Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex

Abstract

Despite their relevance for neuronal Ca(2+)-induced Ca(2+) release (CICR), activation by Ca(2+) of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg(2+)], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg(2+)], and RyR redox state on the Ca(2+) dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg(2+)] up to 1 mM inhibited vesicular [(3)H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg(2+) inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca(2+) dependencies, defined by low, moderate, or high maximal fractional open time (P(o)), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca(2+)] for activation, increased maximal P(o), and shifted channel inhibition to higher [Ca(2+)]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg(2+)] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg(2+)] induced a right shift in Ca(2+) dependence for all channels so that [Ca(2+)] <30 microM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca(2+) at physiological [ATP] and [Mg(2+)]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR.