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Modification of the adenosine 5'‐triphosphate‐sensitive K+ channel by trypsin in guinea‐pig ventricular myocytes.
Author(s) -
Furukawa T,
Fan Z,
Sawanobori T,
Hiraoka M
Publication year - 1993
Publication title -
the journal of physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.802
H-Index - 240
eISSN - 1469-7793
pISSN - 0022-3751
DOI - 10.1113/jphysiol.1993.sp019741
Subject(s) - trypsin , intracellular , biophysics , chemistry , adenosine triphosphate , guinea pig , patch clamp , biochemistry , enzyme , biology , endocrinology , receptor
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.