Localization of an NH(2)-terminal disease-causing mutation hot spot to the "clamp" region in the three-dimensional structure of the cardiac ryanodine receptor

Abstract

A region between residues 414 and 466 in the cardiac ryanodine receptor (RyR2) harbors more than half of the known NH(2)-terminal mutations associated with cardiac arrhythmias and sudden death. To gain insight into the structural basis of this NH(2)-terminal mutation hot spot, we have determined its location in the three-dimensional structure of RyR2. Green fluorescent protein (GFP), used as a structural marker, was inserted into the middle of this mutation hot spot after Ser-437 in the RyR2 sequence. The resultant GFP-RyR2 fusion protein, RyR2(S437-GFP,) was expressed in HEK293 cells and characterized using Ca(2+) release, [(3)H]ryanodine binding, and single cell Ca(2+) imaging studies. These functional analyses revealed that RyR2(S437-GFP) forms a caffeine- and ryanodine-sensitive Ca(2+) release channel that possesses Ca(2+) and caffeine dependence of activation indistinguishable from that of wild type (wt) RyR2. HEK293 cells expressing RyR2(S437-GFP) displayed a propensity for store overload-induced Ca(2+) release similar to that in cells expressing RyR2-wt. The three-dimensional structure of the purified RyR2(S437-GFP) was reconstructed using cryo-electron microscopy and single particle image processing. Subtraction of the three-dimensional reconstructions of RyR2-wt and RyR2(S437-GFP) revealed the location of the inserted GFP, and hence the NH(2)-terminal mutation hot spot, in a region between domains 5 and 9 in the clamp-shaped structure. This location is close to a previously mapped central disease-causing mutation site located in a region between domains 5 and 6. These results, together with findings from previous studies, suggest that the proposed interactions between the NH(2)-terminal and central regions of RyR2 are likely to take place between domains 5 and 6 and that the clamp-shaped structure, which shows substantial conformational differences between the closed and open states, is highly susceptible to disease-causing mutations.

Fig. 1. Insertion of GFP into the RyR2 sequence after residue Ser-437

The linear sequence of RyR2 is denoted by an open rectangle. The NH₂-terminal, central, and COOH-terminal mutation regions, corresponding to CPVT/ARVD2 I and MH/CCD I, CPVT/ARVD2 II and MH/CCD II, and CPVT/ARVD2 III and MH/CCD III, respectively, are indicated by the shaded areas. GFP flanked by two Gly-rich spacers was inserted into the NH₂-terminal region (CPVT/ARVD2 I and MH/CCD I) after Ser-437 and into the central region (CPVT/ARVD2 II and MH/CCD II) after residue Ser-2367 of the RyR2 sequence, as indicated by the filled boxes. The phosphorylation sites (S2030, S2808, and S2814), the calmodulin binding site (CaM, 3614–3643), the proposed cytosolic Ca²⁺ sensor (E3987), and the proposed pore-forming segment (4820–4829) are also shown.

Fig. 2. Expression and functional characterization of RyR2_S437-GFP

(A) HEK293 cells grown on glass coverslips were transfected with RyR2_S437-GFP. Phase-contrast images (a) and GFP-fluorescence (b) of transfected cells were recorded under the fluorescence microscope. (B) The fluorescent intensity of the fluo-3-loaded HEK293 cells transfected with RyR2_S437-GFP cDNA was monitored continuously before and after addition of various concentrations of caffeine (a), or before and after the sequential addition of 0.25 mM caffeine, 100 µM ryanodine, and three successive doses of 2.5 mM caffeine (b). The sharp decreases in fluorescence intensity (b) immediately after the third and forth doses of caffeine were due to fluorescence quenching by caffeine. Similar results were obtained from three separate experiments.

