Dataset Information

Rat brain expresses an alternatively spliced form of the dihydropyridine-sensitive L-type calcium channel alpha 2 subunit.

ABSTRACT: We have cloned and sequenced a cDNA (rB-alpha 2) encoding an alpha 2 subunit of the voltage-sensitive L-type calcium channel (dihydropyridine receptor) of rat brain. The cDNA (3823 base pairs) encodes a protein of 1091 amino acids with a Mr of 123,822. The deduced amino acid sequence of rB-alpha 2 cDNA is highly similar (95% amino acid identity) to that of rabbit skeletal muscle alpha 2 subunit. The rB-alpha 2 protein is distinct from the previously cloned skeletal muscle alpha 2-subunit protein [Ellis, S. B., Williams, M. E., Ways, N. R., Brenner, R., Sharp, A. H., Leung, A. T., Campbell, K. P., McKenna, E., Koch, W., Hui, A., Schwartz, A. & Harpold, M. M. (1988) Science 241, 1661-1664] because it contains an insertion of 7 amino acid residues and a deletion of a 19-amino acid segment between putative transmembrane domains 1 and 2. We show that the rB-alpha 2 and skeletal muscle alpha 2-subunit transcripts are the variants produced by alternative splicing of the primary transcript and that they are differentially expressed in brain and skeletal muscle, respectively.

SUBMITTER: Kim HL

PROVIDER: S-EPMC48844 | biostudies-other | 1992 Apr

REPOSITORIES: biostudies-other

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Rat brain expresses an alternatively spliced form of the dihydropyridine-sensitive L-type calcium channel alpha 2 subunit.

Kim H L HL Kim H H Lee P P King R G RG Chin H H

Proceedings of the National Academy of Sciences of the United States of America 19920401 8

We have cloned and sequenced a cDNA (rB-alpha 2) encoding an alpha 2 subunit of the voltage-sensitive L-type calcium channel (dihydropyridine receptor) of rat brain. The cDNA (3823 base pairs) encodes a protein of 1091 amino acids with a Mr of 123,822. The deduced amino acid sequence of rB-alpha 2 cDNA is highly similar (95% amino acid identity) to that of rabbit skeletal muscle alpha 2 subunit. The rB-alpha 2 protein is distinct from the previously cloned skeletal muscle alpha 2-subunit protein ...[more]

PMID: 1314383

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Project description:BackgroundThe amiloride-sensitive Epithelial Sodium Channel (ENaC) is critical in maintaining Na+ balance, extracellular fluid volume and long term blood pressure control. ENaC is composed of three main subunits alpha, beta, & gamma. While alpha ENaC is critical for channel functionality, beta & gamma ENaC maximize channel function. To date, there are four alternatively spliced forms of the alpha subunit of ENaC (alpha ENaC-a, -b, -c, & -d) that have been published in rats, in addition to the major alpha ENaC transcript. While alpha ENaC-a, -c & -d transcripts are low abundance transcripts compared to full-length alpha ENaC, alpha ENaC-b is a higher abundance and salt-sensitive transcript compared to full-length alpha ENaC.Presentation of the hypothesisalpha ENaC-b protein, which is preferentially produced in Dahl R rats, to a greater extent on high salt diet, exerts a dominant negative effect on full-length alpha ENaC subunit by physically binding to and trapping full-length alpha ENaC subunit in the endoplasmic reticulum, and finally accelerating full-length alpha ENaC proteolytic degradation in a dose-dependent manner.Testing the hypothesis1) To examine the mRNA and protein abundance of alpha ENaC-b relative to alpha ENaC full-length in kidney, lung, and taste tissues of Dahl rats. 2) To compare the expression (mRNA and protein) of alpha ENaC-b in kidneys of Dahl S and R rats on regular and high salt diet. 3) To examine the putative binding of alpha ENaC-b proteins to full-length alpha ENaC in vitro and to determine the impact of such binding on full-length alpha ENaC expression in vitro.Implications of the hypothesisOur studies will be the first to demonstrate the over-expression of salt-sensitive alpha ENaC-b spliced form in kidney tissues of Dahl R rats at the expense of full-length alpha ENaC. The current proposal will provide highly novel insights into the putative mechanisms leading to ENaC hypoactivity in high-salt-fed Dahl R rats. Finally, findings from the present proposal will uncover a new mechanism by which alternative splicing may control the regulation of ENaC expression/function.

Project description:We have examined the regulation of mutually exclusive Cav2.2 exon 37a and b variants by the mouse ?-opioid receptor (mMOR) C-terminal splice variants 1, 1C and 1O in tsA-201 cells. Electrophysiological analyses revealed that both channel isoforms exhibit DAMGO-induced voltage-dependent (G??-mediated) inhibition and its recovery by voltage pre-pulses, as well as a voltage-independent component. However, the two channel isoforms differ in their relative extent of voltage-dependent and independent inhibition, with Cav2.2-37b showing significantly more voltage-dependent inhibition upon activation of the three mMOR receptors studied. In addition, coexpression of either mMOR1 or mMOR1C results in an agonist-independent reduction in the peak current density of Cav2.2-37a channels, whereas the peak current density of Cav2.2-37b does not appear to be affected. Interestingly, this decrease is not due to an effect on channel expression at the plasma membrane, as demonstrated by biotinylation experiments. We further examined the mechanism underlying the agonist-independent modulation of Cav2.2-37a by mMOR1C. Incubation of cells with pertussis toxin did not affect the mMOR1C mediated inhibition of Cav2.2-37a currents, indicating a lack of involvement of Gi/o signaling. However, when a Src tyrosine kinase inhibitor was applied, the effect of mMOR1C was lost. Moreover, when we recorded currents using a Cav2.2-37a mutant in which tyrosine 1747 was replaced with phenylalanine (Y1747F), the agonist independent effects of mMOR1C were abolished. Altogether our findings show that Cav2.2-37a and Cav2.2-37b isoforms are subject to differential regulation by C-terminal splice variants of mMORs, and that constitutive mMOR1C activity and downstream tyrosine kinase activity exert a selective inhibition of the Cav2.2-37a splice variant, an N-type channel isoform that is highly enriched in nociceptors. Our study provides new insights into the roles of the MOR full-length C-terminal variants in modulating Cav2.2 channel isoform activities.

Dataset Information

Rat brain expresses an alternatively spliced form of the dihydropyridine-sensitive L-type calcium channel alpha 2 subunit.

Publications

Rat brain expresses an alternatively spliced form of the dihydropyridine-sensitive L-type calcium channel alpha 2 subunit.

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