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• W2082952610 abstract "Auxiliary β1 subunits of voltage-gated sodium channels have been shown to be cell adhesion molecules of the Ig superfamily. Co-expression of α and β1 subunits modulates channel gating as well as plasma membrane expression levels. We have cloned, sequenced, and expressed a splice variant of β1, termed β1A, that results from an apparent intron retention event. β1 and β1A are structurally homologous proteins with type I membrane topology; however, they contain little to no amino acid homology beyond the shared Ig loop region. β1A mRNA expression is developmentally regulated in rat brain such that it is complementary to β1. β1A mRNA is expressed during embryonic development, and then its expression becomes undetectable after birth, concomitant with the onset of β1 expression. In contrast, β1A mRNA is expressed in adult adrenal gland and heart. Western blot analysis revealed β1A protein expression in heart, skeletal muscle, and adrenal gland but not in adult brain or spinal cord. Immunocytochemical analysis of β1A expression revealed selective expression in brain and spinal cord neurons, with high expression in heart and all dorsal root ganglia neurons. Co-expression of αIIA and β1A subunits in Chinese hamster lung 1610 cells results in a 2.5-fold increase in sodium current density compared with cells expressing αIIA alone. This increase in current density reflected two effects of β1A: 1) an increase in the proportion of cells expressing detectable sodium currents and 2) an increase in the level of functional sodium channels in expressing cells. [3H]Saxitoxin binding analysis revealed a 4-fold increase in B max with no change inK D in cells coexpressing αIIA and β1A compared with cells expressing αIIA alone. β1A-expressing cell lines also revealed subtle differences in sodium channel activation and inactivation. These effects of β1A subunits on sodium channel function may be physiologically important events in the development of excitable cells. Auxiliary β1 subunits of voltage-gated sodium channels have been shown to be cell adhesion molecules of the Ig superfamily. Co-expression of α and β1 subunits modulates channel gating as well as plasma membrane expression levels. We have cloned, sequenced, and expressed a splice variant of β1, termed β1A, that results from an apparent intron retention event. β1 and β1A are structurally homologous proteins with type I membrane topology; however, they contain little to no amino acid homology beyond the shared Ig loop region. β1A mRNA expression is developmentally regulated in rat brain such that it is complementary to β1. β1A mRNA is expressed during embryonic development, and then its expression becomes undetectable after birth, concomitant with the onset of β1 expression. In contrast, β1A mRNA is expressed in adult adrenal gland and heart. Western blot analysis revealed β1A protein expression in heart, skeletal muscle, and adrenal gland but not in adult brain or spinal cord. Immunocytochemical analysis of β1A expression revealed selective expression in brain and spinal cord neurons, with high expression in heart and all dorsal root ganglia neurons. Co-expression of αIIA and β1A subunits in Chinese hamster lung 1610 cells results in a 2.5-fold increase in sodium current density compared with cells expressing αIIA alone. This increase in current density reflected two effects of β1A: 1) an increase in the proportion of cells expressing detectable sodium currents and 2) an increase in the level of functional sodium channels in expressing cells. [3H]Saxitoxin binding analysis revealed a 4-fold increase in B max with no change inK D in cells coexpressing αIIA and β1A compared with cells expressing αIIA alone. β1A-expressing cell lines also revealed subtle differences in sodium channel activation and inactivation. These effects of β1A subunits on sodium channel function may be physiologically important events in the development of excitable cells. Chinese hamster lung hemagglutinin saxitoxin tetrodotoxin polymerase chain reaction Sodium channels isolated from brain are composed of a central pore-forming α subunit and two auxiliary subunits, β1 and β2, which do not form the pore yet play critical roles in channel modulation and expression. A mutation in the β1 gene (SCN1B) has been implicated to play a role in febrile seizures and generalized epilepsy, GEFS+ (1.Wallace R.H. Wang D.W. Singh R. Scheffer I.E. George Jr., A.L. Phillips H.A. Saar K. Reis A. Johnson E.W. Sutherland G.R. Berkovic S.F. Mulley J.C. Nat. Genet. 