INHIBITION OF CLONED HUMAN L-TYPE CARDIAC CALCIUM CHANNELS BY 2,3-BUTANEDIONE MONOXIME DOES NOT REQUIRE PKA-DEPENDENT PHOSPHORYLATION SITES

The oxime derivative 2,3-butanedione monoxime (BDM) is used as an inor ganic phosphatase to probe the phosphorylation state of many cellular proteins including the L-type calcium channel in various tissues. We u sed BDM further to shed light on the controversy surrounding direct ph osphorylation of the L-type Ca2+ channel. We employed a recombinant sy stem that utilizes HEK 293 cells expressing wild type and mutant human heart calcium channels. BDM reversibly reduced the calcium channel cu rrent induced by expression of the wild type channel in a concentratio n-dependent manner with an apparent IC50 value of 15.3 mM. Deletion of part of the carboxyl terminus of the alpha(1) subunit, which contains one putative protein kinase A site, or mutating all of the protein ki nase A consensus sites of the pore forming subunit, did not significan tly change the apparent IC50 value or alter in any other way the block ing effect of BDM on the expressed currents. Our data suggest that BDM produces reversible modifications of the cardiac calcium channel prot ein leading to an expected reduction in the amplitude of the expressed currents, but the site of action must be different from that of the c onsensus sites for protein kinase A dependent phosphorylation. (C) 199 7 Academic Press.