Evidence for a role of the lumenal M3-M4 loop in skeletal muscle Ca2+ release channel (ryanodine receptor) activity and conductance

We tested the hypothesis that part of the lumenal amino acid segment betwee n the two most C-terminal membrane segments of the skeletal muscle ryanodin e receptor (RyR1) is important for channel activity and conductance. Eleven mutants were generated and expressed in HEK293 cells focusing on amino aci d residue I4897 homologous to the selectivity filter of K+ channels and six other residues in the M3-M4 lumenal loop. Mutations of amino acids not abs olutely conserved in RyRs and IP(3)Rs (D4903A and D4907A) showed cellular C a2+ release in response to caffeine, Ca2+-dependent [H-3]ryanodine binding, and single-channel K+ and Ca2+ conductances not significantly different fr om wild-type RyR1. Mutants with an I4897 to A, L, or V or D4917 to A substi tution showed a decreased single-channel conductance, loss of high-affinity [H-3]ryanodine binding and regulation by Ca2+, and an altered caffeine-ind uced Ca2+ release in intact cells. Mutant channels with amino acid residue substitutions that are identical in the RyR and IP3R families (D4899A, D489 9R, and R4913E) exhibited a decreased K+ conductance and showed a loss of h igh-affinity [H-3]ryanodine binding and loss of single-channel pharmacology but maintained their response to caffeine in a cellular assay. Two mutatio ns (G4894A and D4899N) were able to maintain pharmacological regulation bot h in intact cells and in vitro but had lower single-channel K+ and Ca2+ con ductances than the wild-type channel. The results support the hypothesis th at amino acid residues in the lumenal loop region between the two most C-te rminal membrane segments constitute a part of the ion-conducting pore of Ry R1.