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In skeletal muscle, there is bidirectional signaling between the L-type Ca2+ channel (1,4-dihydropyridine receptor; DHPR) and the type 1 ryanodine-sensitive Ca2+ release channel (RyR1) of the sarcoplasmic reticulum (SR). In the case of “orthograde signalling” (i.e., excitation-contraction coupling), the conformation of RyR1 is controlled by depolarization-induced conformational changes of the DHPR resulting in Ca2+ release from the SR. “Retrograde coupling” is manifested as enhanced L-type current. The nature of this retrograde signal, and its dependence on RyR1 conformation, are poorly understood. Here, we have examined L-type currents in normal myotubes after an exposure to ryanodine (200 μM, 1 hour at 37°C) sufficient to lock RyR1 in a non-conducting, inactivated, conformational state. This treatment caused an increase in L-type current at less depolarized test potentials in comparison to myotubes similarly exposed to vehicle as a result of a ~5 mV hyperpolarizing shift in the voltage-dependence of activation. Charge movements of ryanodine-treated mytoubes were also shifted to more hyperpolarizing potentials (~13 mV) relative to vehicle-treated myotubes. Enhancement of the L-type current by ryanodine was absent in dyspedic (RyR1 null) myotubes, indicating that ryanodine does not act directly on the DHPR. Our findings indicate that in retrograde signaling, the functional state of RyR1 influences conformational changes of the DHPR involved in activation of L-type current. This raises the possibility that physiological regulators of the conformational state of RyR1 (e.g., Ca2+, CaM, CaMK, redox potential) may also affect DHPR gating.
Conformational coupling between plasma membrane channels and ER proteins is emerging as an important mechanism by which diverse cell types regulate intracellular Ca2+ levels. “Orthograde” coupling, in which the plasma membrane channel controls ER Ca2+ release independent of the entry of extracellular Ca2+, has been described in both skeletal muscle (Beam and Horowicz 2004) and neurons (Ouardouz et al. 2003; De Crescenzo et al. 2006; Kim et al. 2007). “Retrograde” coupling whereby a resident ER protein modulates the activity of a plasma membrane ion channel was first described between ryanodine receptors and L-type Ca2+ channels in skeletal muscle and not long after in neurons (Nakai et al. 1996; Nakai et al. 1997; Nakai et al. 1998a; Grabner et al. 1999; Avila and Dirksen 2000; Chavis et al. 1996). The nature of retrograde coupling in each of these cell types remains poorly understood. Here, we have examined how conformational state of an ER protein (type 1 ryanodine-sensitive intracellular Ca2+ release channel; RyR1) influences gating transitions of a plasma membrane protein (L-type Ca2+ channel or 1,4-dihydropyridine receptor; DHPR) in skeletal muscle.
In skeletal muscle, the L-type Ca2+ channel serves as the voltage sensor for excitation-contraction (EC) coupling by triggering gating of RyR1 in response to depolarization of the tranverse tubular membrane (Beam and Horowicz 2004). The resultant Ca2+ efflux into the myoplasm from the sarcoplasmic reticulum (SR) engages the contractile filaments. Since communication between the DHPR and RyR1 is rapid and does not require Ca2+ entry via the L-type channel itself, it is believed that there is a physical interaction between the two proteins (Armstrong et al. 1973; Tanabe et al. 1990; Dirksen and Beam 1999). This view is supported by ultrastructural evidence demonstrating that intra-membranous particles in the plasma membrane, which appear to represent DHPRs, are arranged into groups of four (“tetrads”) with a spacing that places them in register with the four subunits of every other RyR1 (Block et al. 1988; Takekura et al. 1994; Protasi et al. 2002; Takekura et al. 2004; Sheridan et al. 2006).
In addition to the orthograde signal that is transmitted from the DHPR to RyR1, a retrograde signal was revealed by the observation that L-type Ca2+ currents of dyspedic (RyR1 null) myotubes were substantially smaller that L-type currents of myotubes harvested from phenotypically normal littermates despite similar membrane expression of the DHPR (Nakai et al. 1996). Since retrograde coupling persists in the presence of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and depends on the integrity of some of the same structural elements of the DHPR CaV1.1 (α1S) subunit that support orthograde coupling (Grabner et al. 1999; Nakai et al. 1998b; Wilkens et al. 2001; Kugler et al. 2004; Takekura et al. 2004; Bannister et al. 2009), it has been postulated that retrograde coupling is supported by protein-protein interactions between RyR1 and the DHPR.
