title: The alpha1B Ca2+ channel amino terminus contributes determinants for beta subunit-mediated voltage-dependent inactivation properties.
abstract: Co-expression of auxiliary beta subunits with the alpha1B Ca2+ channel subunit in COS-7 cells resulted in an increase in current density and a hyperpolarising shift in the mid-point of activation. Amongst the beta subunits, beta2a in particular, but also beta4 and beta1b caused a significant retardation of the voltage-dependent inactivation compared to currents with alpha1B alone, whilst no significant changes in inactivation properties were seen for the beta3 subunit in this system. Prevention of beta2a palmitoylation, by introducing cysteine to serine mutations (beta2a(C3,4S)), greatly reduced the ability of beta2a to retard voltage-dependent inactivation. Deletion of the proximal half of the alpha1B cytoplasmic amino terminus (alpha1BDelta1-55) differentially affected beta subunit-mediated voltage-dependent inactivation properties. These effects were prominent with the beta2a subunit and, to a lesser extent, with beta1b. For beta2a, the major effects of this deletion were a partial reversal of beta2a-mediated retardation of inactivation and the introduction of a fast component of inactivation, not seen with full-length alpha1B. Deletion of the amino terminus had no other major effects on the measured biophysical properties of alpha1B when co-expressed with beta subunits. Transfer of the whole alpha1B amino terminus into alpha1C (alpha1bCCCC) conferred a similar retardation of inactivation on alpha1C when co-expressed with beta2a to that seen in parental alpha1B. Individual (alpha1B(Q47A) and alpha1B(R52A)) and double (alpha1B(R52,54A)) point mutations within the amino terminus of alpha1B also opposed the beta2a-mediated retardation of alpha1B inactivation kinetics. These results indicate that the alpha1B amino terminus contains determinants for beta subunit-mediated voltage-dependent inactivation properties. Furthermore, effects were beta subunit selective. As deletion of the alpha1B amino terminus only partially opposed beta subunit-mediated changes in inactivation properties, the amino terminus is likely to contribute to a complex site necessary for complete beta subunit function.
authors.affiliation: Department of Pharmacology, University College London, London WC1E 6BT, UK.
authors.forename: G J
authors.initials: GJ
authors.lastname: Stephens
authors.forename: K M
authors.initials: KM
authors.lastname: Page
authors.forename: Y
authors.initials: Y
authors.lastname: Bogdanov
authors.forename: A C
authors.initials: AC
authors.lastname: Dolphin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D019556:COS Cells; D015220:Calcium Channels / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D008564:Membrane Potentials; D016297:Mutagenesis, Site-Directed; D020836:Protein Structure, Quaternary; D011817:Rabbits; D051381:Rats; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D017384:Sequence Deletion
chemical_list: D015220:Calcium Channels; D017931:DNA Primers; D011994:Recombinant Proteins