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Calcium-dependent inactivation of L-type calcium channels in planar lipid bilayers
1Department of Pharmacology and Physiology, University of North Carolina at Chapel Hill 27599.
Abstract:
Intracellular Ca2+ can inhibit the activity of voltage-gated Ca channels by modulating the rate of channel inactivation. Ca(2+)-dependent inactivation of these channels may be a common negative feedback process important for regulating Ca2+ entry under physiological and pathological conditions. This article demonstrates that the inactivation of cardiac L-type Ca channels, reconstituted into planar lipid bilayers and studied in the presence of a dihydropyridine agonist, is sensitive to Ca2+. The rates and extents of inactivation, determined from ensemble averages of unitary Ba2+ currents, decreased when the calcium concentration facing the intracellular surface of the channel ([Ca2+]i) was lowered from approximately 10 microM to 20 nM by the addition of Ca2+ chelators. The rates and extents of Ba2+ current inactivation could also be increased by subsequent addition of Ca2+ raising the [Ca2+]i to 15 microM, thus demonstrating that the Ca2+ dependence of inactivation could be reversibly regulated by changes in [Ca2+]i. In addition, reconstituted Ca channels inactivated more quickly when the inward current was carried by Ca2+ than when it was carried by Ba2+, suggesting that local increases in [Ca2+]i could activate Ca(2+)-dependent inactivation. These data support models in which Ca2+ binds to the channel itself or to closely associated regulatory proteins to control the rate of channel inactivation, and are inconsistent with purely enzymatic models for channel inactivation.
Insights
Intracellular calcium (Ca2+) regulates cardiac L-type Ca channels by modulating inactivation rates. Lowering intracellular Ca2+ reduces inactivation, while increasing it enhances it, demonstrating reversible Ca2+ dependent regulation.
Area of Science:
- Cardiology
- Molecular Biology
- Biophysics
Background:
- Voltage-gated calcium channels are crucial for cellular function.
- Intracellular calcium (Ca2+) influences channel activity through feedback mechanisms.
- Ca2+-dependent inactivation is a key regulator of Ca2+ entry.
Purpose of the Study:
- To investigate the Ca2+ sensitivity of cardiac L-type Ca channel inactivation.
- To elucidate the role of intracellular Ca2+ concentration ([Ca2+]i) in channel inactivation.
- To differentiate between direct Ca2+ binding and enzymatic models of inactivation.
Main Methods:
- Reconstitution of cardiac L-type Ca channels into planar lipid bilayers.
- Measurement of unitary Ba2+ currents under varying intracellular Ca2+ concentrations.
- Utilizing Ca2+ chelators and additions to manipulate [Ca2+]i.
- Comparing inactivation rates with Ca2+ versus Ba2+ as charge carriers.
Main Results:
- Cardiac L-type Ca channel inactivation is sensitive to intracellular Ca2+.
- Lowering [Ca2+]i from ~10 microM to 20 nM significantly decreased inactivation rates and extents.
- Increasing [Ca2+]i to 15 microM reversibly increased inactivation.
- Ca2+ currents showed faster inactivation than Ba2+ currents, suggesting local Ca2+ effects.
Conclusions:
- Ca2+ directly binds to the channel or associated proteins to regulate inactivation.
- The findings support direct Ca2+ modulation rather than enzymatic inactivation models.
- Ca2+-dependent inactivation is a critical, reversible feedback mechanism for Ca2+ channel regulation.