Cloning of a sodium channel alpha subunit from rabbit Schwann cells (original) (raw)
Related papers
Journal of Biological Chemistry, 2004
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 ϳ2:1 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.
Genomics, 1996
proximal part of mouse chromosome 2 (13) and on We have used a total of 27 AXB/BXA recombinant the long arm of human chromosome 2 (2q) (1, 8, 12, inbred mouse strains to determine the chromosomal 14). In mouse, the glial-specific voltage-sensitive solocation of a newly identified gene encoding an a-subdium channel gene (Scn7a) is also located on chromounit isoform of the sodium channel from Schwann some 2, just outside of the Scn1a/Scn2a/Scn3a cluscells, Scn9a. Linkage analysis established that Scn9a ter (16). Other sodium channel a-subunit genes ocmapped to the proximal segment of mouse chromocupy a distinct chromosomal position in the mouse some 2. The segregation of restriction fragment length genome (4, 10).
Molecular diversity of voltage-gated sodium channel α and β subunit mRNAs in human tissues
European Journal of Pharmacology, 2006
Voltage-gated Na + channels are composed of one α subunit and one or more auxiliary β subunits. A reverse transcription-polymerase chain reaction assay was used to analyse the expression of the nine known α subunits (Na v 1.1-Na v 1.9) in 20 different human tissues. The mRNA expression of the currently known β subunits (β 1 , β 2 , β 3 and β 4 ) was also assessed. The mRNAs of voltage-gated Na + channel α and β subunits were found in a wide variety of human tissues assayed and were present in neuronal and non-neuronal types of cells. These data suggest that, in addition to its well-established role in skeletal muscle, cardiac cells and neurons, voltage-gated Na + channels might play important, still undetermined local roles in the regulation of cellular functions. These channels could emerge in the next future as potential, new therapeutic targets in the treatment of visceral diseases.
Sodium channel mRNAs I, II and III in the CNS: cell-specific expression
Molecular Brain Research, 1994
The cellular localization of rat brain sodium channel et-subunit mRNAs I, II and III in the central nervous system (CNS) was examined by non-isotope in situ hybridization cytochemistry utilizing two independent sets of isoform-specific RNA probes, one set recognizing sodium channel isoforms in the coding region and the other in the non-coding region of the sodium channel messages. The independent sets of probes demonstrated qualitatively similar patterns of sodium channel mRNA expression. In the hippocampus, sodium channel mRNA I was very weakly expressed in the pyramidal layer and in the granular layer of the dentate gyrus; in contrast, sodium channel mRNA II was strongly expressed by neurons in these regions. Sodium channel mRNA III exhibited low-to-moderate expression in some neurons of the pyramidal layer of the hippocampus and granular layer of the dentate gyrus, and was not detectable in others. In the cerebellum, sodium channel mRNA I was moderately expressed in some Purkinje cells, weakly expressed in scattered cells in the molecular layer and negligibly expressed in the granular layer. Sodium channel mRNA II was strongly expressed in Purkinje and granule cells, and was moderately expressed in some cells in the molecular layer. Sodium channel mRNA III was generally not detectable in the cerebellum. In the spinal cord, motor'neurons and scattered neurons throughout the gray matter exhibited moderate-to-strong expression of both sodium channel mRNA I and II. A population of cells in the spinal zone of Lissauer showed heavy expression of mRNA II, but not mRNA I. Sodium channel mRNA III was not detectable in spinal cord neurons. These observations are consistent with a general regional distribution of sodium channel message isoforms, with mRNA II being preferentially expressed in rostral regions of the CNS and mRNA I in caudal regions. However, the results also indicate that different cell types, within a given region, display different patterns of sodium channel mRNA expression. Moreover, these data suggest that individual neurons may express multiple forms of sodium channel mRNA.
Molecular diversity of voltage-gated sodium channel a and � subunit mRNAs in human tissues
Eur J Pharmacol, 2006
Voltage-gated Na + channels are composed of one α subunit and one or more auxiliary β subunits. A reverse transcription-polymerase chain reaction assay was used to analyse the expression of the nine known α subunits (Na v 1.1-Na v 1.9) in 20 different human tissues. The mRNA expression of the currently known β subunits (β 1 , β 2 , β 3 and β 4) was also assessed. The mRNAs of voltage-gated Na + channel α and β subunits were found in a wide variety of human tissues assayed and were present in neuronal and non-neuronal types of cells. These data suggest that, in addition to its well-established role in skeletal muscle, cardiac cells and neurons, voltage-gated Na + channels might play important, still undetermined local roles in the regulation of cellular functions. These channels could emerge in the next future as potential, new therapeutic targets in the treatment of visceral diseases.
