MODIFICATION OF THE ADENOSINE 5'-TRIPHOSPHATE-SENSITIVE K-PIG VENTRICULAR MYOCYTES( CHANNEL BY TRYPSIN IN GUINEA)

1. The adenosine 5'-triphosphate (ATP)-sensitive K+ channel current wa s recorded in guinea-pig ventricular myocytes using the patch clamp te chnique with inside-out patch configuration. Modification of the chann el activity by intracellular application of an endoprotease trypsin wa s studied. and was related to a possible model of regulation of this c hannel. 2. Maximal ATP-sensitive K+ channel activity was observed imme diately upon formation of inside-out patches in the ATP-free internal solution, thereafter activity declined both spontaneously and graduall y with time ; a phenomenon known as rundown. When trypsin (1 mg/ml) wa s applied to the intracellular side of the membrane upon formation of inside-out patches, spontaneous run-down did not occur, and this tryps in action was irreversible. Neither trypsin (1 mg/ml) applied with try psin inhibitor (0.25 mg/ml) nor heat-denatured trypsin (1 mg/ml) could mimic this effect. When trypsin was applied to the patches after run- down, channels were reactivated at approximately 13 min. 3. Treatment with trypsin did not affect unitary current amplitude, channel gating kinetics, or sensitivity to intracellular ATP. 4. Intracellularly appl ied Ca2+ induced run-down of channel activity in a dose-dependent mann er. In membrane patches that were treated with trypsin (1 mg/ml) for 2 0 min, intracellularly applied Ca2+ Up to 1 mm did not induce run-down of channel activity. 5. Intracellular application of an exopeptidase, carboxypeptidase A (1 mg/ml), but not Leu-aminopeptidase (0.5 mg/ml), prevented spontaneous or Ca2+-induced run-down of channel activity. 6 . As postulated for several other channels, such as Na+ and Ca2+ chann els, there may be a possible 'chemical gate' that is responsible for r un-down of this channel activity. Application of trypsin might somehow modify this 'chemical gate', resulting in prevention of spontaneous o r Ca2+-induced run down. This target site for trypsin may be situated on the carboxy-terminus of the channel proteins, or of associated regu latory units. Because ATP sensitivity remained intact after trypsin tr eatment, the trypsin-selective site for channel inhibition is not rela ted physically to the ATP binding site.