Developing a reconstructing system that compensates for a missing link in the regulation of L-type Ca channels in cardiac myocytes

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The channel activity is augmented by phsophorylation through cAMP-dependent kinase : maximum activation of phosphorylation can increase peak amplitude of Ca^<2+> currents up to 5-6 folds of the control current. The fact that inability to reconstruct the regulation of L-type Ca channels through cAMP-dependent phosphorylation by transducing L-type Ca channel genes in heterologous expression system hampers the development of elucidating molecular mechanisms in the regulation of L-type Ca channels. We assume that heterologous expression systems lack a regulatory factor in controlling the activity of L-type Ca channels from cardiac origin which should exist in native cardiac cells. Thus, our strategy to fully reconstruct the regulation of L-type Ca channel is transducing L-type Ca channel genes into cardiac myocyte itself. In order to achieve these objects, we have two problems to overcome, 1) differentiating function of artificially introduced Ca channels from those originally expressed in … More cardiac myocytes, 2) obtaining functional virus vectors containing L-type Ca channel gene. For the first one, we tested the ion selectivity of mutated L-type Ca channels, such as EEKA or DEKA type of which the portion of the ion selective filter was changed from EEEE of wild type. The results indicated that both mutants have 10 times higher permeability of Na^+ against Cs^+. Ba^<2+> permeability was abolished in DEKA type, while EEKA type has 56 times higher permeability of Ba^<2+> against Cs^+. DEKA type acquired resistance to La^<3+> block. With using this Na^+-selective Ca channel mutants, we would be able to selectively record currents from transduced L-type Ca channels in cardiac myocytes in the presence of tetrodotoxin. For the second one, we could insert Ca_V 1.2 gene in the adenovirus vector that lacks E1/E3 region. Unfortunately, we were unable to amplify virus particles in HEK293 cells due to the size of insert (8kb). A recent report of Ganeasn AN et al.indicated that they could successfully transduce Ca_V1.2 gene in cardiac myocytes by constructing adenovirus vector that lacks fiber gene in addition to E1/E3 region (BBRC 749-754,2005). We are hoping to introduce their technology for further development of our Ca channel study. Less