Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor

Mei, Yingwu; Xu, Le; Mowrey, David D.; Giráldez, Raúl; Wang, Ying; Pasek, Daniel A.; Dokholyan, Nikolay V.; Meissner, Gerhard

doi:10.1074/jbc.m115.659672

Cited by 17 publications

(15 citation statements)

References 42 publications

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“…Although the radial tilting angle and the twisting angle of the S6 helix are accurately reproduced in our model, we note a difference in the lateral tilting angle for the S6 helix ( In our open-channel model, the direction of rotation brings Ile-4937 past Gly-4934. We previously showed that the G4934A mutation reduced the Ca 2ϩ current, whereas the G4934V mutation resulted in loss of function of purified channels (31). In the current study, we find that RyR1-G4934V can form functional channels with reduced Ca 2ϩ current, when avoiding detergent exposure throughout the process of purification.…”

Section: Conformational Changes Of Ryr1 Channel Openingsupporting

confidence: 63%

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Ion-pulling simulations provide insights into the mechanisms of channel opening of the skeletal muscle ryanodine receptor

Mowrey

Mei

et al. 2017

Journal of Biological Chemistry

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The type 1 ryanodine receptor (RyR1) mediates Ca release from the sarcoplasmic reticulum to initiate skeletal muscle contraction and is associated with muscle diseases, malignant hyperthermia, and central core disease. To better understand RyR1 channel function, we investigated the molecular mechanisms of channel gating and ion permeation. An adequate model of channel gating requires accurate, high-resolution models of both open and closed states of the channel. To this end, we generated an open-channel RyR1 model using molecular simulations to pull Ca through the pore constriction site of a closed-channel RyR1 structure determined at 3.8-Å resolution. Importantly, we find that our open-channel model is consistent with the RyR1 and cardiac RyR (RyR2) open-channel structures reported while this paper was in preparation. Both our model and the published structures show similar rotation of the upper portion of the pore-lining S6 helix away from the 4-fold channel axis and twisting of Ile-4937 at the channel constriction site out of the channel pore. These motions result in a minimum open-channel pore radius of ∼3 Å formed by Gln-4933, rather than Ile-4937 in the closed-channel structure. We also present functional support for our model by mutations around the closed- and open-channel constriction sites (Gln-4933 and Ile-4937). Our results indicate that use of ion-pulling simulations produces a RyR1 open-channel model, which can provide insights into the mechanisms of channel opening complementing those from the structural data.

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Section: Conformational Changes Of Ryr1 Channel Openingsupporting

confidence: 63%

“…We previously investigated the single channel properties of purified RyR1-G4934A and RyR1-G4934V mutants (31). We here examine the single channel properties of these mutants in membrane fractions avoiding exposure to detergent during purification of RyR1s.…”

Section: Altering Open-channel Contacts In the S6 Helix Affects Channmentioning

confidence: 99%

Ion-pulling simulations provide insights into the mechanisms of channel opening of the skeletal muscle ryanodine receptor

Mowrey

Mei

et al. 2017

Journal of Biological Chemistry

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“…Dilation of the pore does not occur via bending of S6 at a single ‘hinge’ alone, but rather by bowing and distortion of the S6 helix from a point midway through the membrane (Figure 4B), near the position of G4934 hinge, previously identified to influence gating of both RyR1 and RyR2 (Euden et al, 2013; Mei et al, 2015). Three other notable changes occur in the transmembrane region coincident with pore dilation.…”

