New insights into the gating mechanisms of cardiac ryanodine receptors revealed by rapid changes in ligand concentration.
We have developed a novel technique for incorporation of sheep cardiac sarcoplasmic reticulum (SR) Ca(2+)-release channels into planar phospholipid bilayers in order to investigate changes in [Ca2+] on a physiological time scale and have investigated whether the rate of change of cytosolic [Ca2+] has a direct effect on the gating of the cardiac SR Ca(2+)-release channel. Vesicles of heavy SR were incorporated into planar phospholipid bilayers painted on glass pipettes, and an established technique for rapid solution exchanges at excised membrane patches was modified to allow solution changes to be made at the bilayer within 10 ms. For a given change in [Ca2+], we demonstrate that the open probability (Po) is similar whether the cytosolic [Ca2+] is increased rapidly (10 ms) or slowly (1 s) and appears to be no different from the Po measured under steady state conditions that were recorded by using conventional bilayer techniques. We also demonstrate that no desensitization or inactivation occurs at -40 mV when the channel is activated by Ca2+ alone or in the presence of other channel activators, ATP or EMD 41000. However, at +40 mV, rapid channel activation followed by inactivation was observed. The probability of such voltage-dependent inactivation appears to depend on the mechanism of channel activation.
Sitsapesan
R
R
Department of Cardiac Medicine, National Heart & Lung Institute, London, UK.
Montgomery
R A
RA
Williams
A J
AJ
D000818:Animals; D002118:Calcium / Q000502:physiology; D015220:Calcium Channels / Q000502:physiology*; D006321:Heart / Q000502:physiology*; D008024:Ligands; D008051:Lipid Bilayers; D009124:Muscle Proteins / Q000502:physiology*; D009200:Myocardial Contraction / Q000502:physiology; D019837:Ryanodine Receptor Calcium Release Channel; D012756:Sheep
D015220:Calcium Channels; D008024:Ligands; D008051:Lipid Bilayers; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium