Report

Coupled Gating Between Individual Skeletal Muscle Ca2+ Release Channels (Ryanodine Receptors)

See allHide authors and affiliations

Science  07 Aug 1998:
Vol. 281, Issue 5378, pp. 818-821
DOI: 10.1126/science.281.5378.818

Abstract

Excitation-contraction coupling in skeletal muscle requires the release of intracellular calcium ions (Ca2+) through ryanodine receptor (RyR1) channels in the sarcoplasmic reticulum. Half of the RyR1 channels are activated by voltage-dependent Ca2+ channels in the plasma membrane. In planar lipid bilayers, RyR1 channels exhibited simultaneous openings and closings, termed “coupled gating.” Addition of the channel accessory protein FKBP12 induced coupled gating, and removal of FKBP12 uncoupled channels. Coupled gating provides a mechanism by which RyR1 channels that are not associated with voltage-dependent Ca2+channels can be regulated.

Intracellular Ca2+ release channels, present in the endoplasmic (or sarcoplasmic) reticulum of virtually all cells, are integral to diverse signaling pathways that require translation of electrical or biochemical extracellular signals into intracellular activation of Ca2+-dependent molecules. The Ca2+ release channels in skeletal muscle comprise four 565-kD type 1 RyR subunits and four molecules of the 12-kD protein FKBP12 (1). FKBP12, which stabilizes the RyR1 complex and enables the four subunits to open and close coordinately (2), is a member of the immunophilin family of cis-trans peptidyl-prolyl isomerases that serve as cytosolic receptors for immunosuppressant drugs including rapamycin and FK506 (3).

Recombinant RyR1 expressed in insect (Sf9) cells in the absence of FKBP12 forms channels with multiple subconductance states, consistent with a defect in coordination of the activity of the four channel subunits (2). Addition of FKBP12 to recombinant RyR1 stabilizes the channel complex, resulting in the formation of channels with full conductance (2). This stabilizing effect is reversed by treating the channels with rapamycin or FK506 to remove FKBP12 from RyR1 (2).

The cytosolic domain of RyR1 projects into the space that separates the transverse tubule (T-tubule) and the sarcoplasmic reticulum (SR). A cytosolic domain of the α1 subunit of voltage-dependent Ca2+ channels (VDCCs) in the T-tubule is required for activation of RyR1 during excitation-contraction (E-C) coupling (4). Fragments of this domain can activate or inactivate RyR1 (5–7), indicating that E-C coupling may involve a protein-protein interaction between the two types of Ca2+ channels. Clusters of four VDCCs in the T-tubule overlie only every other RyR1 channel (8). Thus, a cytosolic loop from a VDCC is directly apposed to each subunit of only half of the RyR1 channels.

Recombinant RyR1 coexpressed with FKBP12 in Sf9 cells formed Ca2+-activated Ca2+ channels that exhibited stable openings to 4 pA in planar lipid bilayers (Fig. 1A) (2). A current amplitude histogram (Fig. 1D) revealed two discrete peaks corresponding to closed channels (0 pA) and openings to the full amplitude of a single channel (4 pA). In some experiments (9 of 44), two channels opening and closing (gating) independently in the same bilayer were observed (Fig. 1B). In these experiments, one channel opened to the 4-pA level and a second channel was clearly apparent, opening independently of the first. A current amplitude histogram (Fig. 1E) revealed three discrete peaks corresponding to closed channels (0 pA) and openings to the full amplitude for one channel (4 pA) or for two channels (8 pA).

Figure 1

Coupled gating of channels formed from recombinant skeletal muscle RyR1 coexpressed with FKBP12. (A) A single recombinant RyR1 channel with openings to 4 pA. Liposomes (22) were fused to planar lipid bilayers (23), and channels were activated with 1 mM caffeine. (B) Recording from two independent channels. (C) RyR1 channels opening to 8 pA. The time scales are expanded in the top two traces in (A) through (C) and compressed in the bottom trace in (A) and (C). Recordings were made at 0 mV, channel openings are in the upward direction, and the arrows at the left of the traces in (A) through (C) indicate (from bottom to top, respectively) the 0-, 4-, and 8-pA current levels. (D through F) Current amplitude histograms of channels shown in (A) through (C), respectively. (G) Current-voltage relations for the channel in (A) (▪) and the coupled channels in (C) (○). Data are means ± SD. (H and I) Physical association between RyR1 Ca2+ release channels was demonstrated by centrifugation of high density SR from skeletal muscle, after incubation with [3H]ryanodine and solubilization with CHAPS detergent, through 10 to 32% (w/v) linear sucrose density gradients (2) for 17 or 10 hours, respectively. In (H), the 25% sucrose fraction contained the 30S RyR1 complex (arrow), and ∼60S complexes were in the pellet. In (I), the 30S complex (arrow) was present in the 20% sucrose fraction, and an ∼60S complex (arrowhead) was present in the 24% sucrose fraction. The inset in (I) shows immunoblot analysis of samples from the 20% (30S) and 24% (60S) sucrose fractions with specific antibodies to RyR1 (9). Coomassie staining showed that these fractions contained a single high molecular weight protein (17). Only the bottom portion of each gradient is shown, fraction 1 being the top of the gradient. Data are representative of six similar experiments.

