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

T. Furukawa, Zheng Fan, T. Sawanobori, M. Hiraoka

Research output: Contribution to journalArticle

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Abstract

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.

Original languageEnglish (US)
Pages (from-to)707-726
Number of pages20
JournalJournal of Physiology
Volume466
Issue number1
DOIs
StatePublished - Jul 1 1993
Externally publishedYes

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Adenosine
Trypsin
Muscle Cells
Adenosine Triphosphate
Exopeptidases
Carboxypeptidases A
Intracellular Membranes
Trypsin Inhibitors
Patch-Clamp Techniques
Binding Sites
Membranes

All Science Journal Classification (ASJC) codes

  • Physiology

Cite this

Modification of the adenosine 5'‐triphosphate‐sensitive K+ channel by trypsin in guinea‐pig ventricular myocytes. / Furukawa, T.; Fan, Zheng; Sawanobori, T.; Hiraoka, M.

In: Journal of Physiology, Vol. 466, No. 1, 01.07.1993, p. 707-726.

Research output: Contribution to journalArticle

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abstract = "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.",
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