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. 2005 Aug 15;390(Pt 1):333-43.
doi: 10.1042/BJ20042113.

A recently identified member of the glutathione transferase structural family modifies cardiac RyR2 substate activity, coupled gating and activation by Ca2+ and ATP

Angela F Dulhunty  1 , Pierre Pouliquin, Marjorie Coggan, Peter W Gage, Philip G Board
Affiliations

A recently identified member of the glutathione transferase structural family modifies cardiac RyR2 substate activity, coupled gating and activation by Ca2+ and ATP

Angela F Dulhunty et al. Biochem J. .

Abstract

The recently discovered CLIC-2 protein (where CLIC stands for chloride intracellular channel), which belongs to the ubiquitous glutathione transferase structural family and is expressed in the myocardium, is a regulator of native cardiac RyR2 (ryanodine receptor 2) channels. Here we show that recombinant CLIC-2 increases [3H]ryanodine binding to native and purified RyR channels, enhances substate activity in individual channels, increases the number of rare coupled gating events between associated RyRs, and reduces activation of the channels by their primary endogenous cytoplasmic ligands, ATP and Ca2+. CLIC-2 (0.2-10 microM) added to the cytoplasmic side of RyR2 channels in lipid bilayers depressed activity in a reversible, voltage-independent, manner in the presence of activating (10-100 microM) or sub-activating (100 nM) cytoplasmic Ca2+ concentrations. Although the number of channel openings to all levels was reduced, the fraction and duration of openings to substate levels were increased after exposure to CLIC-2. CLIC-2 reduced increases in activity induced by ATP or adenosine 5'-[beta,gamma-imido]triphosphate. Depression of channel activity by CLIC-2 was greater in the presence of 100 microM cytoplasmic Ca2+ than with 100 nM or 10 microM Ca2+. Further, CLIC-2 prevented the usual approximately 50-fold increase in activity when the cytoplasmic Ca2+ concentration was increased from 100 nM to 100 microM. The results show that CLIC-2 interacts with the RyR protein by a mechanism that does not require oxidation, but is influenced by a conserved Cys residue at position 30. CLIC-2 is one of only a few cytosolic inhibitors of cardiac RyR2 channels, and may suppress their activity during diastole and during stress. CLIC-2 provides a unique probe for substate activity, coupled gating and ligand-induced activation of cardiac RyR channels.

