Characterization ofthetwosizeformsofthealsubunit ofskeletal muscleL-type calcium channels

Abstract

Themolecular properties oftwosize forms of thealsubunit ofpurified skeletal muscle calcium channels wereanalyzed. Theminor, full-length, form, a1212, wasfound tohaveanapparent molecular massof214kDabyFerguson plot analysis, while themajor, truncated, form, nowdesignated al1o, hadanapparent molecular massof193kDa.Antibody mapping oftheC-terminal region ofallg0 with10anti-peptide antibodies placed theC terminus between residues 1685and 1699. Three consensus sites forcAMP-dependent protein phos- phorylation arepresent intheC-terminal region ofa1212 but notinal1g, andtheymaybeimportant fortheregulation of theionconductance activity ofthecalcium channel. Fourclasses ofvoltage-gated calcium channels havebeen defined onthebasis ofelectrophysiological andpharmaco- logical properties (1). L-type calcium channels mediate long- lasting calcium currents that aresensitive toinhibition by dihydropyridines (DHPs). Mostbiochemical studies have concentrated ontheL-type calcium channel fromskeletal muscle transverse tubules (T-tubules), whichcontain 50-to 100-fold moreDHP receptor sites thanother sources. The mostabundant form'4therabbit skeletal muscle calcium channel isacomplex offive subunits: al(175 kDa), a2(143 kDa), , (54kDa), y(30kDa), and8(24-27 kDa)(reviewed in refs. 2-4). Theal,A,andysubunits areproducts ofdistinct genes(5-7), while thea2and8subunits areencoded bythe samegene(8), whoseprotein product isproteolytically processed anddisulfide-linked togive thea28complex (9). Thealsubunit contains thereceptor sites forDHPsandother calcium channel modulating drugs. Thesequence ofits cDNApredicts aprotein of1873 amino acids (212 kDa)whose structure ishomologous tothesodium channel asubunit, with four internally homologous domains eachcontaining six predicted a-helical transmembrane segments (5). Thissub- unit alone issufficient toformfunctional calcium channels whenexpressed inmousefibroblast cells (10). TheDHP-sensitive calcium channel protein inskeletal muscle hasalso beenproposed toserve asthevoltage sensor forexcitation-contr action coupling, aprocess that doesnot