Molecular identification of the ryanodine receptor pore-forming segment.
A sequence motif, GXRXGGGXGD, located in the putative channel-forming domain, is conserved in all known ryanodine receptors and inositol 1,4,5-trisphosphate receptors. The functional significance of this conserved region was investigated by using site-directed mutagenesis together with functional assays consisting of Ca(2+) release measurements, [(3)H]ryanodine binding, and single channel recordings in planar lipid bilayers. We report here that single point mutations introduced into this region of the mouse cardiac ryanodine receptor reduce or abolish high affinity [(3)H]ryanodine binding. Single channel analysis revealed that a single substitution of alanine for glycine 4824 within this region reduced the single channel conductance by 97%, from 798 picosiemens (pS) for the wild type channel to 22 pS. The G4824A mutant channel was modulated by Ca(2+), Mg(2+), ATP, caffeine, ruthenium red, and ryanodine. Co-expression of the wild type and G4824A mutant proteins produced single channels that have intermediate unitary conductances of 516, 256, 176, and 60 pS. These data suggest that this conserved region constitutes an essential part of the ryanodine binding site and the channel conduction pathway of the ryanodine receptor.
Zhao
M
M
Cardiovascular Research Group, Department of Physiology, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Li
P
P
Li
X
X
Zhang
L
L
Winkfein
R J
RJ
Chen
S R
SR
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D008564:Membrane Potentials; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012433:Ryanodine / Q000378:metabolism; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D019837:Ryanodine Receptor Calcium Release Channel; D012433:Ryanodine