1998; 19: 366-370Crossref PubMed Scopus (94) Google Scholar). The primary structure of the β1 subunit predicts an integral membrane glycoprotein with type I transmembrane topology as well as an extracellular Ig fold (2.Isom L.L. DeJongh K.S. Catterall W.A. Neuron. 1994; 12: 1183-1194Abstract Full Text PDF PubMed Scopus (489) Google Scholar, 3.Isom L.L. Catterall W.A. Nature. 1996; 383: 307-308Crossref PubMed Scopus (98) Google Scholar). β1 subunits can be classified as members of the V-set of the Ig superfamily, which includes many cell adhesion molecules. β1 and α subunit co-expression has been well characterized in Xenopus oocytes and in mammalian cells. In oocytes, co-expression of type IIA (SCN2A) or μI (SCN4A) α subunits with β1 increases the proportion of sodium channels that function in a fast gating mode, accelerates the macroscopic rates of activation and inactivation, shifts the voltage dependence of inactivation in the hyperpolarizing direction, and increases the peak current amplitude consistent with increases in channel expression (4.Isom L.L. DeJongh K.S. Patton D.E. Reber B.F.X. Offord J. Charbonneau H. Walsh K. Goldin A.L. Catterall W.A. Science. 1992; 256: 839-842Crossref PubMed Scopus (600) Google Scholar, 5.Patton D.E. Isom L.L. Catterall W.A. Goldin A.L. J. Biol. Chem. 1994; 269: 17649-17655Abstract Full Text PDF PubMed Google Scholar, 6.Bennett Jr., P.B. Makita N. George Jr., A.L. FEBS Lett. 1993; 326: 21-24Crossref PubMed Scopus (73) Google Scholar, 7.Cannon S.C. McClatchey A.I. Gusella J.F. Eur. J. Physiol. 1993; 423: 155-157Crossref PubMed Scopus (79) Google Scholar, 8.Schreibmayer W. Wallner M. Lotan I. Pflügers Arch. 1994; 426: 360-362Crossref PubMed Scopus (22) Google Scholar, 9.Wallner M. Weigl L. Meera P. Lotan I. FEBS Lett. 1993; 336: 535-539Crossref PubMed Scopus (49) Google Scholar). In Chinese hamster lung (CHL)1 cells, stable coexpression of β1 with αIIA results in increased channel expression levels at the plasma membrane as well as moderate hyperpolarizing shifts in the voltage dependence of channel activation and inactivation (10.Isom L.L. Scheuer T. Brownstein A.B. Ragsdale D.S. Murphy B.J. Catterall W.A. J. Biol. Chem. 1995; 270: 3306-3312Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar).β1 mRNA is expressed only after birth in the developing brain (5.Patton D.E. Isom L.L. Catterall W.A. Goldin A.L. J. Biol. Chem. 1994; 269: 17649-17655Abstract Full Text PDF PubMed Google Scholar,11.Sashihara S. Oh Y. Black J.A. Waxman S.G. Brain Res. Mol. Brain Res. 1995; 34: 239-250Crossref PubMed Scopus (30) Google Scholar). However, previous studies showing the developmental time course of β1 protein expression in rat forebrain suggested that multiple β1 subunit isoforms may be present (12.McHugh-Sutkowski E. Catterall W.A. J. Biol. Chem. 1990; 265: 12393-12399Abstract Full Text PDF PubMed Google Scholar). A 26-kDa β1-immunoreactive protein was observed at embryonic day 18. This protein was also expressed in adult adrenal gland, heart, skeletal muscle, and PC12 cells. After birth, there was a dramatic decrease in the level of this protein in brain, and little if any remained by postnatal day 14. The expression time course of this immunoreactive protein was complementary to that of β1 mRNA. Day 18 embryonic brain membranes also exhibited a low level of an immunoreactive peptide that migrated with an apparent molecular mass greater than 42 kDa. This protein was not detected in rat brain after birth. Other excitable tissues expressed multiple size forms of immunoreactive β1-like subunits as well. Adult rat heart and skeletal muscle membrane preparations exhibited 38- and 41-kDa bands on Western blots in addition to the 26-kDa band. The 41-kDa immunoreactive band observed in these studies was shown to be the adult rat brain isoform and was later identified as C1Aa.β1 (4.Isom L.L. DeJongh K.S. Patton D.E. Reber B.F.X. Offord J. Charbonneau H. Walsh K. Goldin A.L. Catterall W.A. Science. 