Studies that have examined the pharmacology of RyR1 in lipid bilayers and/or isolated SR vesicles have demonstrated that lower concentrations of ryanodine (< 1 μM) induce high Po gating (Rosseau et al. 1987; Smith et al. 1988; Buck et al. 1992), while higher concentrations of ryanodine irreversibly lock the channel in a permanently inactivated state (Buck et al. 1992; Zimányi et al. 1992). Significantly, conformational changes induced by high ryanodine result in a 2 nm decrease in distance between adjacent tetradic particles, supporting the hypothesis that DHPRs are docked to RyR1 (Paolini et al. 2004). The observations of Paolini and colleagues (2004) raise the question of whether the conformational state of RyR1 might also regulate gating transitions of the DHPRs. Here, we demonstrate that the high ryanodine-induced conformation of RyR1 enhances skeletal muscle L-type Ca2+ current by shifting activation in the hyperpolarizing direction. Thus, our results indicate that retrograde communication between these channels depends on the conformational state of RyR1 and that, in turn, DHPR gating may be retrogradely influenced by physiological modulators of RyR1.
All procedures involving mice were approved by the University of Colorado-Denver Institutional Animal Care and Use Committee. Primary cultures of phenotypically normal (+/+ or +/mdg) and dyspedic (RyR1−/−) myotubes were prepared as described previously (Beam and Franizini-Armstrong 1997). Cultures were grown for 4–5 days in a humidified 37°C incubator with 5% CO2 in Dulbecco’s Modified Eagle Medium (DMEM; #15-017-CM, Mediatech, Herndon, VA), supplemented with 10% fetal bovine serum/10% horse serum (Hyclone Laboratories, Logan, UT). This medium was then replaced with differentiation medium (DMEM supplemented with 2% horse serum). Ryanodine (#K6017, Sigma, St. Louis, MO) was reconstituted in 40 or 100% EtOH and diluted to either 200 or 10 μM in differentiation medium.
Electrophysiological experiments were performed at room temperature (~25°C) 3–5 days following differentiation. Pipettes were fabricated from borosilicate glass and had resistances of ~2.0 MΩ when filled with internal solution, which consisted of (mM): 140 Cs-aspartate, 10 Cs2-EGTA, 5 MgCl2, and 10 HEPES, pH 7.4 with CsOH. The external solution contained (mM): 145 TEA-Cl, 10 CaCl2, 0.003 tetrodotoxin (TTX), and 10 HEPES, pH 7.4 with TEA-OH. For measurement of intramembrane charge movements, ionic currents were blocked by the addition of 0.5 mM CdCl2 + 0.1 mM LaCl3 to the external solution. Electronic compensation was used to reduce the effective series resistance (usually to < 1 MΩ) and the time constant for charging the linear cell capacitance (usually to < 0.5 ms). L-type currents were corrected for linear components of leak and capacitive current by digital scaling and subtraction of the average of eleven, 30-mV hyperpolarizing pulses from a holding potential of −80 mV. All charge movements were corrected for linear cell capacitance and leakage currents using a -P/6 subtraction protocol. Filtering was at 2 kHz (eight pole Bessel filter; Frequency Devices, Inc.) and digitization was either at 10 kHz (L-type currents) or 20 kHz (charge movements). Voltage clamp command pulses were exponentially rounded with a time constant of 50–500μs and a 1-s prepulse to −20 mV followed by a 50-ms repolarization to −50 mV was administered before the test pulse (prepulse protocol; see Adams et al. 1990) to inactivate low-voltage activated Ca2+ channels and voltage-gated Na+ channels. Cell capacitance was determined by integration of a transient from −80 mV to −70 mV using Clampex 8.0 and was used to normalize current amplitudes (pA/pF).
L-type current-voltage (I-V) curves were fitted according to:
where I is the current for the test potential V, Vrev is the reversal potential, Gmax is the maximum Ca2+ channel conductance, V1/2 is the half-maximal activation potential and kG is the slope factor.
Plots of the integral of the ON charge movement (Qon) as a function of test potential (V) were fitted according to:
where Qmax is the maximal Qon, VQ is the potential causing movement of half the maximal charge, and kQ is a slope parameter.
