TRYPSIN ON CARDIAC ATP-SENSITIVE K+ CHANNELS

Treatment with trypsin of the cytoplasmic surface of excised inside-ou t membrane patches from guinea pig ventricular myocytes altered multip le regulatory properties of ATP-sensitive K+ (K-ATP) channels includin g their sensitivity to intracellular ATP (ATP(i)), intracellular ADP ( ADP(i)), glibenclamide, and cromakalim. The single-channel conductance , reversal potential, and inward rectification (in the presence of int racellular Mg2+) were unaltered after trypsin treatment. K-ATP channel s also remained sensitive to intracellular Ca2+-induced rundown after trypsin treatment (n = 6). The effects of trypsin were not prevented b y including either 15 mM ATP(i) (n = 7), 1 mM ADP(i) (n = 4), or 10 mu M glibenclamide (n = 4) during exposure to trypsin, suggesting that o ccupancy of these binding sites did not prevent access of trypsin to t he proteolytic sites responsible for its effects. Treatment of excised membrane patches with 1 mM phenylglyoxal (n = 4) or 5 mM glyoxal (n = 4), which cleave polypeptides at arginine residues, did not increase the dissociation constant for suppression of K-ATP channels by ATP(i). Because trypsin cleaves peptides at both arginine and lysine residues , these results suggest that modification of the regulatory properties of K-ATP channels by trypsin may result from proteolytic digestion of lysine residues located in cytosolic regions of the channel protein.