Abstract
Spontaneous, rhythmic subsarcolemmal local Ca(2+) releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). Because local Ca(2+) releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca(2+) and cAMP/PKA regulatory components of automaticity are coupled via Ca(2+) activation of AC activity within membrane microdomains. Here we show by quantitative reverse transcriptase PCR that SANC express Ca(2+)-activated AC isoforms 1 and 8, in addition to AC type 2, 5, and 6 transcripts. Immunolabeling of cell fractions, isolated by sucrose gradient ultracentrifugation, confirmed that ACs localize to membrane lipid microdomains. AC activity within these lipid microdomains is activated by Ca(2+) over the entire physiological Ca(2+) range. In intact SANC, the high basal AC activity produces a high level of cAMP that is further elevated by phosphodiesterase inhibition. cAMP and cAMP-mediated PKA-dependent activation of ion channels and Ca(2+) cycling proteins drive sarcoplasmic reticulum Ca(2+) releases, which, in turn, activate ACs. This feed forward "fail safe" system, kept in check by a high basal phosphodiesterase activity, is central to the generation of normal rhythmic, spontaneous action potentials by pacemaker cells.
Highlights
Spontaneous, rhythmic subsarcolemmal local Ca2؉ releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC)
Because local Ca2؉ releases occur beneath the cell surface membrane, near to where adenylyl cyclases (ACs) reside, we hypothesized that the dual Ca2؉ and cAMP/PKA regulatory components of automaticity are coupled via Ca2؉ activation of AC activity within membrane microdomains
Using multiple approaches we show that both Ca2ϩ-regulated ACs reside in lipid microdomains and that Ca2ϩ activation of AC activity occurs within these domains
Summary
Spontaneous, rhythmic subsarcolemmal local Ca2؉ releases driven by cAMP-mediated, protein kinase A (PKA)-dependent phosphorylation are crucial for normal pacemaker function of sinoatrial nodal cells (SANC). CAMP-mediated, PKA-dependent phosphorylation of surface membrane ion channels and SR Ca2ϩ cycling proteins control the SANC basal spontaneous rhythmic firing [2].
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