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

Abstract

The ryanodine receptor (RyR) channel pore is formed by four S6 inner helices, with its intracellular gate located at the S6 helix bundle crossing region. The cytoplasmic region of the extended S6 helix is held by the U motif of the central domain and is thought to control the opening and closing of the S6 helix bundle. However, the functional significance of the S6 cytoplasmic region in channel gating is unknown. Here we assessed the role of the S6 cytoplasmic region in the function of cardiac RyR (RyR2) via structure-guided site-directed mutagenesis. We mutated each residue in the S6 cytoplasmic region of the mouse RyR2 (⁴⁸⁷⁶QQEQVKEDM⁴⁸⁸⁴) and characterized their functional impact. We found that mutations Q4876A, V4880A, K4881A, and M4884A, located mainly on one side of the S6 helix that faces the U motif, enhanced basal channel activity and the sensitivity to Ca²⁺ or caffeine activation, whereas mutations Q4877A, E4878A, Q4879A, and D4883A, located largely on the opposite side of S6, suppressed channel activity. Furthermore, V4880A, a cardiac arrhythmia-associated mutation, markedly enhanced the frequency of spontaneous openings and the sensitivity to cytosolic and luminal Ca²⁺ activation of single RyR2 channels. V4880A also increased the propensity and reduced the threshold for arrhythmogenic spontaneous Ca²⁺ release in HEK293 cells. Collectively, our data suggest that interactions between the cytoplasmic region of S6 and the U motif of RyR2 are important for stabilizing the closed state of the channel. Mutations in the S6/U motif domain interface likely destabilize the closed state of RyR2, resulting in enhanced basal channel activity and sensitivity to activation and increased propensity for spontaneous Ca²⁺ release and cardiac arrhythmias.

Keywords: calcium; calcium channel; calcium imaging; calcium intracellular release; calcium-binding protein; ryanodine receptor; sarcoplasmic reticulum (SR).

FIGURE 1.

Location of the S6 cytoplasmic region in the three-dimensional structure of RyR2. The 3D structure of two RyR2 monomers (30) is shown. The central domain, U motif, CTD, and S6 inner helix are highlighted. The inset is a close-up view showing the cytoplasmic region of the S6 inner helix encompassing residues ⁴⁸⁷⁶QQEQVKEDM⁴⁸⁸⁴.

FIGURE 2.

Effect of mutations in the cytoplasmic region of S6 on caffeine activation of RyR2. A–E and G–K, HEK293 cells were transfected with RyR2 WT (A), Q4877A (B), E4878A (C), Q4879A (D), E4883A (E), Q4876A (G), V4880A (H), K4881A (I), E4882A (J), or M4884A (K). The fluorescence intensity of the Fluo-3-loaded transfected cells before and after repeated additions of increasing concentrations of caffeine (0.025–5 mm) was monitored continuously. F and L, Ca²⁺ release-cumulative caffeine concentration relationships in HEK293 cells transfected with RyR2 WT and mutants that shift the caffeine response curve to the right (F) or mutants that have little effect on caffeine response or shift the caffeine response curve to the left (L). The amplitude of each caffeine peak was normalized to that of the maximum peak for each experiment. Data shown are mean ± S.E. (n = 3–4; *, p < 0.05 versus WT.

FIGURE 3.

Effect of the S6 inner helix mutations on [³H]ryanodine binding to RyR2. A and B, [³H]Ryanodine binding to cell lysate prepared from HEK293 cells expressing RyR2 WT and the S6 mutants Q4877A, E4878A, Q4879A, E4882A, and D4883A (A) or the S6 mutants Q4876A, V4880A, K4881A, and M4884A (B) was carried out at various Ca²⁺ concentrations (∼0.2 nm to 0.1 mm), 800 mm KCl, and 5 nm [³H]ryanodine. The amounts of [³H]ryanodine binding at various Ca²⁺ concentrations were normalized to the maximal binding (100%). C, mutating the cytoplasmic region of the S6 inner helix of RyR2 affects the basal level of [³H]ryanodine binding to RyR2 in the near absence of activating Ca²⁺ (<20 nm). D, the EC₅₀ values of Ca²⁺ activation of [³H]ryanodine binding to RyR2 WT and the S6 mutants. The data points shown are mean ± S.E. from three to five separate experiments (*p < 0.05 versus WT).

FIGURE 4.

Expression of S6 mutants in HEK293 cells. A, HEK293 cells were transfected with RyR2 WT or the S6 inner helix mutants. The same amount of transfected HEK293 cell lysate protein was used for Western blotting (WB) using the anti-RyR antibody (34c) and anti-β-actin antibody. B, besides the use of the same amount of WT and mutant cell lysate protein for loading, the expression levels of the WT and S6 mutants were further normalized to that of β-actin. Data shown are mean ± S.E. from five separate experiments (*, p < 0.05 versus WT; NS, not significant).

FIGURE 5.