1992; 256: 839-842Crossref PubMed Scopus (600) Google Scholar). The immunoreactive peptides identified in the previous study were detected with a polyclonal antibody raised against purified β1 subunits; thus, they could represent β1 subunit isoforms that contained significantly different mRNA sequences from C1Aa.β1 that may not have been detected using previous methods.To test this hypothesis, we screened a rat adrenal cDNA library with a probe encoding only the coding region of β1. The present study reports the molecular cloning and functional expression of β1A, a splice variant of the β1 gene that contains identical amino-terminal and extracellular Ig fold regions as β1 followed by a significantly different extracellular juxtamembrane domain, predicted transmembrane region, and predicted intracellular COOH-terminal domain. β1A mRNA is expressed early in embryonic brain development and then disappears after birth. Western blot analysis of membrane preparations using an antibody to a unique, extracellular region of β1A not found in β1 showed that β1A protein is expressed in adult rat heart, skeletal muscle, and adrenal gland but was not detected in adult rat brain or spinal cord. Immunocytochemical analysis of β1A expression in adult rat tissues revealed high expression in heart and dorsal root ganglion and selective expression in some areas of the brain and spinal cord. β1A functions to increase channel expression at the plasma membrane when coexpressed with αIIA subunits in CHL fibroblasts. Unlike β1, however, mean steady state inactivation curves for αβ1A-expressing cell lines were shifted to more positive potentials than the mean inactivation curves for cells expressing α alone. Previous studies showed that coexpression of α and β1 subunits in CHL cells shifted the voltage dependence of inactivation to more negative potentials compared with α alone (10.Isom L.L. Scheuer T. Brownstein A.B. Ragsdale D.S. Murphy B.J. Catterall W.A. J. Biol. Chem. 1995; 270: 3306-3312Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Therefore, the novel, carboxyl-terminal domains of β1A may be important for electrophysiological function. It has been shown previously that the extracellular domain of β1 is essential for expression and function of the αβ1 complex in Xenopus oocytes (13.Chen C. Cannon S.C. Pflügers Arch. 1995; 431: 186-195Crossref PubMed Scopus (67) Google Scholar, 14.McCormick K.A. Isom L.L. Ragsdale D. Smith D. Scheuer T. Catterall W.A. J. Biol. Chem. 1998; 273: 3954-3962Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). We propose that the extracellular Ig fold, common to β1 and β1A, is essential for the observed increases in channel expression levels. Thus, this report introduces a novel splice variant of β1, β1A, and adds to our understanding of β1 structure-function relationships in terms of channel expression levels and electrophysiology.DISCUSSIONA number of cases of intron retention have been reported in the literature, including alternative splicing of the genes encoding leukocyte-common antigen-related protein tyrosine phosphatase, CD44, effector cell protease receptor-1, the microtubule-associated protein tau, thyrotropin-releasing hormone receptor, and bovine growth hormone (29.Tabiti K. Cui L. Chhatwal V.J. Moochhala S. Ngoi S.S. Pallen C.J. Gene (Amst.). 1996; 175: 7-13Crossref PubMed Scopus (11) Google Scholar, 30.Higashikawa K. Yokozaki H. Ue T. Taniyama K. Ishikawa T. Tarin D. Tahara E. Int. J. Cancer. 1996; 66: 11-17Crossref PubMed Scopus (50) Google Scholar, 31.Zhang J.S. Longo F.M. J. Cell Biol. 1995; 128: 415-431Crossref PubMed Scopus (80) Google Scholar, 32.Altieri D.C. Biochemistry. 1994; 33: 13848-13855Crossref PubMed Scopus (33) Google Scholar, 33.Sadot E. Marx R. Barg J. Behar L. Ginzburg I. J. Mol. Biol. 1994; 241: 325-331Crossref PubMed Scopus (36) Google Scholar, 34.de la Pena P. Delgado L.M. del Camino D. Barros F. J. Biol. Chem. 1992; 267: 25703-25708Abstract Full Text PDF PubMed Google Scholar, 35.Hampson R.K. LaFollette L. Rottman F.M. Mol. Cell. Biol. 1989; 9: 1604-1610Crossref PubMed Scopus (62) Google Scholar). In many cases, the retained intron contains an alternate, in-frame termination codon as well as a polyadenylation signal. Alternative splicing that results in retention of the intron in the primary transcript thus results in an isoform of the protein containing a novel carboxyl terminus. Interestingly, this is not the first report of intron retention in the β1 gene. Waxman and co-workers (36.Oh Y. Waxman S.