Figures were made using the software program SigmaPlot (version 7.0, Systat Software, Inc., Chicago, IL). All data are presented as mean ± SEM. Statistical comparisons were by unpaired, two-tailed t-test, with p < 0.05 considered significant.
Since long exposures to a high concentration of ryanodine persistently block RyR1 (Zimányi et al. 1992) and cause rearrangements of DHPRs within tetrads (Paolini et al. 2004), we probed whether a similar exposure to ryanodine would impact the properties of L-type currents. We found that 200 μM ryanodine for > 1 hour at 37°C caused a small, but consistent, hyperpolarizing shift in the voltage-dependence of activation (Figure 1A and B; Table 1) resulting in an increase in L-type current density at less depolarized test potentials in comparison to myotubes similarly exposed to EtOH vehicle. For example, the treatment with ryanodine caused the average current elicited by a depolarization to +20 mV to increase from −7.6 ± 0.8 pA/pF (n = 16) to −11.3 ± 1.2 pA/pF (n = 28; p < 0.05; Figure 1C). Similar effects were observed in experiments in which myotubes were exposed to 20 μM ryanodine for 12–18 hours at 37°C (data not shown). However, the magnitude and voltage-dependence of L-type currents were not affected by the application of a lesser concentration of ryanodine (10 μM) for 30 minutes at room temperature (Figure 1D; Table 1).
Intramembrane charge movements were measured to investigate further the effects of high ryanodine treatment on DHPR gating. The maximal charge movement (Qmax) in myotubes exposed to 200 μM ryanodine did not differ significantly (p > 0.5) from that of myotubes exposed to the vehicle (Qmax = 4.8 ± 0.4 nC/μF; n = 10 vs. 5.3 ± 0.7 nC/μF; n = 7, respectively; Figure 2A-C). However, ryanodine pre-treatment induced a substantial hyperpolarizing shift in the voltage-dependence of charge movement compared to control myotubes (−15.4 ± 2.2 mV vs. −2.3 ± 2.2 mV, respectively; p < 0.005; Figure 2C). Thus, high ryanodine pre-treatment caused hyperpolarizing shifts in both charge movements and activation of L-type Ca2+ current
As a test of whether the effects of ryanodine treatment on L-type channel gating were a consequence of altered RyR1 conformation or of a direct action of ryanodine on the DHPR, the effects of ryanodine pre-treatment on L-type currents were assessed in dyspedic (RyR1 null) myotubes. At +30 mV, the L-type current density of ryanodine-treated dyspedic mytoubes (−2.0 ± 0.4 pA/pF; n = 5; Figure 3B) was similar to that of vehicle-treated dyspedic myotubes (−1.9 ± 0.3 pA/pF; n = 7; p = 0.9; Figure 3A). Moreover, no shift in channel activation was evident in ryanodine-treated dyspedic myotubes (Figure 3C; Table 1). Thus, ryanodine-mediated potentiation of skeletal muscle L-type currents required the presence of RyR1.
In the present study, we have shown that a > 1 hr exposure to 200 μM ryanodine causes hyperpolarizing shifts in both the activation of skeletal muscle L-type Ca2+ current (Figure 1) and in voltage-dependent charge movement (Figure 2). Importantly, these effects depended on the presence of RyR1 because ryanodine had no effect on L-type Ca2+ current in dyspedic myotubes (Figure 3).
The hypothesis that EC coupling depends on physical linkages between the DHPR and RyR1 implies that manipulations directly affecting the conformation of RyR1 might influence conformational changes of the DHPR (although there is no a priori way to estimate the magnitude or polarity of such possible effects). Electrophysiologically, DHPR conformational changes are reflected in both Ca2+ ionic current (Tanabe et al. 1988) and membrane-bound charge movements (Schneider and Chandler 1973; Ríos and Brum 1987; Adams et al. 1990). In regard to L-type Ca2+ currents, previous studies of amphibian skeletal muscle have reported no effects of ryanodine (García et al. 1991a; Gonzalez and Caputo 1996), except for a for a ~5 mV depolarizing shift in activation (Squecco et al. 2004). This opposite effect (as opposed to the hyperpolarizing shift observed in the present study) may be a consequence of the difference in preparations (amphibian muscle vs. mammalian myotubes). Alternatively, the paradigm used for application of ryanodine in the studies of amphibian muscle (≤100 μM at 10–17°C for ≤ 40 minutes) may not have caused the ryanodine receptor to enter into the deeply inactivated state produced by our use of 200 μM ryanodine applied for >1 hr at 37°C. In this regard, we also observed little effect of 10 μM ryanodine applied to mouse myotubes for 30 minutes at ~25°C (Figure 1D).
