The Predicted TM10 Transmembrane Sequence of the Cardiac Ca2+ Release Channel (Ryanodine Receptor) Is Crucial for Channel Activation and Gating

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.

The Cytoplasmic Region of Inner Helix S6 Is an Important Determinant of Cardiac Ryanodine Receptor Channel Gating

“…1). These structural features are consistent with our previous functional studies demonstrating the critical role of the S6 helix bundle crossing region in the regulation and gating of RyR2 (25)(26)(27)(28)(29). However, the functional significance of the C-terminal (cytoplasmic) region of the S6 inner helix is unknown.…”

Single Channel Properties of Heterotetrameric Mutant RyR1 Ion Channels Linked to Core Myopathies

“…The deletion was identified as a novel mutation in one patient (17). Wang et al (33,34) mutated the residues within the membrane-spanning segment of the cardiac ryanodine receptor (RyR2) to alanine and found that RyR2-V4854A and -I4855A (matching RyR1-V4926 and -I4927 deletion sites) retained function but showed a reduced response to caffeine activation. We previously showed that deletion of RyR1 residues Val-4926 and Ile-4927 results in a nonfunctional channel unable to conduct Ca 2ϩ (19).…”

Inositol Trisphosphate Receptor Ca2+Release Channels

“…The lack of more substantial data in this area begs for additional experimental effort. A number of mutations in the pore region of the RyR have either been described (central core disease mutations) or engineered (114,116,117,155,217,304,500,501,504,522,524,546). The pore region of the cardiac RyR2 channel has been modeled onto the KcsA pore structure (507).…”