Molecular identification of the ryanodine receptor pore-forming segment

A sequence motif, GXRXGGGXGD, located in the putative channel-forming domai n, is conserved in all known ryanodine receptors and inositol 1, 4,5-trisph osphate receptors, The functional significance of this conserved region was investigated by using site-directed mutagenesis together with functional a ssays consisting :of Ca2+ release measurements, [H-3]ryanodine binding, and single channel recordings in planar lipid bilayers, We report here that si ngle point mutations introduced into this region of the mouse cardiac ryano dine receptor reduce or abolish high affinity [H-3]ryanodine binding. Singl e channel analysis revealed that a single substitution of alanine for glyci ne 4824 within this region reduced the single channel conductance by 97%, f rom 798 picosiemens (pS) for the wild type channel to 22 pS, The G4824A mut ant channel was modulated by Ca2+, Mg2+, ATP, caffeine, ruthenium red, and ryanodine, Co-expression of the wild type and G4824A. mutant proteins produ ced 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.