Modification of the adenosine 5'-triphosphate-sensitive K+ channel by trypsin in guinea-pig ventricular myocytes.
1. The adenosine 5'-triphosphate (ATP)-sensitive K+ channel current was recorded in guinea-pig ventricular myocytes using the patch clamp technique with inside-out patch configuration. Modification of the channel activity by intracellular application of an endoprotease trypsin was studied, and was related to a possible model of regulation of this channel. 2. Maximal ATP-sensitive K+ channel activity was observed immediately upon formation of inside-out patches in the ATP-free internal solution, thereafter activity declined both spontaneously and gradually with time; a phenomenon known as rundown. When trypsin (1 mg/ml) was applied to the intracellular side of the membrane upon formation of inside-out patches, spontaneous run-down did not occur, and this trypsin action was irreversible. Neither trypsin (1 mg/ml) applied with trypsin 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 applied Ca2+ induced run-down of channel activity in a dose-dependent manner. In membrane patches that were treated with trypsin (1 mg/ml) for 20 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 Ca(2+)-induced run-down of channel activity. 6. As postulated for several other channels, such as Na+ and Ca2+ channels, there may be a possible 'chemical gate' that is responsible for run-down of this channel activity. Application of trypsin might somehow modify this 'chemical gate', resulting in prevention of spontaneous or Ca(2+)-induced run-down. This target site for trypsin may be situated on the carboxy-terminus of the channel proteins, or of associated regulatory units. Because ATP sensitivity remained intact after trypsin treatment, the trypsin-selective site for channel inhibition is not related physically to the ATP binding site.
Furukawa
T
T
Department of Cardiovascular Diseases, Tokyo Medical and Dental University, Japan.
Fan
Z
Z
Sawanobori
T
T
Hiraoka
M
M
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002268:Carboxypeptidases / Q000494:pharmacology; D043422:Carboxypeptidases A; D004351:Drug Resistance; D004553:Electric Conductivity; D004988:Ethers, Cyclic / Q000494:pharmacology; D006168:Guinea Pigs; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D009206:Myocardium / Q000166:cytology / Q000378:metabolism*; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors; D015221:Potassium Channels / Q000187:drug effects* / Q000378:metabolism; D014357:Trypsin / Q000494:pharmacology*
D004988:Ethers, Cyclic; D015221:Potassium Channels; D019319:Okadaic Acid; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D010749:Phosphoprotein Phosphatases; D002268:Carboxypeptidases; D043422:Carboxypeptidases A; D014357:Trypsin; D002118:Calcium