Ca2+ and voltage inactivate Ca2+ channels in guinea-pig ventricular myocytes through independent mechanisms.
1. L-type Ca2+ currents and Ca2+ channel gating currents were studied in isolated guinea-pig ventricular heart cells using the whole-cell patch-clamp technique, in order to investigate the mechanism of Ca(2+)-dependent inactivation. The effect of altering the intracellular Ca2+ concentration ([Ca2+]i) on these currents was studied through photorelease of intracellular Ca2+ ions using the photolabile Ca2+ chelators DM-nitrophen and nitr-5. 2. We found that step increases in [Ca2+]i produced by photorelease could either increase or decrease the L-type Ca2+ current. Specifically, Ca2+ photorelease from DM-nitrophen almost exclusively caused inactivation of the Ca2+ current. In contrast, Ca2+ photorelease from nitr-5 had a biphasic effect: a small, rapid inactivation of the Ca2+ current was followed by a slow potentiation. These two Ca(2+)-dependent processes seemed to differ in their Ca2+ dependence, as small Ca2+ photoreleases elicited potentiation without a preceding inactivation, whereas larger photoreleases elicited both inactivation and potentiation. 3. The mechanism of the Ca(2+)-dependent inactivation of Ca2+ channels was explored by comparing the effects of voltage and photoreleased Ca2+ on the Ca2+ current and the Ca2+ channel gating current. Voltage was found to reduce both the Ca2+ current and the gating current proportionally. However, Ca2+ photorelease from intracellular DM-nitrophen inactivated the Ca2+ current without having any effect on the gating current. 4. The dephosphorylation hypothesis for Ca(2+)-dependent inactivation was tested by applying isoprenaline to the cells before eliciting a maximal rise of [Ca2+]i (maximal flash intensity, zero external [Na+]i). Isoprenaline could completely prevent Ca(2+)-dependent inactivation under these conditions, even when [Ca2+]i rose so high as to cause an irreversible contracture of the cell. 5. We concluded from these experiments that voltage and Ca2+ ions inactivate the L-type Ca2+ channel through separate, independent mechanisms. In addition, we found that Ca(2+)-dependent inactivation does not result in the immobilization of gating charge, and apparently closes the Ca2+ permeation pathway through a mechanism that does not involve the voltage-sensing region of the channel. Furthermore, we found that Ca(2+)-dependent inactivation is entirely sensitive to beta-adrenergic stimulation. These facts suggest that either Ca(2+)-dependent inactivation results from Ca(2+)-dependent dephosphorylation of the Ca2+ channel, or that Ca(2+)-dependent inactivation is modulated by protein kinase A.
Hadley
R W
RW
Department of Physiology, University of Maryland School of Medicine, Baltimore 21201.
Lederer
W J
WJ
D000085:Acetates / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000187:drug effects / Q000502:physiology*; D002614:Chelating Agents / Q000494:pharmacology; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000494:pharmacology; D005029:Ethylenediamines / Q000494:pharmacology; D006168:Guinea Pigs; D006321:Heart / Q000502:physiology*; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000502:physiology*; D007545:Isoproterenol / Q000494:pharmacology; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D010782:Photolysis; D016276:Ventricular Function
D000085:Acetates; D015220:Calcium Channels; D002614:Chelating Agents; D005029:Ethylenediamines; C052455:nitr 5; C057014:DM-nitrophen; D004533:Egtazic Acid; D007545:Isoproterenol; D002118:Calcium