Calpastatin and nucleotides stabilize cardiac calcium channel activity in excised patches

The activity of single L-type Ca2+ channels is rapidly lost (run-down) when contact between the membrane and cytosol is interrupted. We have now achieved the stabilization of cardiac Ca2+ channel activity of guinea-pig ventricular myocytes by using either cytosol or defined components added to excised patches. The endogenous protease inhibitor, calpastatin, together with nucleotides, ATP + GTP, was found to prevent run-down as effectively as cardiac cytosolic solution. These results suggest the involvement of proteolysis by calpain in run-down of channel activity and enable the study of cardiac Ca2+ channel regulation with free access to both sides of the membrane.