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 IP(3)Rs (D4903A and D4907A) showed cellular Ca(2+) release in response to caffeine, Ca(2+)-dependent [(3)H]ryanodine binding, and single-channel K(+) and Ca(2+) 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 [(3)H]ryanodine binding and regulation by Ca(2+), and an altered caffeine-induced Ca(2+) release in intact cells. Mutant channels with amino acid residue substitutions that are identical in the RyR and IP(3)R families (D4899A, D4899R, and R4913E) exhibited a decreased K(+) conductance and showed a loss of high-affinity [(3)H]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 Ca(2+) 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.

Ryanodine Receptors: Structure, Expression, Molecular Details, and Function in Calcium Release

“…The ion conducting pore has been proposed to be located in the lumenal region and to include the GVRAGGGIGD amino acid sequence (4891 -4900 human RyR1) Du et al 2001) which is conserved between RyRs. The sequence GGIG has been proposed as the selectivity filter (Balshaw et al 1999;Gao et al 2000) based on the similarity with the consensus selectivity filter of K þ channels. During channel opening, massive movements of cytoplasmic and transmembrane masses take place and result in a 4 Å increase of the ion gate (Samsó et al 2009).…”

IP3 Receptors: Toward Understanding Their Activation

“…A conserved acidic residue (D2550 in IP 3 R1) at the luminal end of the selectivity filter (Fig. 2C) contributes to the modest Ca 2þ selectivity of IP 3 R (Boehning et al 2001b;Dellis et al 2008) and RyR (Gao et al 2000;Wang et al 2005;Gillespie 2008), but the structural determinants of ion selectivity and permeation by IP 3 R are otherwise poorly understood. The changes in pore structure that allow it to open are also minimally understood.…”

Different Regions in Skeletal and Cardiac Muscle Ryanodine Receptors Are Involved in Transducing the Functional Effects of Calmodulin

“…Exogenous CaM was added to the cis solution. Electrical signals were filtered at 2 kHz, digitized at 10 kHz, and analyzed as described previously (17). P o values in multichannel recordings were calculated using the equation P o ϭ ⌺ iP o,i /N, where N is the total number of channels, and P o,i is channel open probability of the ith channel.…”