title: Phosphorylation of the consensus sites of protein kinase A on alpha1D L-type calcium channel.
abstract: The novel alpha(1D) L-type Ca(2+) channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation. We recently demonstrated that PKA activation led to increased alpha(1D) Ca(2+) channel activity in tsA201 cells by phosphorylation of the channel protein. Here we sought to identify the phosphorylated PKA consensus sites on the alpha(1) subunit of the alpha(1D) Ca(2+) channel by generating GST fusion proteins of the intracellular loops, N terminus, proximal and distal C termini of the alpha(1) subunit of alpha(1D) Ca(2+) channel. An in vitro PKA kinase assay was performed for the GST fusion proteins, and their phosphorylation was assessed by Western blotting using either anti-PKA substrate or anti-phosphoserine antibodies. Western blotting showed that the N terminus and C terminus were phosphorylated. Serines 1743 and 1816, two PKA consensus sites, were phosphorylated by PKA and identified by mass spectrometry. Site directed mutagenesis and patch clamp studies revealed that serines 1743 and 1816 were major functional PKA consensus sites. Altogether, biochemical and functional data revealed that serines 1743 and 1816 are major functional PKA consensus sites on the alpha(1) subunit of alpha(1D) Ca(2+) channel. These novel findings provide new insights into the autonomic regulation of the alpha(1D) Ca(2+) channel in the heart.
authors.affiliation: Veterans Administration, New York Harbor Healthcare System, Brooklyn, New York 11209, USA.
authors.forename: Omar
authors.initials: O
authors.lastname: Ramadan
authors.forename: Yongxia
authors.initials: Y
authors.lastname: Qu
authors.forename: Raj
authors.initials: R
authors.lastname: Wadgaonkar
authors.forename: Ghayath
authors.initials: G
authors.lastname: Baroudi
authors.forename: Eddy
authors.initials: E
authors.lastname: Karnabi
authors.forename: Mohamed
authors.initials: M
authors.lastname: Chahine
authors.forename: Mohamed
authors.initials: M
authors.lastname: Boutjdir
mesh_terms: D000818:Animals; D015220:Calcium Channels / Q000235:genetics / Q000378:metabolism*; D016384:Consensus Sequence / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000235:genetics / Q000378:metabolism*; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009206:Myocardium / Q000378:metabolism*; D010766:Phosphorylation; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D051381:Rats
chemical_list: D015220:Calcium Channels; D009124:Muscle Proteins; D021122:Protein Subunits; C093712:Cacna1d protein, rat; D017868:Cyclic AMP-Dependent Protein Kinases