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Mechanism of Ca(2+)-sensitive inactivation of L-type Ca2+ channels
1Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
Abstract:
Many high threshold, voltage-gated Ca2+ channels, including the dihydropyridine-sensitive class (L-type), inactivate in response not only to voltage, but also to entry of Ca2+. Despite the physiological importance of this Ca(2+)-sensitive inactivation, its molecular mechanism is understood only in broad outline. We now demonstrate that Ca(2+)-dependent inactivation transpires by a Ca(2+)-induced shift of channel gating to a low open probability mode, distinguished by a more than 100-fold reduction of entry rate to the open state. A gating mechanism that explains this shift quantitatively and enables successful separation of Ca(2+)- and voltage-sensitive forms of inactivation is deduced and tested. Finally, both calmodulin activation and channel (de)phosphorylation are excluded as significant signaling events underlying Ca(2+)-induced mode shifts, leaving direct binding of Ca2+ to the channel as a likely chemical initiation event for inactivation.
Insights
Calcium influx triggers a shift in voltage-gated calcium channel gating to a low-activity state, a process independent of calmodulin or phosphorylation. Direct calcium binding likely initiates this Ca2+-sensitive inactivation.
Area of Science:
- Molecular and Cellular Neuroscience
- Ion Channel Physiology
- Calcium Signaling
Background:
- Voltage-gated calcium channels (VGCCs) are crucial for cellular excitability and calcium signaling.
- VGCCs, including L-type channels, exhibit inactivation in response to both voltage changes and intracellular calcium (Ca2+) influx.
- The precise molecular mechanisms underlying Ca2+-sensitive inactivation remain incompletely understood.
Purpose of the Study:
- To elucidate the molecular mechanism of Ca2+-dependent inactivation in high-threshold VGCCs.
- To differentiate Ca2+-sensitive inactivation from voltage-dependent inactivation.
- To identify the signaling pathways involved in Ca2+-induced gating mode shifts.
Main Methods:
- Quantitative analysis of channel gating kinetics.
- Development and testing of a gating model to separate inactivation components.
- Biochemical assays to assess calmodulin activation and channel phosphorylation states.
Main Results:
- Ca2+-dependent inactivation results from a Ca2+-induced shift in channel gating to a mode with significantly reduced open probability (>100-fold decrease in entry rate).
- A validated gating mechanism successfully distinguished Ca2+-sensitive and voltage-sensitive inactivation.
- Calmodulin activation and channel phosphorylation/dephosphorylation were ruled out as key mediators of Ca2+-induced mode shifts.
Conclusions:
- Direct binding of Ca2+ to the channel is the most probable initiating event for Ca2+-sensitive inactivation.
- This mechanism provides a framework for understanding how calcium influx regulates channel activity.
- The findings offer insights into the fine-tuning of calcium signaling by ion channel properties.