Effects of ATP, Mg2+, and redox agents on the Ca2+ dependence of RyR channels from rat brain cortex.
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.
Bull
Ricardo
R
Facultad de Medicina, Universidad de Chile, Casilla 70005, Santiago 7, Chile. rbull@med.uchile.cl
Finkelstein
José Pablo
JP
Humeres
Alexis
A
Behrens
María Isabel
MI
Hidalgo
Cecilia
C
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000266:Adenylyl Imidodiphosphate / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism*; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism*; D066298:In Vitro Techniques; D015640:Ion Channel Gating / Q000187:drug effects; D008274:Magnesium / Q000378:metabolism*; D008564:Membrane Potentials; D010084:Oxidation-Reduction / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D019163:Reducing Agents / Q000494:pharmacology*; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism*; D013849:Thimerosal / Q000494:pharmacology
D019163:Reducing Agents; D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine; D013849:Thimerosal; D000266:Adenylyl Imidodiphosphate; D000255:Adenosine Triphosphate; D008274:Magnesium; D002118:Calcium; D004229:Dithiothreitol