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Figures

Figure 1
Figure 1. CLIC-2 in the cis solution reversibly decreases the activity of Ca2+-activated cardiac RyR2 channels, by reducing the number of open events and by increasing the fraction of events to substate levels
The cis [Ca2+] was 10 μM. Panels (A) and (B) show 2.5 s segments of activity, and all-points histograms for 27 s and 42 s recordings, at +40 mV under control conditions and after addition of 0.5 μM CLIC-2 respectively. The recordings were selected to illustrate substate levels. Broken lines are through prominent current levels at 17%, 38%, 57%, 77% and 100% of Imax (10.5 pA). Continuous lines (c) show the closed current level. The histograms for 27 s (control) and 42 s (CLIC-2) of activity show a net fall in activity and an increased fraction of substate openings in the presence of CLIC-2, especially at 38% Imax (arrow). (C, D) Analysis of eight experiments in which one channel only was active (with periods of occasional high conductance activity in four of them excluded) and clear substate openings. More than 120 s of activity (more than four 30 s segments) at +40 mV were analysed for control, CLIC-2 (clic) and post-perfusion (washout) conditions (CLIC-2 was added at concentrations between 0.2 and 10 μM). (C) Solid bars show the probability of channel openings to levels greater than ∼2 pA (Po), and hatched bars show the probability of channel opening to the maximum single channel conductance [Po(max)]. (D) Percentage of channel openings to substate levels under each condition. (E, F) All-point histograms for selected segments of activity in (A) and (B) respectively.
Figure 2
Figure 2. Occasional coupled gating events
Occasional synchronized openings to 3*Imax were observed. (A) A 30 s recording with a brief burst of such openings and an otherwise high probability of opening to Imax obtained in the presence of 0.2 μM CLIC-2. (B) All-points histogram for the recording with peaks at the closed level (C), Imax (O1), and 3*Imax (O3), as indicated by arrows, as well as the 2*Imax (O2) level, which does not appear as a sharp peak. Panels (C) and (D) show 100 ms of activity with rapid transitions between C (closed level) and 3*Imax (up arrows), and 3*Imax and C (down arrows), separated by transitions between the three open levels. Panels (E) and (F) show 230 ms of activity with frequent transitions between C (closed level) and 3*Imax and each of the open levels, as well as long openings to the single-channel substate (s) (**) and to Imax (****). In (A) and (C)–(F), the solid line shows the closed level, the dotted lines show O1, O2 and O3, and the dashed line shows a level at ∼40% Imax which is assumed to be a single-channel substate (s).
Figure 3
Figure 3. ATP-induced activation of RyR2 is prevented by CLIC-2
(A)–(F) Recordings (3 s) and all-points histograms (60 s; two 30 s recordings), in the presence of 10 μM cis Ca2+, for control conditions (A), after adding 2 mM ATP (B), after washout of ATP (C), after adding 2.4 μM CLIC-2 (D), after adding 2.4 μM CLIC-2 and then 2 mM ATP (E), and after washout of ATP and CLIC-2 (F). Channels open downward from closed (continuous line) to single (O1) or double (O2) open levels (dotted lines). (G, H) Average mean current (I′). Data at −40 and +40 mV were combined for six experiments with ATP (G) and two with p[NH]ppA (H) (n=4 observations). Means±S.E.M. are given for ATP, and means±S.D. are given for p[NH]ppA, under control conditions (con), after 2 mM ATP (+ATP in G) or p[NH]ppA (+PNP in H), after washout (wash), after adding 2.1–4.5 μM CLIC-2 (+CLIC), after adding CLIC-2 plus 2 mM ATP (+ATP in G) or p[NH]ppA (+PNP in H), and after a second washout (wash). (I, J) Reversibility and specificity of CLIC-2. Data at −40 and +40 mV were combined for eight experiments with 2.1–4.5 μM CLIC-2 (in 40–80 μl of vehicle, i.e. 50 mM Hepes buffer plus 10% glycerol, pH 7) (I) and seven experiments with 40–80 μl of buffer alone (J). Values are means±S.E.M. for control (con), after adding CLIC-2 (I) or buffer (J), then after adding ATP, after washout, and finally after adding 2 mM ATP in the absence of CLIC-2. # indicates significant difference from the preceding condition.
Figure 4
Figure 4. Anti-CLIC-2 antibody prevents the effect of CLIC-2 on ATP-induced activation
