Regulation of the Cardiac Ryanodine Receptor Channel by Luminal Ca 2+ Involves Luminal Ca 2+ Sensing Sites

Abstract

The mechanism of activation of the cardiac calcium release channel/ryanodine receptor (RyR) by luminal Ca 2+ was investigated in native canine cardiac RyRs incorporated into lipid bilayers in the presence of 0.01 μM to 2 mM Ca 2+ (free) and 3 mM ATP (total) on the cytosolic ( cis) side and 20 μM to 20 mM Ca 2+ on the luminal ( trans) side of the channel and with Cs + as the charge carrier. Under conditions of low trans Ca 2+ (20 μM), increasing cis Ca 2+ from 0.1 to 10 μM caused a gradual increase in channel open probability ( P o). Elevating cis Ca 2+ above 100 μM resulted in a gradual decrease in P o. Elevating trans [Ca 2+] enhanced channel activity (EC 50 ≈ 2.5 mM at 1 μM cis Ca 2+) primarily by increasing the frequency of channel openings. The dependency of P o on trans [Ca 2+] was similar at negative and positive holding potentials and was not influenced by high cytosolic concentrations of the fast Ca 2+ chelator, 1,2-bis(2-aminophenoxy)ethane- N, N, N, N-tetraacetic acid. Elevated luminal Ca 2+ enhanced the sensitivity of the channel to activating cytosolic Ca 2+, and it essentially reversed the inhibition of the channel by high cytosolic Ca 2+. Potentiation of P o by increased luminal Ca 2+ occurred irrespective of whether the electrochemical gradient for Ca 2+ supported a cytosolic-to-luminal or a luminal-to-cytosolic flow of Ca 2+ through the channel. These results rule out the possibility that under our experimental conditions, luminal Ca 2+ acts by interacting with the cytosolic activation site of the channel and suggest that the effects of luminal Ca 2+ are mediated by distinct Ca 2+-sensitive site(s) at the luminal face of the channel or associated protein.