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 9985-9989Crossref PubMed Scopus (60) Google Scholar, 37.Dib-Hajj S.D. Waxman S.G. FEBS Lett. 1995; 377: 485-488Crossref PubMed Scopus (19) Google Scholar) have previously reported that intron 5 of β1 can be retained, creating a novel isoform that is expressed in rat brain, optic nerve, sciatic nerve, and skeletal muscle. This isoform contains an 86-nucleotide insert encoded by intron 5 in the 3′-untranslated region. While the intron retention reported previously does not alter the β1 coding sequence, our present data describe a very significant coding sequence change resulting in a novel carboxyl terminus.Effects of β1A on sodium currents and [3H]STX Binding Levels in CHL CellsThe most striking functional consequence of αIIA and β1A coexpression in CHL cells was a 2.5-fold increase in sodium current density compared with cells expressing αIIA subunits alone. This increase in current density reflected two distinct effects of β1A: 1) an increase in the proportion of cells expressing detectable sodium currents and 2) an increase in the level of functional sodium channels in expressing cells. Increases in sodium channel expression with β1A are similar to previous results obtained with the adult β1 isoform in both mammalian cells (10.Isom L.L. Scheuer T. Brownstein A.B. Ragsdale D.S. Murphy B.J. Catterall W.A. J. Biol. Chem. 1995; 270: 3306-3312Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar) and Xenopus oocytes (4.Isom L.L. DeJongh K.S. Patton D.E. Reber B.F.X. Offord J. Charbonneau H. Walsh K. Goldin A.L. Catterall W.A. Science. 1992; 256: 839-842Crossref PubMed Scopus (600) Google Scholar). These observations are consistent with the hypothesis that β1 and β1A subunits facilitate the expression of sodium channels and/or stabilize the channels in the plasma membrane and that the molecular basis for this function resides in the extracellular cell adhesion molecule domain common to the two isoforms. The results of our [3H]STX binding experiments support this hypothesis. We propose that interaction of the extracellular cell adhesion molecule domain (Ig fold) common to β1 and β1A with α may be responsible for the observed effects on channel expression levels. Consistent with this interpretation, two previous studies have shown that the extracellular domain of β1 is essential for modulation of both brain and skeletal muscle α subunits, whereas the intracellular carboxyl-terminal domain is not; truncated β1 subunits lacking the intracellular carboxyl terminus are fully functional in terms of kinetic modulation of brain and skeletal muscle α subunits expressed in Xenopus oocytes (13.Chen C. Cannon S.C. Pflügers Arch. 1995; 431: 186-195Crossref PubMed Scopus (67) Google Scholar, 14.McCormick K.A. Isom L.L. Ragsdale D. Smith D. Scheuer T. Catterall W.A. J. Biol. Chem. 1998; 273: 3954-3962Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Residues predicted to be in the Ig fold of β1 interact with type IIA α subunits (14.McCormick K.A. Isom L.L. Ragsdale D. Smith D. Scheuer T. Catterall W.A. J. Biol. Chem. 1998; 273: 3954-3962Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Thus, the extracellular cell adhesion domain common to β1 and β1A appears to be required for function.Sodium currents in β1A-expressing cell lines also exhibited subtle functional differences compared with the parent SNaIIA cell line. For example, inactivation curves in SNaIIAβ1A cell lines were shifted to slightly more positive potentials than inactivation curves for SNaIIA cells. In contrast as shown both here and in a previous study (10.Isom L.L. Scheuer T. Brownstein A.B. Ragsdale D.S. Murphy B.J. Catterall W.A. J. Biol. Chem. 1995; 270: 3306-3312Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar), coexpression of β1 with αIIA in CHL cells shifted inactivation to potentials approximately 10 mV more negative than for cells expressing αIIA alone. Perhaps the differences between these two β subunit isoforms located in the putative juxtamembrane and/or transmembrane domains are responsible for these subtle distinctions in functional effects. Makita et al. (38.Makita N. Bennett P.B. George Jr., A.L. J. Neurosci. 