Role of the proposed pore-forming segment of the Ca2+ release channel (ryanodine receptor) in ryanodine interaction.
In earlier studies we showed that point mutations introduced into the proposed pore-forming segment, GVRAGGGIGD (amino acids 4820-4829), of the mouse cardiac ryanodine receptor reduced or abolished high affinity [3H]ryanodine binding. Here we investigate the effects of these mutations on the affinity and dissociation properties of [3H]ryanodine binding and on ryanodine modification of the ryanodine receptor channel at the single channel and whole cell levels. Scatchard analysis and dissociation studies reveal that mutation G4824A decreases the equilibrium dissociation constant (K(d)) and the dissociation rate constant (k(off)), whereas mutations G4828A and D4829A increase the K(d) and k(off) values. The effect of ryanodine on single G4828A and D4829A mutant channels is reversible on the time scale of single channel experiments, in contrast to the irreversible effect of ryanodine on single wild-type channels. Ryanodine alone is able to induce a large and sustained Ca2+ release in HEK293 cells transfected with the R4822A or G4825A mutant cDNA at the resting cytoplasmic Ca2+ but causes little or no Ca2+ release in cells transfected with the wild-type cDNA. Mutation G4826C diminishes the functional effect of ryanodine on Ca2+ release but spares caffeine-induced Ca2+ release in HEK293 cells. Co-expression of the wild-type and G4826C mutant proteins produces single channels that interact with ryanodine reversibly and display altered conductance and ryanodine response. These results are consistent with the view that the proposed pore-forming segment is a critical determinant of ryanodine interaction. A putative model of ryanodine-ryanodine receptor interaction is proposed.
Chen
S R Wayne
SR
Cardiovascular Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada. swchen@ucalgary.ca
Li
Pin
P
Zhao
Mingcai
M
Li
Xiaoli
X
Zhang
Lin
L
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002110:Caffeine / Q000494:pharmacology; D002122:Calcium Chloride / Q000494:pharmacology; D020013:Calcium Signaling / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D007473:Ion Channels / Q000502:physiology*; D007668:Kidney; D007700:Kinetics; D015636:Magnesium Chloride / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D020239:Receptor Cross-Talk / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012433:Ryanodine / Q000494:pharmacology*; D019837:Ryanodine Receptor Calcium Release Channel / Q000235:genetics / Q000502:physiology*
D007473:Ion Channels; D011994:Recombinant Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D015636:Magnesium Chloride; D012433:Ryanodine; D002110:Caffeine; D002122:Calcium Chloride