Molecular basis of Ca(2)+ activation of the mouse cardiac Ca(2)+ release channel (ryanodine receptor)

Abstract

Activation of the cardiac ryanodine receptor (RyR2) by Ca(2)+ is an essential step in excitation-contraction coupling in heart muscle. However, little is known about the molecular basis of activation of RyR2 by Ca(2)+. In this study, we investigated the role in Ca(2)+ sensing of the conserved glutamate 3987 located in the predicted transmembrane segment M2 of the mouse RyR2. Single point mutation of this conserved glutamate to alanine (E3987A) reduced markedly the sensitivity of the channel to activation by Ca(2)+, as measured by using single-channel recordings in planar lipid bilayers and by [(3)H]ryanodine binding assay. However, this mutation did not alter the affinity of [(3)H]ryanodine binding and the single-channel conductance. In addition, the E3987A mutant channel was activated by caffeine and ATP, was inhibited by Mg(2)+, and was modified by ryanodine in a fashion similar to that of the wild-type channel. Coexpression of the wild-type and mutant E3987A RyR2 proteins in HEK293 cells produced individual single channels with intermediate sensitivities to activating Ca(2)+. These results are consistent with the view that glutamate 3987 is a major determinant of Ca(2)+ sensitivity to activation of the mouse RyR2 channel, and that Ca(2)+ sensing by RyR2 involves the cooperative action between ryanodine receptor monomers. The results of this study also provide initial insights into the structural and functional properties of the mouse RyR2, which should be useful for studying RyR2 function and regulation in genetically modified mouse models.

Figure 1

Cloning and functional expression of the mouse RyR2 cDNA. (A) The strategy and outline for the cloning of cDNA encoding the mouse RyR2. Four overlapping clones (mCRR1-mCRR4, solid boxes) covering the entire coding region of the mouse RyR2 cDNA (open box) were obtained by screening a mouse cardiac cDNA library using six short PCR fragments as probes. (B) Caffeine-induced Ca²+ release in HEK293 cells transfected with 6 μg wt RyR2 cDNA (mRyR2 (wt)) (B, panel a), 6 μg mutant (E3987A) cDNA (B, panel b), or 6 μg control (pCDNA3) DNA (B, panel c). Fluorescence intensity of fluo-3 loaded HEK293 cells was monitored continuously before and after addition of 2 mM caffeine (arrows). Decreases in fluorescence upon additions of caffeine are due to fluorescence quenching by caffeine. (C) Immunoblotting of sucrose density gradient fractions of wt or E3987A mutant RyR2 proteins. An aliquot of 10 μl each sucrose gradient fraction was used for immunoblotting. The wt and mutant RyR2 proteins were detected by using the anti-RyR mAb 34C. The top (right) and bottom (left) of the gradient were labeled.

Figure 2

Ca² + response of single wt and E3987A mutant RyR2 channels. Single-channel activities of the wt (mRyR2(wt)) (A) and the E3987A mutant (B) were recorded in a symmetrical recording solution containing 250 mM KCl, 25 mM Hepes, pH 7.4, and varying concentrations of cytoplasmic free Ca²+. The orientation of each single channel detected in the bilayer was determined at the beginning of each experiment by addition of an aliquot of EGTA solution to either the cis or the trans chamber. The trans chamber was connected to the input of the headstage amplifier and the cis chamber was held at virtual ground. Single-channel activities shown in A and B were inhibited by addition of 0.1 mM EGTA to the cis chamber, indicating that the cytoplasmic side of the incorporated channel was facing the cis chamber. All subsequent additions were made to the cytoplasmic side of the channel (cis chamber). The relationships between Po and Ca²+ concentrations of single wt (solid circles) and E3987A mutant (open circles) channels are shown in C. Data points shown are individual Po measurements from 22 single wt channels and 16 single E3987A mutant channels. Curve shown represents fit of data from single wt channels using the Hill equation. Data points at Ca²+ concentrations >1 mM from three single wt channels that did not show significant Ca²+-dependent inactivation were not included in the fitting for Ca²+ inactivation. The parameters of the fitting are indicated in the text. Note that the sensitivities of single wt channels to activation by Ca²+ are similar, with a threshold for activation ∼100 nM, whereas the sensitivities to inactivation by Ca²+ vary significantly among single wt channels. The threshold for activation of single E3987A mutant channels by Ca²+ is ∼0.2 mM. The maximum Po of the Ca²+-activated E3987A mutant channels ranges from 0.01 to 13%. The open probability (Po), arithmetic mean open time (To), and the arithmetic mean closed time (Tc) at each Ca²+ level are indicated on the top of each panel. A short line to the right of each current trace indicates the base line. The holding potential for both the wt and E3987A mutant channels was +20 mV.