1996; 16: 7117-7127Crossref PubMed Google Scholar) reported that a β1/β2 subunit chimeric construct containing the extracellular region plus the first 6 residues of the transmembrane domain of β1 was sufficient to modulate sodium channel skeletal muscle α subunits expressed in oocytes. Interestingly, this construct included an additional segment of β1 (ANRDMASIVSEIMMYVL) that is located carboxyl-terminal to the intron 3 splice site and is therefore not present in β1A. In contrast, β1A contains a novel juxtamembrane region that is 55 amino acids larger than that found in β1. This structural difference may be responsible for the opposite effects on steady state inactivation by β1versus β1A.In addition to opposite effects on steady state inactivation, the voltage dependence of activation and the rate of channel inactivation were also different in one of the three β1A-expressing cell lines, compared with the parent SNaIIA cell line. Thus, whole cell electrophysiological data suggest that β1A subunits may subtly modulate various aspects of sodium channel function. Nevertheless, the differences between cell lines with and without β1A subunits in the properties of whole cell sodium currents were very small and/or not observed in all cell lines. Therefore, additional analysis, perhaps at the single channel level, will be necessary to resolve whether these small differences actually reflect modulation of sodium channel function by β1A or some other source of variability between cell lines.TTX-sensitive sodium channel α subunits expressed in brain (SCN1A, Ref. 39.Smith R.D. Goldin A.L. J. Neurosci. 1998; 18: 811-820Crossref PubMed Google Scholar; SCN2A, Refs. 4.Isom L.L. DeJongh K.S. Patton D.E. Reber B.F.X. Offord J. Charbonneau H. Walsh K. Goldin A.L. Catterall W.A. Science. 1992; 256: 839-842Crossref PubMed Scopus (600) Google Scholar and 10.Isom L.L. Scheuer T. Brownstein A.B. Ragsdale D.S. Murphy B.J. Catterall W.A. J. Biol. Chem. 1995; 270: 3306-3312Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar; SCN3A, Ref. 5.Patton D.E. Isom L.L. Catterall W.A. Goldin A.L. J. Biol. Chem. 1994; 269: 17649-17655Abstract Full Text PDF PubMed Google Scholar; SCN8A, Ref. 40.Smith M.R. Smith R.D. Plummer N.W. Meisler M.H. Goldin A.L. J. Neurosci. 1998; 18: 6093-6102Crossref PubMed Google Scholar) and skeletal muscle (SCN4A, Ref. 9.Wallner M. Weigl L. Meera P. Lotan I. FEBS Lett. 1993; 336: 535-539Crossref PubMed Scopus (49) Google Scholar) have been shown to be modulated by co-expression of β1 subunits in heterologous systems. In contrast, TTX-resistant sodium channel α subunits expressed in cardiac myocytes (SCN5A, Ref. 41.Qu Y. Isom L.L. Westenbroek R.E. Rogers J.C. Tanada T.N. McCormick K.A. Scheuer T. Catterall W.A. J. Biol. Chem. 1995; 270: 25696-25701Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar) and peripheral nerve (SNS/PN3/SCN10A, Refs. 42.Sangameswaran L. Delgado S.G. Fish L.M. Koch B.D. Jakeman L.B. Stewart G.R. Sze P. Hunter J.C. Eglen R.M. Herman R.C. J. Biol. Chem. 1996; 271: 5953-5956Abstract Full Text Full Text PDF PubMed Scopus (396) Google Scholar, 43.Klugbauer N. Lubica L. Flockerzi V. Hofmann F. EMBO J. 1995; 14: 1084-1090Crossref PubMed Scopus (274) Google Scholar, 44.Akopian A.N. Sivilotti L. Wood J.N. Nature. 1996; 379: 257-262Crossref PubMed Scopus (908) Google Scholar) are much less sensitive or insensitive to modulation by β1 when co-expressed either in Xenopus oocytes or mammalian cells. A transcript encoding a predicted TTX-insensitive sodium channel (NaN/SNS-2/SCN11A) has been cloned but not yet expressed (45.Dib-Hajj S.D. Tyrrell L. Black J.A. Waxman S.G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 8963-8968Crossref PubMed Scopus (458) Google Scholar). Interestingly, heart, skeletal muscle, and dorsal root ganglia express both TTX-sensitive and -resistant sodium channel α subunits. (Primary cultures of cardiac myocytes express SCN1A, as assessed by Western blot analysis.) 