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

In myocardial ischemia, pHi and [ATP] fall, whereas the free [Ca2+] and [Mg2+] rise. The effects of these changes on cardiac Ca2+ release channel (ryanodine receptor) activity were investigated in [3H]ryanodine binding and single-channel measurements, using isolated membrane and purified channel preparations. In the absence of the two channel ligands Mg2+ and ATP, cardiac Ca2+ release channels were half-maximally activated at pH 7.4 by approximately 4 mumol/L cytosolic Ca2+ and half-maximally inhibited by approximately 9 mmol/L cytosolic Ca2+. Regulation of channel activity by Ca2+ was modulated by Mg2+ 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. [3H]Ryanodine binding measurements also indicated that acidosis impairs cardiac Ca2+ release channel activity. Mg2+ and adenine nucleotide concentrations regulated the extent of inhibition and the Ca2+ dependence of binding. In the presence of 5 mmol/L Mg2+ and 5 mmol/L beta, gamma-methyleneadenosine 5'-triphosphate (AMPPCP, a nonhydrolyzable ATP analogue), the free [Ca2+] for half-maximal [3H]ryanodine binding was increased from 1.9 mumol/L at pH 7.3 to 36 mumol/L at pH 6.5 and to 89 mumol/L at pH 6.2. These results suggest that ionic and metabolic changes that might be expected to affect sarcoplasmic reticulum Ca2+ release channel activity in ischemic myocardium include an altered Ca2+ sensitivity of the channel, a fall in pH, and a loss of the high-energy adenine nucleotide pool, leading to an increased inhibition by Mg2+.