Searching responsible sites for the regulation of the L-type Ca channel through phosphorylation

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[Overview]The ultimate aim of this study is to identify responsible sites for the regulations of L-type Ca channels in the heart, such as intracellular Mg^<2+> block, or enhancement of channel activity accelerated by phsphorylation. As a result, we found that something is lacking to fully reconstruct channel activity only using L-type Ca channels with non-cardiac cells. Especcially, Mg^<2+> response was totally different from the behavior found in native cardiac cells. Depletion of intracellular Mg^<2+> led to the rapid wash out of channel activity in reconstructed systems, while in native cardiac cells, depletion of intracellular Mg^<2+> normally causes prominent increase in Ca channel current (I_<ca>). This indicates the requirement of extensive improvement of reconstruction systems regarding host cell conditions[Results]1. Increase in I_<ca> responding to reduction in intracellular Mg^<2+> was disturbed in guinea-pig cardiac cells, when temperature was below 28℃. This temperature dependency was not explained by simple thermodynamic activities.2. We identified whole length of cDNAs encoding α_1, β_<2C> and α_<2/δ> subunits of the L-type Ca channel in frog ventricular cells. They exhibited more than 80 % homology of those reported in rat or mouse. Co-expression of all subunits of α_1, β_<2C> and α_<2/δ> in BHK cells exhibited functional L-type Ca channel activity.3. Reconstructed Ca channels in BHK cells rapidly ran down, when cells were dialysed with log Mg^<2+> (below 10^<-5>M) patch solutions below 10^<-5>M. This is in contrast to data found in native cardiac cells. We need to search unkown proteins to accommodate the lacking of important regulations of the channel.