Related Experiment Videos
Interaction between permeant ions and voltage sensor during inactivation of N-type Ca2+ channels
1Department of Molecular Biophysics and Physiology, Rush University, 1750 W. Harrison Street, Chicago, IL 60612, USA. rshiroko@rush.edu
Abstract:
1. Inactivation of neuronal N-type Ca2+ channels transiently expressed in human kidney tSA-201 cells was studied at the level of whole-cell Ca2+ current and intramembrane charge movement. 2. Prolonged (5 s) depolarization to 40 mV shifted the voltage distribution of intramembrane charge movement from a transition potential (mid-point voltage) of 9.5 +/- 3.8 mV to -55.4 +/- 8.2 mV. Because of the large negative shift, it was possible to record intramembrane charge movement from unblocked inactivated channels and determine the effect of Ca2+ influx on inactivation of intramembrane charge movement. 3. In unblocked channels, the rate of inactivation of charge movement (21 +/- 3 s-1 at 0 mV) was close to that of Ca2+ current decay during the conditioning pulse. However, in blocked channels inactivation was significantly slower (4 +/- 1 s-1 at 0 mV). In unblocked channels, the availability of Ca2+ current was minimal and charge movement from inactivated channels was maximal after conditioning to about 10 mV. After the block of ionic current, inactivation of charge movement gradually increased with voltage. 4. Although the rate of Ca2+ current run-down was not affected by 10-15 microM free Ca2+ in the pipette solution, inactivation of Ca2+ currents during depolarization was about two times faster in high intracellular Ca2+. 5. The present results favour the current-dependent mechanism of inactivation of N-type channels. They also suggest that Ca2+ acting in the permeation pathway and transmembrane voltage are the proximate causes of the same inactivation transitions of voltage sensing moieties in these channels.
Insights
Neuronal N-type calcium channels inactivate faster with calcium influx, suggesting voltage and calcium ions are key to this process in voltage-gated channels.
Area of Science:
- Neuroscience
- Molecular Biology
- Biophysics
Background:
- Neuronal N-type Ca2+ channels are crucial for neurotransmitter release and neuronal excitability.
- Understanding their inactivation mechanisms is vital for comprehending neuronal function and dysfunction.
Purpose of the Study:
- To investigate the inactivation mechanisms of neuronal N-type Ca2+ channels.
- To differentiate between current-dependent and voltage-dependent inactivation processes.
- To elucidate the roles of intracellular Ca2+ and transmembrane voltage in channel inactivation.
Main Methods:
- Whole-cell patch-clamp electrophysiology was used to record Ca2+ currents and intramembrane charge movement.
- Experiments were conducted on human kidney tSA-201 cells expressing N-type Ca2+ channels.
- Intramembrane charge movement was measured under conditions of blocked and unblocked ionic currents.
Main Results:
- Prolonged depolarization induced a significant negative shift in the voltage dependence of intramembrane charge movement, indicating voltage sensor rearrangement.
- In unblocked channels, the rate of charge movement inactivation correlated with Ca2+ current decay, but was slower in blocked channels.
- High intracellular Ca2+ accelerated the inactivation of Ca2+ currents during depolarization.
- Inactivation of charge movement was voltage-dependent, particularly after ionic current block.
Conclusions:
- The findings support a current-dependent mechanism for N-type Ca2+ channel inactivation.
- Both intracellular Ca2+ concentration and transmembrane voltage act as proximate triggers for inactivation transitions in voltage-sensing domains.