Evidence for a Role of the Lumenal M3-M4 Loop in Skeletal Muscle Ca²⁺ Release Channel (Ryanodine Receptor) Activity and Conductance

Abstract

We tested the hypothesis that part of the lumenal amino acid segment between the two most C-terminal membrane segments of the skeletal muscle ryanodine receptor (RyR1) is important for channel activity and conductance. Eleven mutants were generated and expressed in HEK293 cells focusing on amino acid 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 absolutely conserved in RyRs and IP₃Rs (D4903A and D4907A) showed cellular Ca²⁺ release in response to caffeine, Ca²⁺-dependent [³H]ryanodine binding, and single-channel K⁺ and Ca²⁺ conductances not significantly different from wild-type RyR1. Mutants with an I4897 to A, L, or V or D4917 to A substitution showed a decreased single-channel conductance, loss of high-affinity [³H]ryanodine binding and regulation by Ca²⁺, and an altered caffeine-induced Ca²⁺ release in intact cells. Mutant channels with amino acid residue substitutions that are identical in the RyR and IP₃R families (D4899A, D4899R, and R4913E) exhibited a decreased K⁺ conductance and showed a loss of high-affinity [³H]ryanodine binding and loss of single-channel pharmacology but maintained their response to caffeine in a cellular assay. Two mutations (G4894A and D4899N) were able to maintain pharmacological regulation both in intact cells and in vitro but had lower single-channel K⁺ and Ca²⁺ conductances than the wild-type channel. The results support the hypothesis that amino acid residues in the lumenal loop region between the two most C-terminal membrane segments constitute a part of the ion-conducting pore of RyR1.