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Voltage-dependent effects of Ca(2+)/calmodulin on Cl(-) channel in cardiac sarcoplasmic reticulum
1Department of Cardiovascular Diseases Medical Research Institute Tokyo Medical and Dental University. seiko.card@mri.tmd.ac.jp
Abstract:
A new kind of chloride channels in the cardiac sarcoplasmic reticulum, 116 pS Cl(-) channel (500 mM Cl(-) in the cis and 50 mM Cl(-) in the trans chamber solutions), which is activated by protein-kinase-A-dependent phosphorylation, has been determined to conduct adenine nucleotide as a transporter between cytosol and SR lumen. We investigated the voltage-dependent gating of this Cl(-) channel by recording single-channel activities using the planar lipid bilayer-vesicle fusion technique. The channel activities did not change at different membrane potentials (-100 mV to +50 mV) or different Ca(2+) concentrations (1 nM to 1 mM) in cis solution. In the presence of calmodulin (CaM) (0.1 microM /microg SR vesicles), however, Ca(2+) added to the cis solution at 0 mV inhibited channel openings in a Ca(2+) -concentration-dependent manner. These effects were prevented by the addition of CaM inhibitors. The blocking effects of CaM differed depending on the membrane potentials at negative potentials below -20 mV. With CaM and 3 microM Ca(2+), the values of opening probability were 0 at -80 mV, 0.2 at -40 mV, 0.3 at -20 mV, 0.71 at 0 mV and 0.92 at +20 mV. These results may indicate the membrane potential affects the action of Ca(2+) /CaM complex
Insights
A novel cardiac sarcoplasmic reticulum chloride channel transports adenine nucleotides. Its function is modulated by calcium and calmodulin, with voltage-dependent effects observed at negative membrane potentials.
Area of Science:
- Cardiovascular Physiology
- Ion Channel Function
- Molecular Transport
Background:
- Cardiac sarcoplasmic reticulum (SR) chloride channels play roles in excitation-contraction coupling.
- A specific 116 pS Cl(-) channel activated by protein kinase A has been identified in cardiac SR.
- This channel functions as an adenine nucleotide transporter between the cytosol and SR lumen.
Purpose of the Study:
- To investigate the voltage-dependent gating of the 116 pS Cl(-) channel.
- To determine the influence of calcium (Ca2+) and calmodulin (CaM) on channel activity.
- To elucidate the interplay between membrane potential, Ca2+/CaM, and channel gating.
Main Methods:
- Single-channel recordings using the planar lipid bilayer-vesicle fusion technique.
- Systematic variation of membrane potentials from -100 mV to +50 mV.
- Application of varying Ca2+ concentrations (1 nM to 1 mM) and CaM (0.1 microM) to the cis chamber.
Main Results:
- Channel activity was independent of membrane potential and Ca2+ concentration alone.
- In the presence of CaM, Ca2+ inhibited channel openings in a concentration-dependent manner at 0 mV.
- CaM inhibition was voltage-dependent, with reduced blocking effects at negative potentials, suggesting Ca2+/CaM complex interaction with the channel.
Conclusions:
- The 116 pS cardiac SR chloride channel's gating is not directly voltage-dependent but is modulated by Ca2+/CaM.
- Ca2+/CaM binding to the channel is influenced by membrane potential, altering its inhibitory effect.
- This suggests a novel regulatory mechanism for adenine nucleotide transport in the cardiac SR.