Regulation of the Cardiac Ryanodine Receptor Channel by Luminal Ca²⁺ Involves Luminal Ca²⁺ Sensing Sites

Abstract

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 μM to 2 mM Ca²⁺ (free) and 3 mM ATP (total) on the cytosolic (cis) side and 20 μM 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 μM), increasing cis Ca²⁺ from 0.1 to 10 μM caused a gradual increase in channel open probability (P_o). Elevating cis Ca²⁺ above 100 μM resulted in a gradual decrease in P_o. Elevating trans [Ca²⁺] enhanced channel activity (EC₅₀ ≈ 2.5 mM at 1 μM cis Ca²⁺) primarily by increasing the frequency of channel openings. The dependency of P_o 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 P_o 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.