Abstract
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.
Highlights
Muscle contraction is initiated by the release of Ca2ϩ from the sarcoplasmic reticulum
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 Ca2؉
By assessing the Ca2؉ dependence of [3H]ryanodine binding to RyR2 wild type and TM10 mutants we found that mutations in the TM10 sequence altered the sensitivity of the channel to activation by Ca2؉ and enhanced the basal activity of [3H]ryanodine binding
Summary
RyR, ryanodine receptor; CHAPS, 3-[(3cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; HEK, human embryonic kidney; TM, transmembrane; wt, wild type. Based on sequence analysis and mutational studies, Williams et al (25) have suggested that the RyR pore may share a basic common architecture with that of potassium channels, and they proposed a structural model for the pore-forming region of RyR using the three-dimensional pore structure of KcsA as a framework In this hypothetical model, the TM8 and TM10 sequences are thought to correspond, respectively, to the outer and inner helices of the KcsA pore, whereas the luminal loop linking TM8 and TM10 contain motifs equivalent to the pore helix and selectivity filter of KcsA. These studies demonstrate that mutations in the TM10 sequence can alter the sensitivity of the channel to activation by caffeine and Ca2ϩ, the basal channel activity, and the gating properties, indicating that the TM10 sequence plays an important role in RyR channel activation and gating
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