Phosphorylation of the Consensus Sites of Protein Kinase A on α1D L-type Calcium Channel

Mohamed Boutjdir,Mohamed Chahine,Raj Wadgaonkar,Ghayath Baroudi,Yongxia Qu,Eddy Karnabi,Omar Ramadan

doi:10.1074/jbc.m809132200

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.

Highlights

The novel ␣1D L-type Ca2؉ channel is expressed in supraventricular tissue and has been implicated in the pacemaker activity of the heart and in atrial fibrillation
The distal C terminus of the ␣1 subunit of ␣1D Ca2ϩ channel showed phosphorylation when probed with the anti-PKA substrate (Fig. 3A) and miniwhich of the potential PKA consensus sites is phosphorylated mal when probed with anti-phosphoserine antibodies (Fig. 3B)
By PKA, constructs of GST fusion protein were designed in These experiments were repeated four times, each giving identhree more non-PKA consensus sites not identified by scansite and these were: serine 1703, serine

Summary

EXPERIMENTAL PROCEDURES

Subcloning of Intracellular Loops, N Terminus, Proximal or Distal C Terminus of the ␣1 Subunit of the Rat ␣1D Ca2ϩ Channel into pGEX-6P-1 Vector—pCMV6b/rat ␣1D plasmid was kindly provided by Dr Susumu Seino from Kobe University, Japan. Two sets of primers were used to amplify each of the intracellular loops, proximal, or distal C terminus of ␣1 subunit of rat ␣1D Ca2ϩ channel. The mixture consisted of 5 ␮l of 5ϫ cloning of the N terminus of ␣1 subunit of rat ␣1D Ca2ϩ channel reaction buffer, 2 ng of recombinant PKA catalytic subunit, 7.5 into pGEX 6P-1 vector, the forward primer had a BamHI site, ␮l of double distilled water, and diluted 1:1 magnesium/ATP and the reverse primer had an EcoRI site. The side as described before (Handbook of GST Gene Fusion Sys- membrane was probed with mouse monoclonal anti-PKA subtem, 18-1157-58, Amersham Biosciences). The following mutations were introduced by mutagenic primers: S1743A, S1816A, and S1964A

The presence of mutations was confirmed by sequencing at the

RESULTS

Mass spectrometry covered

DISCUSSION