The recordings show 1 s of continuous activity and all-points histograms for 60 s of channel activity (two 30 s recordings) with cis [Ca2+]=10 μM under control conditions (A), after adding 12 μM CLIC-2 (B), then 2 mM ATP (C), then 5 μl of antibody (D), and finally after washout of ATP, antibody and CLIC-2 (E). Channel opening is downward from the closed level (continuous line) to the maximum single-channel conductance (dotted line). Opening of a second channel is seen in (D) and (E): the first dotted line (O1) is Imax and the second dotted line (O2) is I when the two channels are open. (F) Coomassie Blue-stained gel (left panel) and after staining with the anti-CLIC antiserum (right panel). Arrows point to the high-molecular-mass RyR2 and the lower-molecular-mass CLIC-2. The positions of selected molecular mass markers are shown on the left in kDa. CLIC-2 lies just above the 31 kDa marker. (G) Relative open probability under control conditions, and after adding 7 μM CLIC-2 to the cis chamber (n=4 experiments) or the trans chamber (n=5 experiments).
Figure 5
Figure 5. Effects of ATP on current parameters in the absence and presence of CLIC-2
Each group of two histograms shows average parameters at −40 mV (left) and +40 mV (right). The data are given as means±S.E.M. for open probability (A), mean open time (B) and closed time (C). Data in the absence of CLIC-2 are shown under control conditions with 10 μM cis Ca2+ (con) and after addition of 2 mM ATP (ATP). Data in the presence of CLIC-2 are shown under control conditions (con) and after addition of 4–12 μM CLIC-2 (C2) or CLIC-2 plus 2 mM ATP (ATP). # indicates significant difference from the preceding condition.
Figure 6
Figure 6. CLIC-2 inhibits RyR2 at resting cytoplasmic Ca2+ concentrations and reduces Ca2+-induced activation of the channel
Panels (A)–(E) show 10 s recordings and all-points histograms for 60 s of activity (two 30 s recordings), at +40 mV (left panels) and −40 mV (right panels). Cis [Ca2+] was 100 nM under control conditions. In (A)–(D), channels opened from the closed level (C; continuous line) to the maximum single-channel conductance (O1; dotted line). In the last recording in each panel, openings of one, two or three channels can be seen to levels O1, O2 and O3 respectively in the recordings and histograms. Recordings are shown under control conditions (A), with 9 μM CLIC-2 (B), after increasing cis [Ca2+] to 100 μM (C), after washout and return to 100 nM cis Ca2+ (D), and then after addition of 100 μM Ca2+ in the absence of CLIC-2 (E). (F, G) Data at −40 and +40 mV are combined for 11 experiments with 9 μM CLIC-2 (in 50 μl of vehicle, i.e. 50 mM Hepes buffer plus 200 mM NaCl, pH 7.5) (F) (n=22 observations) and for six experiments with 50 μl of buffer alone (G) (n=12 observations). The bars show averages±S.E.M. of the mean current (I′), with 100 nM cis Ca2+ (CON), with 2–9 μM CLIC-2 [or 50 μl of buffer alone (BUFF)], after increasing [Ca2+] to 100 μM (+Ca), and after washout. # indicates significant difference from the preceding condition.
Figure 7
Figure 7. Effects of increasing the cis Ca2+ concentration from 100 nM to 100 μM on current parameters in the absence and presence of CLIC-2
(A, B) Each group of two histograms shows average parameters at −40 mV (left) and +40 mV (right). Data are shown as means+S.E.M. In the absence of CLIC-2, control data with 100 nM cis Ca2+ (con) and data with 100 μM Ca2+ (Ca2+) are shown. Data with CLIC-2 are control with 100 nM cis Ca2+ (con), after addition of 2–9 μM CLIC-2 (C2), and in the presence of CLIC-2 plus 100 μM Ca2+ (Ca2+). # indicates significant difference from the preceding condition. (C) Data at different Ca2+ concentrations, in the presence (solid bars) or absence (hatched bars) of 0.2–12 μM CLIC-2, plotted as a function of [Ca2+]. In the absence of CLIC-2, n=100 for 100 nM Ca2+, n=42 for 10 μM Ca2+ and n=38 for 100 μM Ca2+. In the presence of CLIC-2, n=22 for 100 nM Ca2+, n=12 for 10 μM Ca2+ and n=22 for 100 μM Ca2+.
Figure 8
Figure 8. CLIC-2 decreases maximum channel openings and enhances substate activity in purified RyR channels
Panels (A) and (B) show 5 s current recordings from a single purified RyR channel at +40 mV, before (A) and after (B) the addition of 8.6 μM CLIC-2 (100 μl) to the cis solution. The closed current level is indicated by the solid line (C), Imax by the dotted line (O), and the dominant substate level by the broken line (S). (C, D) All-points histograms for 30 s of activity before (C) and after (D) addition of CLIC-2. The arrow in (D) points to the dominant substate level.
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