ABSTRACT The sarcoplasmic reticulum (SR) Ca^sup 2+^ release channel (RyR1) from malignant hyperthermia-susceptible (MHS) porcine skeletal muscle has a decreased sensitivity to inhibition by Mg^sup 2+^. This diminished Mg^sup 2+^ inhibition has been attributed to a lower Mg^sup 2+^ affinity of the inhibition (I) site. To determine whether alterations in the Ca^sup 2+^ and Mg^sup 2+^ affinity of the activation (A) site contribute to the altered Mg^sup 2+^ inhibition, we estimated the Ca^sup 2+^ and Mg^sup 2+^ affinities of the A- and I-sites of normal and MHS RyR1. Compared with normal SR, MHS SR required less Ca^sup 2+^ to half-maximally activate [^sup 3^H]ryanodine binding (K^sub A,Ca^: MHS = 0.17 +/- 0.01 (mu)M; normal = 0.29 +/- 0.02 AM) and more Ca^sup 2+^ to half-maximally inhibit ryanodine binding (K^sub I,Ca^: MHS = 519.3 - 48.7 AM; normal = 293.3 - 24.2 AM). The apparent Mg^sup 2+^ affinity constants of the MHS RyR1 A- and I-sites were approximately twice those of the A- and I-sites of the normal RyR1 (KA,,: MHS = 44.36 + 4.54 AM; normal = 21.59 + 1.66 (mu)M; K^sub I,Mg^: MHS = 660.8 +/- 53.0 (mu)M; normal = 299.2 +/- 24.5 (mu)M). Thus, the reduced Mg^sup 2+^ inhibition of the MHS RyR1 compared with the normal RyR1 is due to both an enhanced selectivity of the MHS RyR1 A-site for Ca^sup 2+^ over Mg^sup 2+^ and a reduced Mg^sup 2+^ affinity of the I-site.
INTRODUCTION
Supported by grants from the National Institutes of Health (GM-31382 to C.F.L.) and the American Heart Association, Northland Affiliate (9704662A to E.M.B.).
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Edward M. Balog, Bradley R. Fruen, Nirah H. Shomer, and Charles F. Louis
Department Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455 USA
Received for publication 27 October 2000 and in final form 11 July 2001. Address reprint requests to Dr. Edward M. Balog, Department of Biochemistry, Molecular Biology, and Biophysics, University of Minnesota, 6-155 Jackson Hall, 321 Church St., S.E., Minneapolis, MN 55455. Tel.: 612-- 625-3292; Fax: 612-625-2163; E-mail: balog004@tc.umn.edu.
Copyright Biophysical Society Oct 2001
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