The single-channel properties of recombinant RyR1 coexpressed with FKBP12 were identical to those of native RyR1 from SR vesicles (2). The native RyR1 exhibited the typical current amplitude of 4 pA (Fig. 2, A and B). In some experiments (12 of 56), two channels were observed in the bilayer (Fig. 2B); channel openings to the 4-pA level and a second channel opening to 8 pA were apparent. A current amplitude histogram (Fig. 2E) revealed three discrete peaks corresponding to closed channels (0 pA) and openings to 4 and 8 pA.

Figure 2

Coupled gating between native skeletal muscle RyR1 channels (23). (A) High density SR vesicles were fused to planar lipid bilayers, and RyR1 channels opening to 4 pA were activated with 1 mM caffeine. (B) Two independent 4-pA RyR1 channels active in the bilayer. (C) RyR1 channels opening to 8 pA. (Dthrough F) Current amplitude histograms of channels shown in (A) through (C), respectively. Recordings were made at 0 mV, and channel openings are in the upward direction. The arrows at the left of the traces in (A) through (C) indicate 0, 4, and 8 pA.

In ∼10% of experiments with either recombinant (4 of 44) (Fig. 1C) or native (5 of 56) (Fig. 2C) RyR1, channels were observed that opened to 8 pA, twice the normal current amplitude. Current amplitude histograms (Figs. 1F and 2F) revealed two discrete peaks corresponding to closed channels (0 pA) and openings to 8 pA. RyR1 channels exhibit a conductance of ∼100 pS when Ca2+ (50 mM) is the current carrier at 0 mV (9). The conductances were 93 ± 18 pS for the single-amplitude openings and 180 ± 20 pS for the double-amplitude openings (Fig. 1G).

If both the 4-pA and the 8-pA openings represented activity of two independent RyR1 channels in the bilayer, then the binomial distribution of open probabilities would provide a calculated open probability (P1 calc) for the 4-pA current equal to the experimental value P1. The probability of the 4-pA openings in Fig. 1B predicted by a binomial distribution (P1 calc) equalled the experimentally observed value P1 (P > 0.05, Student's ttest). The same analysis applied to the open probabilities of currents in Fig. 1C showed that P1 calc did not match the experimentally observed value for P1 (P < 0.001, Student's t test). The failure of a binomial distribution based on the open probability of the 8-pA currents (P2) in Fig. 1C to predict the open probability of the 4-pA currents (P1) indicates that the 8-pA currents did not result from openings of two independent channels (10). Thus, the gating of two channels in Fig. 1C was likely coupled. Application of the binomial distribution is limited by the fact that it cannot distinguish between the presence of two interdependent cooperative channels each exhibiting a 4-pA current only, and that of a single channel with current amplitudes of 4 and 8 pA. However, if the actual current amplitude for RyR1 is 8 pA, the conductance of the channel would be exactly twice that measured for RyR1 in previous studies (2, 11, 12). Moreover, the 8-pA currents showed the same biophysical properties (including dwell times and open probabilities) and responded to pharmacological modulators of RyR1 in exactly the same manner as did the 4-pA currents (13).

Ultrastructural studies of skeletal muscle have shown that RyR1 channels are tightly clustered square structures arrayed in regular rows, and that the corners of adjacent channels contact one another (14, 15). Coupled gating was observed in ∼10% of experiments with either recombinant or native RyR1 channels. Electron micrographs of purified RyR1 homotetramers show that ∼10% of these structures are physically connected to form contacting pairs (14). We incubated high density SR with [3H]ryanodine and, after solubilization, subjected the preparation to sucrose density gradient centrifugation under conditions designed to separate individual 30S RyR1 homotetramers from dimers of two physically associated channels that sediment at ∼60S (16). Approximately 10% of the RyR1 channels from high density SR preparations sedimented at ∼60S, corresponding to two physically associated RyR1 channels (Fig. 1, H and I). Specific [3H]ryanodine binding was also detected in fractions with sedimentation coefficients of >60S, suggesting that more than two RyR1 homotetramers can remain physically connected during purification. We have observed 12-, 16-, and 20-pA openings, indicative of coupled gating of three, four, and five channels, respectively (17). The physical association of RyR1 channels was not affected by removal of FKBP12 (17).

