Alcohol Decreases the Activity of Native Ryanodine Receptors from Rat Heart

Abstract

Alcohol intoxication can decrease cardiac contractility, yet the underlying mechanisms are not fully known. Type2 ryanodine receptors (RyR2s) release calcium from the sarcoplasmic reticulum, which is critical for heart excitation-contraction coupling. We showed that intoxicating concentrations of ethanol (10-100 mM) reduced the activity of recombinant RyR2 expressed in HEK cells and reconstituted into lipid bilayers (Ye et al, 2014). Here we address whether ethanol action persists when native RyRs from heart muscle are probed. Rat ventricles were crushed and centrifuged to obtain an RyR-enriched membrane preparation. RyR was reconstituted into bilayers made of phosphatidyl-oleyl-phosphatidyl-ethanolamine:phosphatidyl-oleyl-phosphatidyl-serine:phosphatidyl-oleyl-phosphatidyl-choline (5:3:2 molar ratio), and currents were recorded using cesium as permeant ion. As index of activity, we used the product of the number of channels in the bilayer (N) and the channel open probability (Po). The RyR2 phenotype was verified from NPo increases evoked by either 1 mM caffeine or increased cytosolic calcium (0.1-100 microM), and the appearance of a long-lived subconductance triggered by 10 microM ryanodine. As found with recombinant RyR2, NPo decreased by 50-100 mM ethanol, with maximal inhibition occurring at 50 mM ethanol (−49.5%). Data suggest that regulatory proteins such as Fkbp12.6, known to remain associated with the native cardiac RyR but absent when recombinant RyR2 are expressed in HEK cells (Xiao et al., 2004;2005), do not significantly regulate ethanol action. Ethanol-induced inhibition of native RyR was modified by cytosolic calcium, being maximal at 100 microM calcium. Amplification of ethanol-induced inhibition, however, was also induced by raising pre-ethanol NPo with 1 mM caffeine. Data suggest that ethanol inhibits cardiac RyR primarily by destabilizing the channel active state(s) rather than stabilizing RyR inactive state(s). Upcoming experiments are aimed at determining the mechanism(s) and structural bases underlying ethanol actions on cardiac RyR2. Support:R37-AA11560(AMD).