Fig. 3. Ca²⁺- and caffeine-dependent activation and response to Ca²⁺ overload of RyR2_S437-GFP

(A) [³H]ryanodine binding to cell lysates prepared from HEK293 cells transfected with RyR2-wt (filled circles) or RyR2_S437-GFP (open circles) was carried out at various concentrations of Ca²⁺ in the presence of 5 nM [³H]ryanodine. (B) [³H]ryanodine binding to cell lysates prepared from HEK293 cells transfected with RyR2-wt or RyR2_S437-GFP was carried out at various concentrations of caffeine in the presence of ~43 nM (pCa=7.37) Ca²⁺ and 5 nM [³H]ryanodine. (C) Stable, inducible HEK293 cells expressing RyR2-wt or RyR2_S437-GFP were grown on glass coverslips. The cells were induced with tetracycline for 24 hours and loaded with 5 mM fura-2-AM in KRH buffer for 20 min at room temperature. The cells were continuously perfused with KRH buffer containing 0.1, 0.2, 0.3, 0.5, or 1.0 mM CaCl₂. The fluorescent intensity of individual cells was continuously monitored using single cell Ca²⁺ imaging. The fractions of RyR2-wt and RyR2_S437-GFP cells that displayed Ca²⁺ oscillations at various [Ca²⁺]_o are shown. Data shown are mean ± SEM from 3–6 separate experiments.

Fig. 4. Immuno-blotting of and [³H]ryanodine binding to the purified RyR2_S437-GFP

(A) The RyR2_S437-GFP protein was purified from cell lysate by affinity chromatography using GST-FKBP12.6 as the affinity ligand. The purified RyR2_S437-GFP and RyR2-wt proteins were solubilized, separated in 6% SDS-PAGE, and transferred to nitrocellulose membranes. The membrane was probed either with an anti-RyR antibody or an anti-GFP antibody for Western blotting. Note that the RyR2_S437-GFP protein migrated at a slightly slower rate in SDS-PAGE than did the RyR2-wt, due to the insertion of GFP. Also note that the anti-GFP antibody reacted only with RyR2_S437-GFP, but not with RyR2-wt. (B) [³H]ryanodine binding to purified RyR2_S437-GFP protein (open circles) was carried out at various concentrations of Ca²⁺ in the presence of 5 nM [³H]ryanodine. Data shown are mean ± SEM from 3 separate experiments.

Fig. 5. Cryo-electron microscopy of RyR2_S437-GFP

A portion of a cryo-EM micrograph of the purified RyR2_S437-GFP proteins embedded in a thin layer of vitreous ice is shown. Several individual RyR2_T1874-GFP particles are marked with white circles. The scale bar represents 500Å.

Fig. 6. Two-dimensional averages of RyR2_S437-GFP and RyR2-wt

(A) Two-dimensional average of RyR2_S437-GFP (n=263 particle images) “top” view; (B) The top view of the two-dimensional average of RyR2-wt (n=269 particle images); (C) Difference map obtained by subtracting (B) from (A). The top view represents the projection of the channel as seen from the cytoplasmic side. The largest differences shown in (C), corresponding to the additional masses due to the GFP insertion, are seen as bright white areas, one of which is circled, in the clamp-shaped domains. (D) Map of statistically significant regions of difference obtained by t-test; map is displayed at >99.9% confidence level. The width of each frame is 544 Å.

Fig. 7. Three-dimensional surface representation of RyR2_S437-GFP and difference map

(A) The three-dimensional reconstruction of RyR2_S437-GFP is shown in green. (B) Difference map (RyR2_S437-GFP – RyR2-wt) shown in green is superimposed on the three-dimensional reconstruction of the RyR2-wt (in blue). The three-dimensional reconstructions are shown in three views: left, top views from the cytoplasmic surface, which in situ would face the transverse-tubule; middle, views towards the bottom of the channel (i.e., as it would appear if viewed from the lumen of the sarcoplasmic reticulum); right, side views. The numerals on the cytoplasmic assembly indicate the distinguishable domains. (C) A detailed view of the clamp-shaped structures, showing the relative positions of the inserted GFPs at Ser-437 and Ser-2367, the inserted GST at the amino terminus (in red), and the docking of a domain formed by residues 216–572 in RyR1, as indicated by a dash circle.