Regulation of the cardiac ryanodine receptor channel by luminal Ca2+ involves luminal Ca2+ sensing sites.
The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca2+ was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 microM to 2 mM Ca2+ (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 microM to 20 mM Ca2+ on the luminal (trans) side of the channel and with Cs+ as the charge carrier. Under conditions of low trans Ca2+ (20 microM), increasing cis Ca2+ from 0.1 to 10 microM caused a gradual increase in channel open probability (Po). Elevating cis Ca2+ above 100 microM resulted in a gradual decrease in Po. Elevating trans [Ca2+] enhanced channel activity (EC50 approximately 2.5 mM at 1 microM cis Ca2+) primarily by increasing the frequency of channel openings. The dependency of Po on trans [Ca2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N,N,N, N-tetraacetic acid. Elevated luminal Ca2+ enhanced the sensitivity of the channel to activating cytosolic Ca2+, and it essentially reversed the inhibition of the channel by high cytosolic Ca2+. Potentiation of Po by increased luminal Ca2+ occurred irrespective of whether the electrochemical gradient for Ca2+ supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca2+ through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca2+ acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca2+ are mediated by distinct Ca2+-sensitive site(s) at the luminal face of the channel or associated protein.
Györke
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Department of Physiology, Texas Tech University Health Sciences Center, Lubbock, Texas 79430 USA.
Györke
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D000494:Allosteric Regulation; D000818:Animals; D001665:Binding Sites; D055592:Biophysical Phenomena; D001703:Biophysics; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D008051:Lipid Bilayers / Q000378:metabolism; D008564:Membrane Potentials; D008861:Microsomes / Q000378:metabolism; D009206:Myocardium / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism
D008051:Lipid Bilayers; D019837:Ryanodine Receptor Calcium Release Channel; D002118:Calcium