Regulation of Cardiac Ca 2+ Release Channel (Ryanodine Receptor) by Ca 2+ , H + , Mg 2+ , and Adenine Nucleotides Under Normal and Simulated Ischemic Conditions

Abstract

In myocardial ischemia, pH i and [ATP] fall, whereas the free [Ca 2+ ] and [Mg 2+ ] rise. The effects of these changes on cardiac Ca 2+ release channel (ryanodine receptor) activity were investigated in [ 3 H]ryanodine binding and single-channel measurements, using isolated membrane and purified channel preparations. In the absence of the two channel ligands Mg 2+ and ATP, cardiac Ca 2+ release channels were half-maximally activated at pH 7.4 by ≈4 μmol/L cytosolic Ca 2+ and half-maximally inhibited by ≈9 mmol/L cytosolic Ca 2+ . Regulation of channel activity by Ca 2+ was modulated by Mg 2+ and ATP. Single-channel activities were more sensitive to a change of cytosolic pH than SR lumenal pH. Reduction in lumenal and/or cytosolic pH from 7.3 to 6.5 and 6.0 resulted in decreased single-channel activities without a change in single-channel conductance. [ 3 H]Ryanodine binding measurements also indicated that acidosis impairs cardiac Ca 2+ release channel activity. Mg 2+ and adenine nucleotide concentrations regulated the extent of inhibition and the Ca 2+ dependence of binding. In the presence of 5 mmol/L Mg 2+ and 5 mmol/L β,γ-methyleneadenosine 5′-triphosphate (AMPPCP, a nonhydrolyzable ATP analogue), the free [Ca 2+ ] for half-maximal [ 3 H]ryanodine binding was increased from 1.9 μmol/L at pH 7.3 to 36 μmol/L at pH 6.5 and to 89 μmol/L at pH 6.2. These results suggest that ionic and metabolic changes that might be expected to affect sarcoplasmic reticulum Ca 2+ release channel activity in ischemic myocardium include an altered Ca 2+ sensitivity of the channel, a fall in pH, and a loss of the high-energy adenine nucleotide pool, leading to an increased inhibition by Mg 2+ .