Insights into the Regulation of the Ryanodine Receptor: Differential Effects of Mg 2+ and Ca 2+ on ATP Binding ² Jose ´ M. Dias, ‡ Csaba Szegedi, §,| Istvan Jo ´na, § and Pia D. Vogel* ,‡ Department of Biological Sciences, Southern Methodist UniVersity, 6501 Airline Road, Dallas, Texas 75275, MHSC, MMRI, and Department of Physiology, UniVerity of Debrecen, H-4012 Debrecen, P.O. Box 22, Hungary, and Cell Physiological Research Group of the Hungarian Academy of Sciences, H-4012 Debrecen, P.O. Box 22, Hungary ReceiVed March 29, 2006; ReVised Manuscript ReceiVed April 27, 2006 ABSTRACT: Calcium ions are frequently used second messengers in most living organisms. Members of the family of ryanodine sensitive calcium channels (ryanodine receptors, RyRs) are responsible for many important Ca 2+ signaling events in both excitable and nonexcitable cells. The biological activity of these membrane proteins is modulated and regulated by a great variety of different cellular and extracellular effectors, proteins, and small molecules. However, very little is still understood about how the modulators work on a molecular level. The very large size of more than 2 million Da per functional tetrameric RyR unit and its membrane association have made more detailed biochemical and structural analysis extremely challenging. In this study, we have investigated the effects of the main cellular effectors of RyR, 1 Ca 2+ and Mg 2+ , on the properties for binding of the channel’s third cellular modulator, ATP, to RyR. Employing an innovative approach using ESR spectroscopy and an ATP analogue that carries an ESR-active reporter group, spin-labeled ATP, we directly observed for the first time that a total of eight ATP sites exist on the tetrameric RyR1 purified from rabbit back muscle. We show that the number of ATP sites that are occupied by the ATP analogue and the respective dissociation constants directly depend on the presence and concentrations of Ca 2+ and Mg 2+ ions. The plant alkaloid ryanodine had no effect on ATP binding under activating Ca 2+ conditions. Our findings will have strong implications for the current models for the regulation of cellular Ca 2+ signaling. Ryanodine receptors are homotetrameric protein com- plexes with a molecular mass of ∼560 kDa per subunit, forming the largest known ion channel protein. There are three different isoforms of this Ca channel protein that are found in different mammalian tissues. RyR1 is mostly located in the skeletal muscle; RyR2 is associated with heart muscle, and RyR3 is found in different cell types that do not necessarily constitute contractile cells (1). Although the Ca channel activity of the three different isoforms of RyR is highly regulated by many physiologically and pharmacologi- cally important agents and proteins (2-5), it is assumed that Ca 2+ , Mg 2+ , and ATP are key cellular regulators of the channel (for a recent review, see ref 6). A strong dependence of the channel opening events on different concentrations of either ion or both ions was observed (7-13). Isolated RyR1 was shown to be activated in the presence of 1 µM Ca 2+ and inhibited in the presence of 1 mM cytoplasmic Ca 2+ (3), while inhibition of RyR by Mg 2+ may be the result of two independent mechanisms (14). It is believed that Mg 2+ competes with Ca 2+ for the activating sites (15) or, alterna- tively, that Mg 2+ can bind to low-affinity, inhibitory, non- ion-selective sites that can also mediate Ca 2+ inhibition of the channel (15, 16). A reduced Mg 2+ inhibition and increased Ca sensitivity (5) were found in malignant hypo- thermia, linking this important disease to a malfunctioning regulation of the ryanodine receptor (17). ATP was also previously shown to strongly modulate the Ca 2+ channel activities. Even in the absence of cytoplasmic, activating Ca 2+ ions, ATP strongly activates RyR1. Full activation then results from the presence of both Ca 2+ and ATP. The dissociation constants for ATP that have been reported previously (10, 11, 15, 18) to be in the high micromolar and lower millimolar range will result in maximal RyR activation at cellular ATP concentrations of ∼8 mM in muscle cells (19). RyR2 from cardiac muscle is not activated by ATP to the same extent as RyR1 in the absence of Ca 2+ (20, 21), but ATP clearly aids the activation of RyR2 by Ca 2+ . These different effects of adenine nucleotides in the cardiac muscle RyR are deemed to be part of the primarily important regulation events during contrac- tion of the heart (21, 22). ² This work was supported by a Beginning Grant-In-Aid from the American Heart Association, Texas Affiliate, and a grant from the Southern Methodist University Research Council to P.D.V. and a grant from OTKA K 61442 to I.J. * To whom correspondence should be addressed: Department of Biological Sciences, Southern Methodist University, 6501 Airline Rd., Dallas, TX 75275. Telephone: (214) 768-1790. Fax: (214) 768-3955. E-mail: pvogel@mail.smu.edu. ‡ Southern Methodist University. § Univerity of Debrecen. | Cell Physiological Research Group of the Hungarian Academy of Sciences. 1 Abbreviations: ESR, electron spin resonance spectroscopy; RyR, ryanodine sensitive Ca channel; SL-ATP, 2′,3′-(2,2,5,5-tetramethyl-3- pyrroline-1-oxyl-3-carboxylic acid ester) ATP, where 2′,3′ depicts a rapid equilibrium of the ester bond between the 2′ and 3′ positions of the ribose. 9408 Biochemistry 2006, 45, 9408-9415 10.1021/bi060623a CCC: $33.50 © 2006 American Chemical Society Published on Web 07/15/2006