TRYPSIN ON CARDIAC ATP-SENSITIVE K+ CHANNELS

Treatment with trypsin of the cytoplasmic surface of excised inside-out membrane patches from guinea pig ventricular myocytes altered multiple regulatory properties of ATP-sensitive K+ (K-ATP) channels including 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 intracellular Mg2+) were unaltered after trypsin treatment. K-ATP channels also remained sensitive to intracellular Ca2+-induced rundown after trypsin treatment (n = 6). The effects of trypsin were not prevented by 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 occupancy of these binding sites did not prevent access of trypsin to the 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.