67 within alpha subunit isoforms are important in the assembly of receptors comprised of alphabeta and alphabetagamma subunits. Deletion of these residues from the alpha1 or alpha6 subunits results in retention of either alpha subunit isoform in the endoplasmic reticulum on coexpression with the beta3, or beta3 and gamma2 subunits. Immunoprecipitation revealed that residues 58-67 mediated oligomerization of the alpha1 and beta3 subunits, but were without affect on the production of alpha/gamma complexes. Within this domain, glutamine 67 was of central importance in mediating the production of functional alpha1beta3 receptors. Mutation of this residue resulted in a drastic decrease in the cell surface expression of alpha1beta3 receptors and the resulting expression of beta3 homomers. Sucrose density gradient centrifugation revealed that this residue was important for the production of a 9S alpha1beta3 complex representing functional GABA(A) receptors. Therefore, our studies detail residues that specify GABA(A) receptor alphabeta subunit interactions. This domain, which is conserved in all alpha subunit isoforms, will therefore play a critical role in the assembly of GABA(A) receptors composed of alphabeta and alphabetagamma subunits.