Consistent with our present results, Balog and Gallant (1999) observed a hyperpolarizing shift in L-type current activation, and a small increase in current density, when ryanodine (1 mM in the patch pipette) was dialyzed into normal mouse myotubes. Our current results both confirm and extend their results in that: i) the ryanodine-induced effects on the L-type current were dependent on the expression of RyR1 (Figure 3), and ii) the increased density was not due to insertion of additional DHPRs into the plasma membrane (Figure 2; see below). Balog and Gallant (1999) postulated that the ryanodine-induced shift in L-type current activation resulted from a decrease in triadic Ca2+ concentration since dialysis of BAPTA produced a similar hyperpolarizing shift. It should be noted, however, that in our experiments on both control and ryanodine-treated myotubes, EGTA (10 mM) was dialyzed for >5 minutes prior to recording L-type currents (and even longer for charge movements). Consequently, resting Ca2+ levels in both experimental groups should have been nearly identical. Thus, we propose an alternative explanation based on previous work showing that treatment with high ryanodine results in a ~2 nm decrease in distance between adjacent tetradic particles (Paolini et al. 2004). Specifically, this altered disposition of tetrads, which is one indicator of altered protein-protein interactions between RyR1 and the DHPR, also affects the gating behaviour of the DHPR. The observation that the effects of ryanodine on the L-type current required the presence of RyR1 also supports the view that the alterations in DHPR gating were a consequence of altered conformational coupling between the two channels.
Several previous studies have examined the effects of ryanodine on charge movements in both amphibian and mammalian preparations. With respect to Qβ the major component of charge movement in amphibian muscle, a number of studies (García et al. 1991a; Gonzalez and Caputo 1996; Huang 1996; Huang 1998; Squecco et al. 2004) have reported no appreciable effect of ryanodine (≤ 200 μM at 2–17°C for 25–120 minutes). However, Gonzales and Caputo (1996) reported that the recovery from depolarization-induced immobilization of Qβ is inhibited by ryanodine treatment (100 μM, ~25 minutes at 10°C). García et al. (1991a) reported that ryanodine (100 μM, ~40 minutes at 17°C) eliminated Qγ, the “hump” of extra charge that is superimposed on the falling phase of Qβ at voltages near the threshold for eliciting Ca2+ release. If, as some have hypothesized (Csernoch et al. 1991; García et al. 1991b), Qγ is a result of the released Ca2+ causing the movement of additional Qβ, then the block of Qγ could be explained by ryanodine block of Ca2+ release. In contrast to the block of Qγ observed by García et al. (1991a), Squecco et al. (2004) reported that 100 μM ryanodine shifted amphibian Qγ in the depolarizing direction without abolishing it. In rat extensor digitorum longus fibres, where only a single component of charge movement is evident, 10 μM ryanodine had little effect on the total charge moved (Fryer et al. 1989). Our current results are consistent with this observation in that a higher concentration of ryanodine did not significantly affect Qmax (Figure 2; Table 2). In addition, we observed a substantial (~13 mV) hyperpolarizing shift in gating charge relative to control myotubes (Figure 2; Table 2). Such a shift provides a mechanistic basis for the concurrent shift in the I-V relationship (Figure 1; Table 1) and larger L-type current amplitude at weaker test potentials.
Our work now shows that opening transitions of the skeletal L-type Ca2+ channel are directly affected by the conformational state of RyR1 such that the ryanodine-inactivated RyR1 enhances entry of the CaV1.1-containing channel into the open state as reflected both in shifted voltage-dependence of activation and increased steady-state current at weaker test depolarizations. The observation that pharmacological modulation of RyR1 conformation affects DHPR gating raises the possibility that physiological modulation of RyR1 (e.g., by Ca2+, CaM, CaMK, PKA, redox potential; for a review, see Meissner 2002) may also influence DHPR activity and thus the cellular processes which are dependent on voltage-driven conformational changes of the DHPR.
We thank Dr. D.C. Sheridan and Mr. J.D. Ohrtman for insightful discussion. This work was supported in part by National Institutes of Health Grants NS24444 and AR44750 (to K.G.B.). R.A.B. was supported by a Developmental Grant from Muscular Dystrophy Association (MDA4155).