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MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

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

⁴Centro Fondo de Investigación Avanzada en Areas Prioritarias (FONDAP) de Estudios Moleculares de la Célula, Facultad de Medicina, Universidad de Chile; ¹Programa de Fisiología y Biofísica, Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile; ²Departamento de Neurología y Neurocirugía, Hospital Clínico, Universidad de Chile; and ³Programa 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 Ca²⁺-induced Ca²⁺ release (CICR), activation by Ca²⁺ of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg²⁺], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg²⁺], and RyR redox state on the Ca²⁺ dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg²⁺] up to 1 mM inhibited vesicular [³H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg²⁺ inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca²⁺ 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 [Ca²⁺] for activation, increased maximal P_o, and shifted channel inhibition to higher [Ca²⁺]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg²⁺] 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 [Mg²⁺] induced a right shift in Ca²⁺ dependence for all channels so that [Ca²⁺] <30 µM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca²⁺ at physiological [ATP] and [Mg²⁺]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR.

Ca²⁺-induced Ca²⁺ release; Ca²⁺ release channels; endoplasmic reticulum; thimerosal; 2,4-dithiothreitol; ryanodine receptor

This article has been cited by other articles:

				R. Bull, J. P. Finkelstein, J. Galvez, G. Sanchez, P. Donoso, M. I. Behrens, and C. Hidalgo Ischemia Enhances Activation by Ca2+ and Redox Modification of Ryanodine Receptor Channels from Rat Brain Cortex J. Neurosci., September 17, 2008; 28(38): 9463 - 9472. [Abstract] [Full Text] [PDF]