Molecular Brain Research, 1997
The expressicn of sodium channel o-subunit mRNAs I, II, III. NaG, Na6 and hNE (PNI) was examined in developing (E17-P30) hippocampus, cerebellum, spinal cord and dorsal root ganglia using non-isotopic in situ hybridization cytochemistry. The results showed distinct patterns of expression for each of the sodium channel tnRNAs with maturation of the nervous system. In the hippocampus. sodium channel mRNA I was not detected at any developmental time, while mRNA II showed increasing hybridization signal between El7 and P30. Sodium channel mRNA III was more prevalent at late embryonic and early postnatal times. and was barely detectable at P30. The transcript for NaG showed transient expression between P2 and PIS, being expressed at low levels at El7 and not being detectable at P30. Sodium channel mRNA Na6 exhibited a high level of expression between El7 and PI5 in the bippocampal formation, with an attenuation of the stgnal by P30. hNE (PNI) mRNA was not detected in the hippocampus at any time examined. In the cerebellum, sodium channel mRNA I was not detected at El7 or P7, but became detectable in Purkinje cells at PlS and continued to show a low level of expression in these cells at P30. mRNA I was not detected at any time examined in c oranule cells of the cerebellum. Sodium channel mRNA II exhibited increasing expression in the developing cerebellum. and showed increasin, c 0 \iunal in Purkinge cells beginning on P2 and granule cells on P15. Sodium channel mRNA III was down-regulated with development in the cerebellum. although mRNA III was readily detected at E17. it was not detected in any layers of the cerebelluni by PIS. NaG mRNA showed a peak of expression at P2. and was present at low levels at El7 and P15 and not detectable at P30. Na6 mRNA was highly expressed in the El7 cerebellum; this mRNA was present at high levels in Purkinjc cells throughout development. although in granule cells the signal v.as attenuated at PlS-P30. Sodium channel hNE (PNI) mRNA was not detected in the cerebellum at any time in development. In the spinal cord, sodium channel mFaA I showed increasing expression beginning at P2 and was highly expressed, particuharly in ventral motor neurons, by P30. Sodium channel II mRNA was detected at all stages of development in the spinal cord; in contrast, mRNA Ii! was detected at El7 and P2, but showed very low levels of expression by P30. NaG mRNA exhibited a transient expression in spinal cord at P2, but was not detectable at El7 and P30. Na6 mRNA was detectable at vety low levels at El7 and became highly expressed at P2. prior to a reduction of the signal at PlS and P30. hNE (PNI) mRNA was not detected in the spinal cord at any time in development. In the dorsal root ganglia, sodium channel I mRNA hybridization signal was detected in DRG neurons at P2, with slightly increased levels at PI5 and P30. Sodium channel II mRNA exhibited a relatively constant, moderate level of expression at all developmental ages. Sodium channel III mRNA was highly expressed in DRG neurons at El7 but was down-regulated with further development so that it was not detectable by P30. NaG mRNA was strongly expressed by some DRG neurons at all stages of development from El7 to P30; in general the level of NaG labelling was greater in larger neurons than in smaller neurons. Na6 mRNA showed increasing expression with developlnent in DRG neurons; at E17, low levels of Na6 mRNA were detected and by Pl5 to P30 high levels of expression were present in some neurons. hNE (PNI) mRNA was present in DRG neurons at P2, and was up-regulated with further development so that by P30 hNE (PN! ! was expressed in all DRG neurons sizes. These results demonstrate that sodium channel a-subunit m ..RXAs I, II, III, NaG. Na6 and hNE (PNl) exhibit distinct spatial and temporal patterns of expression in nervous tissue. and suggest that the expression of the sodium channel a-subunits is differentially regulated. The expression of NaG and hNE iPN1) mRNAs in P30 DRG, but not otller tissues. may provide a correlate for the presence in dorsal root ganglia neu.,,k., ~'x-s of unique sodium currents.
Comparative distribution of voltage-gated sodium channel proteins in human brain
Molecular Brain Research, 2001
Antisera directed against unique peptide regions from each of the human brain voltage-gated sodium channel a subunits were generated. In immunoblots these were found to be highly specific for the corresponding recombinant polypeptides and to recognise the native holoprotein in human brain membrane preparations. These antisera were used to perform a comparative immunohistochemical distribution analysis of all four brain sodium channel subtypes in selected human CNS regions. Distinct but heterogeneous distribution patterns were observed for each of the a subunits. In general, these were complimentary to that previously shown for the corresponding human mRNAs. A high degree of conservation with respect to the distribution found in rat was also evident. The human a subunit proteins exhibited distinct subcellular localisation patterns. Types I, III and VI immunoreactivity was predominantly in neuronal cell bodies and proximal processes, whereas type II was concentrated along axons. This is similar to rat brain and suggests the different the sodium channel subtypes have distinct functions which are highly conserved between human and rodents. A notable difference was that the type III protein was detected in all human brain regions examined, unlike in rat brain where expression in adults is very restricted. Also in contrast to rat brain, the human type VI protein was not detected in axons of unmyelinated neurons. These differences may reflect true species variation and could have important implications for understanding the function of the sodium channel subtypes and their roles in human disease.
