The predicted TM10 transmembrane sequence of the cardiac Ca2+ release channel (ryanodine receptor) is crucial for channel activation and gating.
The predicted TM10 transmembrane sequence, (4844)IIFDITFFFFVIVILLAIIQGLII(4867), has been proposed to be the pore inner helix of the ryanodine receptor (RyR) and to play a crucial role in channel activation and gating, as with the inner helix of bacterial potassium channels. However, experimental evidence for the involvement of the TM10 sequence in RyR channel activation and gating is lacking. In the present study, we have systematically investigated the effects of mutations of each residue within the 24-amino acid TM10 sequence of the mouse cardiac ryanodine receptor (RyR2) on channel activation by caffeine and Ca(2+). Intracellular Ca(2+) release measurements in human embryonic kidney 293 cells expressing the RyR2 wild type and TM10 mutants revealed that several mutations in the TM10 sequence either abolished caffeine response or markedly reduced the sensitivity of the RyR2 channel to activation by caffeine. By assessing the Ca(2+) dependence of [(3)H]ryanodine binding to RyR2 wild type and TM10 mutants we also found that mutations in the TM10 sequence altered the sensitivity of the channel to activation by Ca(2+) and enhanced the basal activity of [(3)H]ryanodine binding. Furthermore, single I4862A mutant channels exhibited considerable channel openings and altered gating at very low concentrations of Ca(2+). Our data indicate that the TM10 sequence constitutes an essential determinant for channel activation and gating, in keeping with the proposed role of TM10 as an inner helix of RyR. Our results also shed insight into the orientation of the TM10 helix within the RyR channel pore.
Cardiovascular Research Group, Department of Physiology and Biophysics, University of Calgary, Calgary, Alberta T2N 4N1, Canada.
Ruiwu
R
Wang
Jeff
J
Bolstad
Huihui
H
Kong
Lin
L
Zhang
Cindy
C
Brown
S R Wayne
SR
Chen
D000595:Amino Acid Sequence; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004247:DNA / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009206:Myocardium / Q000378:metabolism*; D015221:Potassium Channels / Q000737:chemistry; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D019837:Ryanodine Receptor Calcium Release Channel / Q000737:chemistry / Q000502:physiology*; D013997:Time Factors; D014162:Transfection
D015221:Potassium Channels; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D004247:DNA; D002118:Calcium