2J. D. Malhotra and L. L. Isom, unpublished results. We have shown in the present study that these tissues also express β1A subunits. It will be interesting in the future to determine whether the spinal cord and brain contain unique populations of neurons expressing TTX-resistant channels and whether TTX-resistant channels are modulated by β1A. We also show that β1A subunits appear to be expressed in nonexcitable tissues such as the lung, where sodium channel α subunits would not be expected to be expressed. This finding raises the intriguing hypothesis that auxiliary β subunits may be expressed independently of α subunits to function as cell adhesion molecules. Recent results form our laboratory have shown that β1 and β2 interact homophilically in the absence of α subunits, resulting in cell aggregation and recruitment of the ankyrin cytoskeleton to the plasma membrane. 3J. D. Malhotra, M. Hortsch, and L. L. Isom, unpublished results. It will be interesting in the future to test the function of β1A as a cell adhesion molecule. Sodium channels isolated from brain are composed of a central pore-forming α subunit and two auxiliary subunits, β1 and β2, which do not form the pore yet play critical roles in channel modulation and expression. A mutation in the β1 gene (SCN1B) has been implicated to play a role in febrile seizures and generalized epilepsy, GEFS+ (1.Wallace R.H. Wang D.W. Singh R. Scheffer I.E. George Jr., A.L. Phillips H.A. Saar K. Reis A. Johnson E.W. Sutherland G.R. Berkovic S.F. Mulley J.C. Nat. Genet. 1998; 19: 366-370Crossref PubMed Scopus (94) Google Scholar). The primary structure of the β1 subunit predicts an integral membrane glycoprotein with type I transmembrane topology as well as an extracellular Ig fold (2.Isom L.L. DeJongh K.S. Catterall W.A. Neuron. 1994; 12: 1183-1194Abstract Full Text PDF PubMed Scopus (489) Google Scholar, 3.Isom L.L. Catterall W.A. Nature. 1996; 383: 307-308Crossref PubMed Scopus (98) Google Scholar). β1 subunits can be classified as members of the V-set of the Ig superfamily, which includes many cell adhesion molecules. β1 and α subunit co-expression has been well characterized in Xenopus oocytes and in mammalian cells. In oocytes, co-expression of type IIA (SCN2A) or μI (SCN4A) α subunits with β1 increases the proportion of sodium channels that function in a fast gating mode, accelerates the macroscopic rates of activation and inactivation, shifts the voltage dependence of inactivation in the hyperpolarizing direction, and increases the peak current amplitude consistent with increases in channel expression (4.Isom L.L. DeJongh K.S. Patton D.E. Reber B.F.X. Offord J. Charbonneau H. Walsh K. Goldin A.L. Catterall W.A. Science. 1992; 256: 839-842Crossref PubMed Scopus (600) Google Scholar, 5.Patton D.E. Isom L.L. Catterall W.A. Goldin A.L. J. Biol. Chem. 1994; 269: 17649-17655Abstract Full Text PDF PubMed Google Scholar, 6.Bennett Jr., P.B. Makita N. George Jr., A.L. FEBS Lett. 1993; 326: 21-24Crossref PubMed Scopus (73) Google Scholar, 7.Cannon S.C. McClatchey A.I. Gusella J.F. Eur. J. Physiol. 1993; 423: 155-157Crossref PubMed Scopus (79) Google Scholar, 8.Schreibmayer W. Wallner M. Lotan I. Pflügers Arch. 1994; 426: 360-362Crossref PubMed Scopus (22) Google Scholar, 9.Wallner M. Weigl L. Meera P. Lotan I. FEBS Lett. 1993; 336: 535-539Crossref PubMed Scopus (49) Google Scholar). In Chinese hamster lung (CHL)1 cells, stable coexpression of β1 with αIIA results in increased channel expression levels at the plasma membrane as well as moderate hyperpolarizing shifts in the voltage dependence of channel activation and inactivation (10.Isom L.L. Scheuer T. Brownstein A.B. Ragsdale D.S. Murphy B.J. Catterall W.A. J. Biol. Chem. 1995; 270: 3306-3312Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). β1 mRNA is expressed only after birth in the developing brain (5.Patton D.E. Isom L.L. Catterall W.A. Goldin A.L. J. Biol. Chem. 1994; 269: 17649-17655Abstract Full Text PDF PubMed Google Scholar,11.Sashihara S. Oh Y. Black J.A. Waxman S.G. Brain Res. Mol. Brain Res. 1995; 34: 239-250Crossref PubMed Scopus (30) Google Scholar). However, previous studies showing the developmental time course of β1 protein expression in rat forebrain suggested that multiple β1 subunit isoforms may be present (12.McHugh-Sutkowski E. Catterall W.A. J. Biol. Chem. 1990; 265: 12393-12399Abstract Full Text PDF PubMed Google Scholar). A 26-kDa β1-immunoreactive protein was observed at embryonic day 18. This protein was also expressed in adult adrenal gland, heart, skeletal muscle, and PC12 cells. After birth, there was a dramatic decrease in the level of this protein in brain, and little if any remained