title: Residue Gln4863 within a predicted transmembrane sequence of the Ca2+ release channel (ryanodine receptor) is critical for ryanodine interaction.
abstract: Despite the pivotal role of ryanodine in ryanodine receptor (RyR) research, the molecular basis of ryanodine-RyR interaction remains largely undefined. We investigated the role of the proposed transmembrane helix TM10 in ryanodine interaction and channel function. Each amino acid residue within the TM10 sequence, 4844IIFDITFFFFVIVILLAIIQGLII4867, of the mouse RyR2 was mutated to either alanine or glycine. Mutants were expressed in human embryonic kidney 293 cells, and their properties were assessed. Mutations D4847A, F4850A, F4851A, L4858A, L4859A, and I4866A severely curtailed the release of intracellular Ca2+ in human embryonic kidney 293 cells in response to extracellular caffeine and diminished [3H]ryanodine binding to cell lysates. Mutations F4846A, T4849A, I4855A, V4856A, and Q4863A eliminated or markedly reduced [3H]ryanodine binding, but cells expressing these mutants responded to extracellular caffeine by releasing stored Ca2+. Interestingly these two groups of mutants, each with similar properties, are largely located on opposite sides of the predicted TM10 helix. Single channel analyses revealed that mutation Q4863A dramatically altered the kinetics and apparent affinity of ryanodine interaction with single RyR2 channels and abolished the effect of ryanodol, an analogue of ryanodine, whereas the single channel conductance of the Q4863A mutant and its responses to caffeine, ATP, and Mg2+ were comparable to those of the wild type channels. Furthermore the effect of ryanodine on single Q4863A mutant channels was influenced by the transmembrane holding potential. Together these results suggest that the TM10 sequence and in particular the Q4863 residue constitute an important determinant of ryanodine interaction.
authors.affiliation: Cardiovascular Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
authors.forename: Ruiwu
authors.initials: R
authors.lastname: Wang
authors.forename: Lin
authors.initials: L
authors.lastname: Zhang
authors.forename: Jeff
authors.initials: J
authors.lastname: Bolstad
authors.forename: Ni
authors.initials: N
authors.lastname: Diao
authors.forename: Cindy
authors.initials: C
authors.lastname: Brown
authors.forename: Luc
authors.initials: L
authors.lastname: Ruest
authors.forename: William
authors.initials: W
authors.lastname: Welch
authors.forename: Alan J
authors.initials: AJ
authors.lastname: Williams
authors.forename: S R Wayne
authors.initials: SR
authors.lastname: Chen
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D003001:Cloning, Molecular; D004594:Electrophysiology; D005973:Glutamine; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D012433:Ryanodine / Q000378:metabolism*; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D014162:Transfection
chemical_list: D008565:Membrane Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D005973:Glutamine; D012433:Ryanodine