Modification of the adenosine 5'-triphosphate-sensitive K+ channel by trypsin in guinea-pig ventricular myocytes

• Published in: The Journal of physiology 1993-07, Vol.466 (1), p.707-726
• Main Authors: Furukawa, T, Fan, Z, Sawanobori, T, Hiraoka, M
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - analogs & derivatives; Adenosine Triphosphate - pharmacology; Animals; Biological and medical sciences; Calcium - pharmacology; Carboxypeptidases - pharmacology; Carboxypeptidases A; Drug Resistance; Electric Conductivity; Ethers, Cyclic - pharmacology; Fundamental and applied biological sciences. Psychology; Guinea Pigs; Heart; Heart Ventricles - cytology; Heart Ventricles - metabolism; In Vitro Techniques; Kinetics; Myocardium - cytology; Myocardium - metabolism; Okadaic Acid; Phosphoprotein Phosphatases - antagonists & inhibitors; Potassium Channels - drug effects; Potassium Channels - metabolism; Trypsin - pharmacology; Vertebrates: cardiovascular system
• ISSN: 0022-3751; 1469-7793
• DOI: 10.1113/jphysiol.1993.sp019741

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.