Role of phosphorylation of the alpha one subunit in cyclic adenosine monophosphate dependent modulation of skeletal muscle calcium channels

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Role of phosphorylation of the alpha one subunit in cyclic adenosine monophosphate dependent modulation of skeletal muscle calcium channels

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Title: Role of phosphorylation of the alpha one subunit in cyclic adenosine monophosphate dependent modulation of skeletal muscle calcium channels
Author: Brousal, Jeffrey P
Abstract: Phosphorylation of the skeletal muscle $\alpha\sb1$ subunit of the L-type calcium channel has been proposed to regulate the calcium current, allowing increased calcium flux during tetanus and during $\beta$-adrenergic activation. This stimulation has been shown to increase skeletal muscle contraction and calcium channel activity through cAMP-dependent phosphorylation of serine and threonine amino acid residues. The molecular mechanism of this phosphorylation has been studied by altering the serine and threonine residues to alanine to prevent phosphate transfer. These mutants have been analysed biochemically and physiologically for changes in calcium channel regulation.Oligonucleotide directed mutagenesis was used to alter serine and threonine amino acid residues to alanine in consensus sequences for phosphorylation by cAMP-dependent protein kinase. This prevented the incorporation of phosphate into this molecule. These wild type and mutant cDNAs were transiently tranfected into a heterologous expression system with the rat brain L-type calcium channel $\beta\sb{\rm 1b}$ subunit and the rabbit skeletal muscle L-type calcium channel $\alpha\sb2/\delta$ subunit in human embryonic kidney cells expressing the large T antigen. This system allowed analysis of protein expression, protein phosphorylation and electrophysiology.The calcium channels expressed dihydropyridine binding sites with K$\sb{\rm d}$ at 136-215 pM and $\beta$max at 141 to 718 fmol/mg protein. The wild type protein had a molecular mass of 212 kDa and the truncated mutant protein had a molecular mass of 190 kDa in western blot analysis. The wild type protein had normal in vivo and in vitro cAMP-dependent protein kinase phosphorylation. A mutant protein containing 17 amino acid changes was not phosphorylated by cAMP-dependent protein kinase in vivo or in vitro.The functional properties of wild-type and mutant channels and their regulation by cAMP-dependent protein kinase were studied by analysis of tail currents with barium as the permeant ion. These measurements showed no change in the wild type or mutant channel in the absence or presence of the activating agent, dcl-cBIMPS. This shows that the $\alpha\sb1$ subunit of the rabbit skeletal muscle calcium channel is not the target of phosphorylation for the phenomena of increased calcium channel activity due to $\beta$-adrenergic stimulation.
Description: Thesis (Ph. D.)-University of Washington, 1998
URI: http://hdl.handle.net/1773/6305

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