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 current to a level similar to beta-adrenergic stimulation. Using transiently transfected HEK 293 cells, we studied the molecular mechanism underlying calcium channel stimulation by proteolytic treatment. Perfusion of HEK cells, coexpressing the human cardiac (hHT) alpha(1), alpha(2), and beta(3) subunits, with 1 mg/ml of trypsin or carboxypeptidase A, increased the peak amplitude of the calcium channel current 3-4-fold without affecting the voltage dependence. Similar results were obtained in HEK cells cotransfected with hHT alpha(1) and alpha(2) or with alpha(1) alone, suggesting that modification of the alpha(1) subunit itself is responsible for the current enhancement by proteolysis. To further characterize the modification of the alpha(1) subunit by trypsin, we expressed a deletion mutant in which part of the carboxyl-terminal tail up to amino acid 1673 was removed. The expressed calcium channel currents no longer responded to intracellular application of the proteases; however, a 3-fold higher current density as well as faster inactivation compared with the wild type was observed. The results provide evidence that a specific region of the carboxyl-terminal tail of the cardiac alpha(1) subunit is an important regulatory segment that may serve as a critical component of the gating machinery that influences both inactivation properties as well as channel availability.