Mutations to Gly2370, Gly2373 or Gly2375 in malignant hyperthermia domain 2 decrease caffeine and cresol sensitivity of the rabbit skeletal-muscle Ca2+-release channel (ryanodine receptor isoform 1)

Abstract

and K3940A were made in two potential ATPbinding motifs (amino acids 2370-2375 and 3937-3940) In the presence of 1 mM AMP-PCP, Ca# + -activation curves were shifted to higher affinity and maximal binding was increased to a similar extent for wild-type RyR1 and mutants. ATP sensitivity of channel opening was also similar for wild-type INTRODUCTIONCa# + -release channels of sarcoplasmic reticulum (ryanodine receptors, or RyRs) are large homotetrameric molecules with molecular masses of $ 2.26 MDa [1][2][3][4]. These channels are modulated by endogenous and exogenous ligands such as Ca# + , ATP, caffeine, calmodulin, Mg# + , Ruthenium Red and ryanodine, but the extent of modulation varies among RyR1, the isoform expressed in skeletal muscle, RyR2, expressed in cardiac muscle, and RyR3, expressed more ubiquitously [5][6][7]. Ca# + is a basic modulator of Ca# + -release channel function. At low concentrations, Ca# + activates both skeletal-and cardiac-muscle Ca# + -release channels ; at high concentrations, it inactivates the skeletal-muscle Ca# + -release channel. Ca# + activation and inactivation sites are localized in the C-terminal one-fifth of the RyR1 molecule region [8][9][10][11][12]. This C-terminal region of RyR also forms the Ca# + -conducting pore and contains both high-and low-affinity binding sites for ryanodine [8,[13][14][15]. The molecular mechanisms underlying the interactions of modulators with RyRs are poorly understood.Adenine nucleotides activate Ca# + -release channels with EC &! values in the millimolar range. Activation is probably through a molecular site different from, but interacting with, the Ca# + -activation or Mg# + -binding site [5][6][7]. Motifs identifying potential Abbreviations used : RyR, ryanodine receptor ; RyR1, skeletal-muscle RyR isoform ; RyR2, cardiac-muscle RyR isoform ; AMP-PCP, adenosine 5h-[β,γ-methylene]triphosphate ; MH, malignant hyperthermia ; CCD, central core disease ; pCa, klog [Ca].

Structural Insights Into Disease Mutations of the Ryanodine Receptor

“…In addition, RyR1 I404M expressed in dyspedic mice myotubes exhibited resting [Ca 2+ ] levels and SR Ca 2+ content comparable to that of the wild type [85]. Furthermore, expressions of RyR1 D3987A and G3939A rabbit cDNA in HEK-293 cells, which correspond to the positions of MH-associated mutations D3986E and G3938D in humans, respectively, showed responses to activation by [Ca 2+ ], caffeine, and 4-CmC comparable to that of wild type [92,95]. For these mutations where no clear functional effect has been observed, it is of course possible that they are not causative of the disease but, instead, simply represent polymorphisms.…”

“…Commonly used is a [ 3 H]ryanodine binding assay ( Figure 4C) [19,51,54,60,67,77,79,86,90,92,93,95,96,103,105,108,113,129] As opposed to whole-cell measurements, activities of a single channel can be recorded using planar lipid bilayer electrophysiology ( Figure 4D) [19, 53, 54, 67, 69, 77, 79, 99-101, 105, 107, 108, 111, 112, 114]. Recombinant or endogenous RyR from crude SR vesicle preparations or from purified material are fused into an artificial lipid bilayer formed across two chambers.…”

Recognition and Activation of Ryanodine Receptors by Purines

“…Other possible ATP binding sites suggested for RyR1 are similar to those present in chaperon proteins such as GroES [35].…”

“…Studies utilizing mutations, deletions, or chimeras of the RyR1 have suggested the existence of regions that regulate the caffeine binding sites [35,48]. Substitution of all 3 Gly, in the motif from amino acids 2370 to 2375, by Ala showed that this motif played a role in the sensitivity of the RyR1 for caffeine.…”

“…Substitution of all 3 Gly, in the motif from amino acids 2370 to 2375, by Ala showed that this motif played a role in the sensitivity of the RyR1 for caffeine. This site is within domain 2, which is associated with the hypersensitivity to anesthetics in Malignant Hyperthermia [35].…”