by postnatal day 14. The expression time course of this immunoreactive protein was complementary to that of β1 mRNA. Day 18 embryonic brain membranes also exhibited a low level of an immunoreactive peptide that migrated with an apparent molecular mass greater than 42 kDa. This protein was not detected in rat brain after birth. Other excitable tissues expressed multiple size forms of immunoreactive β1-like subunits as well. Adult rat heart and skeletal muscle membrane preparations exhibited 38- and 41-kDa bands on Western blots in addition to the 26-kDa band. The 41-kDa immunoreactive band observed in these studies was shown to be the adult rat brain isoform and was later identified as C1Aa.β1 (4.Isom L.L. DeJongh K.S. Patton D.E. Reber B.F.X. Offord J. Charbonneau H. Walsh K. Goldin A.L. Catterall W.A. Science. 1992; 256: 839-842Crossref PubMed Scopus (600) Google Scholar). The immunoreactive peptides identified in the previous study were detected with a polyclonal antibody raised against purified β1 subunits; thus, they could represent β1 subunit isoforms that contained significantly different mRNA sequences from C1Aa.β1 that may not have been detected using previous methods. To test this hypothesis, we screened a rat adrenal cDNA library with a probe encoding only the coding region of β1. The present study reports the molecular cloning and functional expression of β1A, a splice variant of the β1 gene that contains identical amino-terminal and extracellular Ig fold regions as β1 followed by a significantly different extracellular juxtamembrane domain, predicted transmembrane region, and predicted intracellular COOH-terminal domain. β1A mRNA is expressed early in embryonic brain development and then disappears after birth. Western blot analysis of membrane preparations using an antibody to a unique, extracellular region of β1A not found in β1 showed that β1A protein is expressed in adult rat heart, skeletal muscle, and adrenal gland but was not detected in adult rat brain or spinal cord. Immunocytochemical analysis of β1A expression in adult rat tissues revealed high expression in heart and dorsal root ganglion and selective expression in some areas of the brain and spinal cord. β1A functions to increase channel expression at the plasma membrane when coexpressed with αIIA subunits in CHL fibroblasts. Unlike β1, however, mean steady state inactivation curves for αβ1A-expressing cell lines were shifted to more positive potentials than the mean inactivation curves for cells expressing α alone. Previous studies showed that coexpression of α and β1 subunits in CHL cells shifted the voltage dependence of inactivation to more negative potentials compared with α alone (10.Isom L.L. Scheuer T. Brownstein A.B. Ragsdale D.S. Murphy B.J. Catterall W.A. J. Biol. Chem. 1995; 270: 3306-3312Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Therefore, the novel, carboxyl-terminal domains of β1A may be important for electrophysiological function. It has been shown previously that the extracellular domain of β1 is essential for expression and function of the αβ1 complex in Xenopus oocytes (13.Chen C. Cannon S.C. Pflügers Arch. 1995; 431: 186-195Crossref PubMed Scopus (67) Google Scholar, 14.McCormick K.A. Isom L.L. Ragsdale D. Smith D. Scheuer T. Catterall W.A. J. Biol. Chem. 1998; 273: 3954-3962Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). We propose that the extracellular Ig fold, common to β1 and β1A, is essential for the observed increases in channel expression levels. Thus, this report introduces a novel splice variant of β1, β1A, and adds to our understanding of β1 structure-function relationships in terms of channel expression levels and electrophysiology. DISCUSSIONA number of cases of intron retention have been reported in the literature, including alternative splicing of the genes encoding leukocyte-common antige" @default.
• W2082952610 created "2016-06-24" @default.
• W2082952610 creator A5004990133 @default.
• W2082952610 creator A5043756865 @default.
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• W2082952610 date "2000-01-01" @default.
• W2082952610 modified "2023-09-26" @default.
• W2082952610 title "Cloning, Localization, and Functional Expression of Sodium Channel β1A Subunits" @default.
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