Regulatory role of voltage-gated Na+ channel β subunits in sensory neurons
Frontiers in Pharmacology, 2011
Voltage-gated sodium Na + channels are membrane-bound proteins incorporating aqueous conduction pores that are highly selective for sodium Na + ions. The opening of these channels results in the rapid influx of Na + ions that depolarize the cell and drive the rapid upstroke of nerve and muscle action potentials. While the concept of a Na + -selective ion channel had been formulated in the 1940s, it was not until the 1980s that the biochemical properties of the 260-kDa and 36-kDa auxiliary β subunits (β 1 , β 2 ) were first described. Subsequent cloning and heterologous expression studies revealed that the α subunit forms the core of the channel and is responsible for both voltage-dependent gating and ionic selectivity. To date, 10 isoforms of the Na + channel α subunit have been identified that vary in their primary structures, tissue distribution, biophysical properties, and sensitivity to neurotoxins. Four β subunits (β 1 -β 4 ) and two splice variants (β 1A , β 1B ) have been identified that modulate the subcellular distribution, cell surface expression, and functional properties of the α subunits. The purpose of this review is to provide a broad overview of β subunit expression and function in peripheral sensory neurons and examine their contributions to neuropathic pain.
Localization of Nav1.5 sodium channel protein in the mouse brain
Neuroreport, 2002
Na v 1.5 or SCN5A is a member of the voltage-dependent family of sodium channels. The distribution of Na v 1.5 protein was investigated in the mouse brain using immunohistochemistry. Immunostaining with a Na v 1.5-specific antibodyrevealed that Na v 1.5 protein was localizedin certain distinctregions of brain including the cerebral cortex, thalamus, hypothalamus, basalganglia, cerebellum and brain stem. Notably, we found that Na v 1.5 protein co-localized with neurofilaments and clustered at a high density in the neuronal processes, mainly axons.These results suggest that Na v 1.5 protein may play a role in the physiology of the central nervous system (generation and propagation of electrical signals by axons).
The Journal of Physiology, 1995
1. The rat brain type IIA Nae channel a-subunit was stably expressed in Chinese hamster ovary (CHO) cells. Current through the expressed Na+ channels was studied using the whole-cell configuration of the patch clamp technique. The transient Na+ current was sensitive to TTX and showed a bell-shaped peak current vs. membrane potential relation. 2. Na+ current inactivation was better described by the sum of two exponentials in the potential range-30 to +40 mV, with a dominating fast component and a small slower component. 3. The steady-state inactivation, hoo, was related to potential by a Boltzmann distribution, underlying three states of the inactivation gate. 4. Recovery of the channels from inactivation at different potentials in the range-70 to-120 mV were characterized by an initial delay which decreased with hyperpolarization. The time course was well fitted by the sum of two exponentials. In this case the slower exponential was the major component, and both time constants decreased with hyperpolarization. 5. For a working description of the Nae channel inactivation in this preparation, with a minimal deviation from the Hodgkin-Huxley model, a three-state scheme of the form O = II = I2 was proposed, replacing the original two-state scheme of the Hodgkin-Huxley model, and the rate constants are reported. 6. The instantaneous current-voltage relationship showed marked deviation from linearity and was satisfactorily fitted by the constant-field equation. 7. The time course of activation was described by an mx model. However, the best-fitted value of x varied with the membrane potential and had a mean value of 2. 8. Effective gating charge was determined to be 4-7e from the slope of the activation plot, plotted on a logarithmic scale. 9. The rate constants of activation, am and /3n were determined. Their functional dependence on the membrane potential was investigated. Neuronal excitability is mediated by ion-specific channel proteins through which membrane currents flow. The rising phase of the action potential is caused by an influx of Na+ ions through voltage-activated Na+ channels (Hille, 1992). The mammalian brain Na+ channel is a heterotrimeric protein consisting of a large glycosylated ax-subunit of 230-270 kDa and two smaller /31and ,82-subunits (36 and 33 kDa, respectively). Different cDNA clones encoding highly homologous subtypes (types I, II, IIA, and III) of the a-subunit have been isolated from rat brain. The type II form is most predominant in embryonic and neonatal brain, whereas the alternatively spliced form of type II, i.e. type IIA, is most abundant in adult brain (Beckh, Noda, Liibbert & Numa, 1989). The cDNA for the rat brain Nae channel /31-subunit has also been cloned (Isom et al. 1992). The mRNA encoded by the Nae channel type IIA ac-subunit, when injected in Xenopus oocytes, directs the synthesis of functional Na+ channels, but with slower inactivation properties. Co-expression of the /31-subunit increases the expression level and accelerates the decay of Nae current (Catterall, 1992). Similar results were obtained with the a-subunit of the rat muscle ,ul sodium channel