Section: Resultsmentioning

confidence: 84%

Structural Basis for Gating and Activation of RyR1

Georges

Clarke

Zalk

et al. 2016

Cell

325

566

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Summary The type-1 ryanodine receptor (RyR1) is an intracellular calcium (Ca2+) release channel required for skeletal muscle contraction. Here we present cryo-EM reconstructions of RyR1 in multiple functional states revealing the structural basis of channel gating and ligand-dependent activation. Binding sites for the channel activators Ca2+, ATP and caffeine were identified at interdomain interfaces of the C-terminal domain. Either ATP or Ca2+ alone induce conformational changes in the cytoplasmic assembly (‘priming’), without pore dilation. In contrast, in the presence of all three activating ligands, high-resolution reconstructions of open and closed states of RyR1 were obtained from the same sample, enabling analyses of conformational changes associated with gating. Gating involves global conformational changes in the cytosolic assembly accompanied by local changes in the transmembrane domain, which include bending of the S6 transmembrane segment and consequent pore dilation, displacement and deformation of the S4-S5 linker, and conformational changes in the pseudo-voltage-sensor domain.

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“…Single-channel studies were done using purified RyR2 channel preparations. We reported that replacement of RyR1-Gly-4934 and Gly-4941 with Ala altered RyR1 channel function in single-channel measurements using purified RyR1 preparations (15). A mutation further increasing the side-chain volume at Gly-4934 (G4934V) resulted in loss of function.…”

Section: Discussionmentioning

confidence: 99%

“…Mutagenesis and single-channel measurements showed that negatively charged luminal residues Asp-4899 and Glu-4900 and the cytosolic residues Asp-4938, Glu-4942, and Asp-4945 in the pore-lining S6 helix impact RyR1 ion permeation and selectivity (13,14). The RyR1 S6 pore-lining helix has also two conserved glycines, Gly-4934 and Gly-4941, whose replacement with uncharged residues with an increased sidechain volume altered RyR1 channel gating and ion permeation (15). High-resolution cryoelectron microscopy (cryo-EM) using open RyR1 ( Fig.…”

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confidence: 99%

G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to cytosolic and luminal Ca2+

Mowrey

Chirasani

et al. 2018

Journal of Biological Chemistry

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The ryanodine receptor ion channel RyR1 is present in skeletal muscle and has a large cytoplasmic N-terminal domain and smaller C-terminal pore-forming domain comprising six transmembrane helices, a pore helix, and a selectivity filter. The RyR1 S6 pore-lining helix has two conserved glycines, Gly-4934 and Gly-4941, that facilitate RyR1 channel gating by providing S6 flexibility and minimizing amino acid clashes. Here, we report that substitution of Gly-4941 with Asp or Lys results in functional channels as indicated by caffeine-induced Ca release response in HEK293 cells, whereas a low response of the corresponding Gly-4934 variants suggested loss of function. Following purification, the RyR1 mutants G4934D, G4934K, and G4941D did not noticeably conduct Ca in single-channel measurements. Gly-4941 replacement with Lys resulted in channels having reduced K conductance and reduced selectivity for Ca compared with wildtype. RyR1-G4941K did not fully close at nanomolar cytosolic Ca concentrations and nearly fully opened at 2 μm cytosolic or sarcoplasmic reticulum luminal Ca, and Ca- and voltage-dependent regulation of RyR1-G4941K mutant channels was demonstrated. Computational methods and single-channel recordings indicated that the open G4941K variant results in the formation of a salt bridge to Asp-4938. In contrast, wildtype RyR1 was closed and not activated by luminal Ca at low cytosolic Ca levels. A model suggested that luminal Ca activates RyR1 by accessing a recently identified cytosolic Ca-binding site in the open channel as the Ca ions pass through the pore.

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Channel Gating Dependence on Pore Lining Helix Glycine Residues in Skeletal Muscle Ryanodine Receptor

Cited by 17 publications

References 42 publications

Ion-pulling simulations provide insights into the mechanisms of channel opening of the skeletal muscle ryanodine receptor

Ion-pulling simulations provide insights into the mechanisms of channel opening of the skeletal muscle ryanodine receptor

Structural Basis for Gating and Activation of RyR1

G4941K substitution in the pore-lining S6 helix of the skeletal muscle ryanodine receptor increases RyR1 sensitivity to cytosolic and luminal Ca2+

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