INVOLVEMENT OF THE CARBOXYL-TERMINAL REGION OF THE ALPHA(1) SUBUNIT IN VOLTAGE-DEPENDENT INACTIVATION OF CARDIAC CALCIUM CHANNELS

Intracellular application of proteases increases cardiac calcium curre nt to a level similar to beta-adrenergic stimulation. Using transientl y transfected HEK 293 cells, we studied the molecular mechanism underl ying calcium channel stimulation by proteolytic treatment. Perfusion o f HEK cells, coexpressing the human cardiac (hHT) alpha(1), alpha(2), and beta(3) subunits, with 1 mg/ml of trypsin or carboxypeptidase A, i ncreased the peak amplitude of the calcium channel current 3-4-fold wi thout affecting the voltage dependence. Similar results were obtained in HEK cells cotransfected with hHT alpha(1) and alpha(2) or with alph a(1) alone, suggesting that modification of the alpha(1) subunit itsel f is responsible for the current enhancement by proteolysis. To furthe r characterize the modification of the alpha(1) subunit by trypsin, we expressed a deletion mutant in which part of the carboxyl-terminal ta il up to amino acid 1673 was removed. The expressed calcium channel cu rrents no longer responded to intracellular application of the proteas es; however, a 3-fold higher current density as well as faster inactiv ation compared with the wild type was observed. The results provide ev idence that a specific region of the carboxyl-terminal tail of the car diac alpha(1) subunit is an important regulatory segment that may serv e as a critical component of the gating machinery that influences both inactivation properties as well as channel availability.