Effect of the disease-causing RyR2 mutation V4880A on the gating of single RyR2 channels. A and B, single-channel activities of the RyR2 WT (A) and the V4880A mutant (B) were recorded in a symmetrical recording solution containing 250 mm KCl and 25 mm Hepes (pH 7.4). EGTA was added to either the cis or trans chamber to determine the orientation of the incorporated channel. The side of the channel to which an addition of EGTA inhibited the activity of the incorporated channel presumably corresponds to the cytosolic (cyto) face. The Ca²⁺ concentration on both the cytosolic and the luminal face of the channel was adjusted to ∼45 nm. Recording potentials were −20 mV. Openings are downward, and baselines are indicated (short bars). C–F, the event frequency (C), Po (D), To (E), and Tc (F) of single RyR2 WT and V4880A (VA) mutant channels at 45 nm cytosolic Ca²⁺ are shown. Data shown are mean ± S.E. from nine RyR2 WT and 11 V4880A single channels (**, p < 0.01; *, p < 0.05 versus WT; NS, not significant).

FIGURE 6.

Effect of V4880A on the cytosolic Ca²⁺ activation of single RyR2 channels. A and B, single-channel activities of RyR2 WT (A) and the V4880A (B) mutant were recorded in a symmetrical recording solution containing 250 mm KCl and 25 mm Hepes (pH 7. 4). The Ca²⁺ concentration on the cytoplasmic (cyto) and luminal face of the channel was first adjusted to ∼45 nm. The cytosolic Ca²⁺ concentration was then increased from 45 nm to various levels by addition of aliquots of CaCl₂ solution. C, the relationships between open probability and pCa of single RyR2 WT (solid circles) and the V4880A mutant (open circles). The data points shown are mean ± S.E. from four RyR2-WT and 11 V4880A single channels.

FIGURE 7.

Effect of V4880A on the luminal Ca²⁺ activation of single RyR2 channels. A and B, single-channel activities of RyR2 WT (A) and V4880A mutant (B) were recorded in a symmetrical recording solution containing 250 mm KCl and 25 mm Hepes (pH 7.4). The Ca²⁺ concentration on the cytoplasmic (cyto) and the luminal face of the channel was first adjusted to ∼45 nm. The luminal Ca²⁺ concentration was then increased to various levels by addition of aliquots of CaCl₂ solution. C, the relationships between open probability and luminal pCa of single RyR2 WT (solid circles) and the V4880A mutant (open circles). The data points shown are mean ± S.E. from five RyR2-WT and four V4880A single channels.

FIGURE 8.

Effect of the V4880A mutation on the propensity for SOICR. A and B, stable, inducible HEK293 cells expressing RyR2 WT (A) and V4880A (B) were loaded with Fura-2/AM. The cells were then perfused continuously with KRH buffer containing increasing levels of extracellular Ca²⁺ (0–2 mm) to induce SOICR. Fura-2 ratios were recorded using epifluorescence single-cell Ca²⁺ imaging. Caff, caffeine. C, percentages of RyR2 WT (220 cells) and V4880A (171 cells) cells that display Ca²⁺ oscillations at various extracellular Ca²⁺ concentrations. Data shown are mean ± S.E. (n = 3; **, p < 0.01 versus WT).

FIGURE 9.

Effect of V4880A on SOICR activation and termination thresholds. A and B, stable, inducible HEK293 cell lines expressing RyR2 WT (A) and V4880A (B) were transfected with the FRET-based ER luminal Ca²⁺-sensing protein D1ER 48 h before single-cell FRET imaging. Expression of the RyR2 WT and mutant was induced 24 h before imaging. The cells were perfused with KRH buffer containing increasing levels of extracellular Ca²⁺ (0–2 mm) to induce SOICR. This was followed by the addition of 1.0 mm tetracaine to inhibit SOICR and then 20 mm caffeine to deplete the ER Ca²⁺ stores. FRET recordings from representative RyR2 WT (total 57 cells, A) and mutant V4880A cells (total 53 cells, B) are shown. C and D, to minimize the influence of CFP/YFP cross-talk, we used relative FRET measurements to calculate the activation threshold (C) and termination threshold (D) using the equations shown in A. F_SOICR indicates the FRET level at which SOICR occurs, whereas F_termi represents the FRET level at which SOICR terminates. E, the fractional Ca²⁺ release was calculated by subtracting the termination threshold from the activation threshold. The maximum FRET signal F_max is defined as the FRET level after tetracaine treatment. The minimum FRET signal F_min is defined as the FRET level after caffeine treatment. F, the store capacity was calculated by subtracting F_min from F_max. Data shown are mean ± S.E. (n = 4; *, p < 0.05 versus WT; NS, not significant).

FIGURE 10.

Residues in the cytoplasmic region of S6 that are potentially involved in S6/U-motif interactions. Images show the 3D structures of the U motif, the CTD, and the S6 inner helix of RyR2 (30). A, mutations that enhance the activity of RyR2, including Q4876A, V4880A, K4881A, and M4884A, are located on one side of the S6 helix that faces the U motif. B, mutations that suppress RyR2 activity are located on the opposite side of the S6 helix that faces the conduction pore. C, potential hydrophobic interactions among residues Val⁴⁸⁸⁰ in the S6 helix and Ile⁴¹⁷³, Phe⁴¹⁷⁴, and Val⁴¹⁷⁷ in the U motif of the central domain (14, 30).