In experiments in which RyR1 was expressed in insect cells in the absence of FKBP12 (Sf9 cells do not contain FKBP12), subconductance states (openings to <4 pA) (2) were observed. In some of these experiments (3 of 44), both subconductance states and rare openings with currents of >4 pA were observed (Fig. 3A). A current amplitude histogram (Fig. 3E) revealed a distribution of events with currents from 1 to 8 pA, consistent with the presence of two uncoupled channels. Addition of FKBP12 eliminated subconductance states and induced stable openings to 8 pA (Fig. 3B). A current amplitude histogram (Fig. 3F) revealed two discrete peaks corresponding to closed channels (0 pA) and openings to 8 pA, consistent with coupled gating of two channels.

Figure 3

Requirement of FKBP12 for coupled gating of RyR1 channels. (A) Two recombinant RyR1 channels (expressed without FKBP12) activated with 1 mM caffeine. (B) The same two channels as in (A) 30 min after addition of FKBP12. (C) Native RyR1 activated with 1 mM caffeine and exhibiting 8-pA openings. (D) Subconductance states of the same two channels as in (C) 10 min after addition of rapamycin (2 μM). (E through H) Current amplitude histograms of channels shown in (A) through (D), respectively. Recordings were made at 0 mV, and channel openings are in the upward direction. The arrows at the left of the traces in (A) through (D) indicate 0, 4, and 8 pA. Data are representative of three experiments with three preparations of recombinant or native channels.

Amplitude histograms (Fig. 3, E and F) showed that the probability of 8-pA openings increased markedly in the presence of FKBP12. Effective removal of FKBP12 by the addition of rapamycin (2 μM) induced two coupled RyR1 channels (Fig. 3, C and G) to uncouple (Fig. 3, D and H). The low probability of 8-pA openings in the absence of FKBP12, compared with the high probability of eight such openings in its presence, suggests that coupled gating is a function of FKBP12. These data indicate that FKBP12 has two effects: (i) it permits the four subunits of the RyR1 channel to gate coordinately, thereby eliminating subconductance states, and (ii) it enhances functional coupling between two RyR1 channels, resulting in 8-pA openings. Indeed, a recent study of mice lacking FKBP12 demonstrated subconductance states and no evidence of coupled gating for both RyR1 channels from skeletal muscle and RyR2 channels from cardiac muscle (18). Addition of the channel blocker ruthenium red (5 μM), which is often used to establish the identity of RyR1 channels, to 8-pA channels resulted in a marked decrease in channel activity (Fig. 4). Despite the evidence in favor of coupled gating, it was possible that the 8-pA openings were mediated by previously unidentified type of channel, a rare mode of the normal 4-pA RyR1 channel, or a partially degraded form of the normal channel. These possibilities were rendered less likely by the fact that coupled gating was not observed in 10 experiments with 30S RyR1 channels isolated by sucrose density gradient centrifugation, but it was observed in four experiments with purified RyR1 channels reconstituted from the 60S complex (17).

Figure 4

Inhibition of coupled native skeletal muscle RyR1 channel activity by ruthenium red (5 μM). Vertical arrows indicate the addition of ruthenium red. Recordings were made at 0 mV, and channel openings are in the upward direction. Arrows at the left indicate 0, 4, and 8 pA. Time calibration is 10 ms in upper traces and 500 ms in lower traces.

Coupled gating, modulated by FKBP12, is compatible with previous observations supporting a role for Ca2+-induced Ca2+ release in skeletal muscle (19–21). The movement of Ca2+ through one RyR1 channel should increase the Ca2+ concentration on the cytoplasmic side of neighboring channels and might play a role in activating channels that are not associated with a VDCC. However, coupled gating is observed in the absence of luminal Ca2+, such as when Ba2+is the current carrier (17), suggesting that Ca2+ cannot be the only signal required for coupled gating.

Taken together, our data show that physical and functional association between RyR1 channels in skeletal muscle can be preserved during isolation. The physical association between RyR1 homotetramers is not dependent on FKBP12 (17). Two or more physically connected RyR1 channels can open and close simultaneously. The functional association between RyR1 channels, which we have termed coupled gating, is enhanced by FKBP12. In skeletal muscle, the T-tubule–SR junctions are discontinuous such that each junction forms a discrete subset of all of the RyR1 channels in a skeletal myotube. Through coupled gating, activation of one RyR1 channel might activate all of the RyR1 channels in a junction. In skeletal muscle, the T-tubule–SR junctions are discontinuous such that each junction forms a discrete subset of all the RyR1 channels in a skeletal myotube. Through coupled gating, activation of one RyR1 channel might activate all of the RyR1 channels in a junction. Coupled gating provides a mechanism for the concerted activation of RyR1 Ca2+ release channels during skeletal muscle E-C coupling.

  • * To whom correspondence should be addressed. E-mail: arm42{at}columbia.edu

REFERENCES AND NOTES

View Abstract

Navigate This Article