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

• Published in: American Journal of Physiology: Cell Physiology Vol. 293; no. 1; p. C162
• Main Authors: Bull, Ricardo, Finkelstein, Jose Pablo, Humeres, Alexis, Behrens, Maria Isabel, Hidalgo, Cecilia
• Format: Journal Article
• Language: English
• Published: United States 01.07.2007
• Subjects: Adenosine Triphosphate - metabolism; Adenylyl Imidodiphosphate - pharmacology; Animals; Binding, Competitive; Calcium - metabolism; Cerebral Cortex - drug effects; Cerebral Cortex - metabolism; Dithiothreitol - pharmacology; Endoplasmic Reticulum - drug effects; Endoplasmic Reticulum - metabolism; In Vitro Techniques; Ion Channel Gating - drug effects; Magnesium - metabolism; Membrane Potentials; Oxidation-Reduction - drug effects; Rats; Rats, Sprague-Dawley; Reducing Agents - pharmacology; Ryanodine - metabolism; Ryanodine Receptor Calcium Release Channel - drug effects; Ryanodine Receptor Calcium Release Channel - metabolism; Thimerosal - pharmacology
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00518.2006

4 Centro Fondo de Investigación Avanzada en Areas Prioritarias (FONDAP) de Estudios Moleculares de la Célula, Facultad de Medicina, Universidad de Chile; 1 Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile; 2 Departamento de Neurología y Neurocirugía, Hospital Clínico, Universidad de Chile; and 3 Programa de Biología Celular y Molecular, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile Submitted 4 October 2006 ; accepted in final form 12 March 2007 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 µM 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. Ca 2+ -induced Ca 2+ release; Ca 2+ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor Address for reprint requests and other correspondence: R. Bull, ICBM, Facultad de Medicina, Universidad de Chile, Casilla 70005, Santiago 7, Chile (e-mail: rbull{at}med.uchile.cl )