The regulation of cardiac L-type voltage-dependent calcium channel function by cAMP-dependent protein kinase mediated phosphorylation: Biochemical studies of heterologously expressed and native channels.

摘要

Cardiac L-type Ca{dollar}sp{lcub}+2{rcub}{dollar} channels were the first ion channels discovered to regulated by neurotransmitters and have since become a paradigm for protein kinase-mediated neurotransmitter regulation of ion channel function. However, due to the low density of L-type channels in cardiac and neuronal tissues, very little information is known regarding the specific biochemical reactions involved in regulation of these channels by protein phosphorylation. In this study we have expressed the predicted cardiac isoforms of L-type channel subunits in Sf9 insect cells by infection with recombinant baculoviruses. Heterologous expression of channel proteins allowed us to overcome the problem of low channel density in native tissues, and thus facilitated heretofore difficult biochemical studies. Initial studies demonstrated that each channel subunit was appropriately expressed by insect cells and, upon co-expression, assembled into complexes with properties of functional channels. The expressed {dollar}alphasb{lcub}rm 1C{rcub}{dollar} and {dollar}betasb{lcub}rm 2a{rcub}{dollar} subunits were both shown to be phosphorylated by purified PKA and PKC in vitro at stoichiometric levels. These results suggested that either or both the {dollar}alphasb{lcub}rm 1C{rcub}{dollar} and {dollar}betasb{lcub}rm 2a{rcub}{dollar} subunit may be involved in protein kinase-mediated regulation of channel function. The subunit composition of native cardiac L-type channels in freshly isolated rabbit ventricular myocytes was studied using a panel of subunit-specific antibodies. Subsequently, native cardiac {dollar}alphasb{lcub}rm 1C{rcub}{dollar} and {dollar}betasb2{dollar} subunits were characterized as substrates for PKA in intact myocytes by back phosphorylation. Finally, using both cardiac myocytes and the Sf9 cell expression system, critical issues regarding the status of the {dollar}alphasb{lcub}rm 1C{rcub}{dollar} carboxyl-terminus were addressed. In particular, our studies suggested that the observed carboxyl-terminal truncation of a majority of the native {dollar}alphasb{lcub}rm 1C{rcub}{dollar} protein is not likely a result of artifactual proteolysis that occurs upon myocyte lysis. In vitro proteolytic cleavage assays of the expressed {dollar}alphasb{lcub}rm 1C{rcub}{dollar} subunit were developed and may provide a method by which to investigate the potential mechanisms involved in processing of the native {dollar}alphasb{lcub}rm 1C{rcub}{dollar} carboxyl-terminus and the implications of processing on channel function. In summary, our results provide important new information regarding the ability of channel proteins to serve as substrates for protein kinases and the status of the {dollar}alphasb{lcub}rm 1C{rcub}{dollar} carboxyl-terminus in native cardiac L-type Ca{dollar}sp{lcub}+2{rcub}{dollar} channels.