Figure 3

Ligand gating properties of single wt (A) and E3987A mutant (B) RyR2 channels. Single-channel recordings were performed as described in the legend to Fig. 2. (A) Control a was performed in the presence of 171 nM cytoplasmic free Ca²+. Subsequent additions of ATP, caffeine, Mg²+, and ryanodine were made to the cytoplasmic side of the same channel. (B) The concentration of free Ca²+ in control a is 1.7 mM, and the recording solution in control d contains 1.7 mM Ca²+, 0.5 mM EGTA, 2 mM ATP, and 4 mM caffeine. Current recordings in a–c are from the same channel, whereas those in d–f are from a different channel. Base lines are indicated. The holding potential was +20 mV for all recordings shown.

Figure 4

Mutation E3987A does not alter the affinity of [³H]ryanodine binding to RyR2. [³H]ryanodine binding to cell lysate prepared from HEK293 cells transfected with wt or mutant E3987A cDNA was performed in the presence of 25 mM Tris, 50 mM HEPES, pH 7.4, 500 mM KCl, CaCl₂ (0.2 mM for wt and 1.0 mM for mutant E3987A), 0.1–30 nM [³H]ryanodine, and various protease inhibitors at 37°C for 2 h. Data shown are from a representative experiment, which has been repeated three to four times. Inset shows a Scatchard plot.

Figure 5

Mutation E3987A reduced dramatically the sensitivity of single mouse RyR2 channel to Ca² + activation. The Ca²+ responses of single wt (A) and E3987A mutant (B and C) channels were determined in the presence of 2 mM ATP and 4 mM caffeine. Other conditions for recordings were the same as described in Fig. 2. According to their maximum Po, single E3987A mutant channels could be divided into two groups with high (B) and low Po (C). The Po-pCa relationships of the wt and E3987A mutant channels are shown in D. Data points shown are individual Po measurements from five single wt channels (solid circles), five E3987A mutant channels with high Po (open triangles), and five E3987A mutant channels with low Po (solid triangles). Lines represent curve fits using the Hill equation. The yielded parameters of fitting are indicated in the text. The holding potential for all recordings shown was +20 mV. Base lines are indicated.

Figure 6

Ca² + dependence of [³H]ryanodine binding to wt and mutant E3987A proteins [³H]ryanodine binding to cell lysate prepared from HEK293 cells transfected with wt or mutant E3987A cDNA was performed at various concentrations of Ca²+, as described in materials and methods. The concentration of [³H]ryanodine used was 10 nM. Data shown are individual measurements from six separate binding experiments.

Figure 7

Ca² + responses of single channels produced by coexpression of the wt and E3987A mutant RyR2 proteins in HEK293 cells. HEK293 cells were transfected with an equal amount (6 μg) of wt and E3987A mutant RyR2 cDNA. The Ca²+ responses of single hybrid channels with high Po (A), medium Po (B), and low Po (C) were determined as described in the legend to Fig. 2. Single-channel activities shown in A and B were inhibited by addition of 0.1 mM EGTA to the cis chamber, indicating that the cytoplasmic side of the incorporated channel was facing the cis chamber. On the other hand, single-channel activities shown in C were inhibited by addition of 0.1 mM EGTA to the trans chamber, indicating that the cytoplasmic side of the incorporated channel was facing the trans chamber. To measure currents in the same direction, from the luminal to the cytoplasmic side of the channel, a +20-mV holding potential was applied in A and B and −20 mV in C. The Po-pCa relationships of single hybrid channels are shown in D. Data points represent individual measurements. According to their Ca²+ sensitivities to activation, single hybrid channels could be divided into at least five groups (I–V). Group I (solid circles) and group II (solid triangles) displayed Ca²+ sensitivity to activation similar to that of the wt and E3987A mutant channel, respectively. Group III (solid squares), group IV (open circles), and group V (open squares) showed sensitivities to Ca²+ activation in between those of the wt and E3987A mutant channels. These different Ca²+ sensitivities most probably resulted from hybrid channels with different compositions of wt and E3987A mutant subunits. Note that two single channels are present in recordings shown in C and Po indicates the average open probability.

Figure 8

Single mouse and canine RyR2 channels share similar Ca² + response. Single-channel activities of the canine RyR2 were recorded as described in materials and methods and in the legend to Fig. 2. Single-channel activities shown in A were inhibited by the addition of 0.1 mM EGTA to the cis chamber, indicating that the cytoplasmic side of the incorporated channels was facing the cis chamber. All subsequent additions were made to the cytoplasmic side of the channel (cis chamber). Current traces (21 nM–0.27 μM) shown on the left and those (2–18 mM) shown on the right were from separate channels. The relationships between Po and Ca²+ concentrations of single canine (open circles, solid line) and mouse (dashed line, from Fig. 2) RyR2 channels are shown in B. Data points shown are individual Po measurements from nine single canine RyR2 channels. The holding potential was +20 mV for Ca²+ concentrations at 21 nM, 64 nM, 126 nM, 190 nM, 0.27 μM, and 2 mM, and +40 mV for Ca²+ concentrations at 5.5 and 18 mM. The open probability (Po), arithmetic mean open time (To) and the arithmetic mean closed time (Tc) at each Ca²+ level are indicated on the top of each panel. A short line to the right of each current trace indicates the base line.