Functional Impact of the Ryanodine Receptor on the Skeletal Muscle L-Type Ca²⁺ Channel

Skeletal and cardiac muscle dihydropyridine receptors (DHPRs) are voltage-dependent L-type calcium channels (L-channels) (Tanabe et al. 1988; Mikami et al. 1989) that control the activity of intracellular Ca²⁺ release channels, ryanodine receptors (RyRs), during excitation–contraction (EC) coupling (McPherson and Campbell 1993). In cardiac muscle, the release of intracellular Ca²⁺ from the sarcoplasmic reticulum (SR) depends on the influx of extracellular calcium through cardiac DHPRs (Nabauer et al. 1989). However, influx of extracellular calcium through voltage-gated Ca²⁺ channels is not a requirement for SR Ca²⁺ release or contraction in skeletal muscle (Armstrong et al. 1972; Garcia and Beam 1994; Dirksen and Beam 1999). Rather, EC coupling in skeletal muscle is thought to involve a mechanical interaction between sarcolemmal DHPRs and the skeletal muscle RyR isoform (RyR1) (Schneider and Chandler 1973). During EC coupling in skeletal muscle, the DHPRs undergo voltage-driven conformational changes that result in the activation of SR Ca²⁺ release channels (“orthograde signal”). Thus, the DHPR in skeletal muscle functions as both a voltage sensor for EC coupling (Ríos and Brum 1987) and as a voltage-gated L-channel (Tanabe et al. 1988).

Interestingly, the presence of the RyR1 protein promotes the Ca²⁺ conducting activity and accelerates the activation of skeletal L-channels (“retrograde signal”) (Fleig et al. 1996; Nakai et al. 1996). This finding was based, in part, on experiments involving cultured skeletal myotubes derived from mice homozygous for a disrupted RyR1 gene (RyR1-knockout or dyspedic mice; Takeshima et al. 1994; Nakai et al. 1996). Compared with normal myotubes, dyspedic myotubes exhibit a dramatic reduction in the density of L-current (Fleig et al. 1996; Nakai et al. 1996). Moreover, expression of RyR1 in dyspedic myotubes enhances L-current in the absence of a change in intramembrane charge movement (Nakai et al. 1996). These observations led to the conclusion that RyR1 promotes the Ca²⁺ channel activity of the skeletal muscle DHPR in a manner that is independent of L-channel expression. Moreover, this ability seems to be a unique property of RyR1 since the cardiac isoform (RyR2) is incapable of restoring either EC coupling or robust skeletal L-currents in dyspedic myotubes (Nakai et al. 1997). Thus, the signaling between the skeletal muscle DHPR and RyR1 is bidirectional, such that the channel activity associated with each protein is strongly dependent upon this unique interaction.

Interaction between intracellular signaling molecules, such as G-proteins (for review, see Dolphin 1998) and proteins of the synaptic core complex (SNARE proteins) (for review, see Catterall 1999), markedly alter the functional properties of voltage-dependent Ca²⁺ channels. Thus, the reciprocal nature of the interaction between the DHPR and RyR in skeletal muscle appears to represent an additional example of a voltage-gated Ca²⁺ channel that is functionally modulated by interaction with an intracellular signaling partner. The orthograde and retrograde signals of skeletal muscle EC coupling appear to be mediated by the intracellular loop that links the second and third internal homology repeats (II-III loop) of the skeletal muscle DHPR (Tanabe et al. 1990; Nakai et al. 1998a; Grabner et al. 1999). However, results from the expression of chimeric RyRs into dyspedic myotubes indicate that these two signals are localized to distinct regions within RyR1 (Nakai et al. 1998b). Thus, although dyspedic myotubes provide an excellent model for probing structure/function relationships of RyR1 on skeletal muscle EC coupling, the precise influence of RyR1 on the fundamental properties of skeletal L-channels has yet to be fully elucidated. Here, we describe experiments designed to characterize the influence of the RyR1 protein on the biophysical and pharmacological properties of the skeletal L-channel. Our data demonstrates that L-currents recorded from normal and dyspedic myotubes exhibit two distinct kinetic components that are equivalently enhanced by RyR1. In addition, our results indicate that RyR1 influences L-channel expression, activation kinetics, modulation by DHP agonists, and divalent conductance.

Primary cultures of myotubes were prepared from skeletal muscle of newborn normal and dyspedic mice as described previously (Nakai et al. 1996). The presence (or absence) of RyR1 mutant alleles was determined by PCR analysis on tail snip tissue obtained from each animal used to generate cultures. Thus, each individual culture was identified as homozygous normal (+/+), heterozygous normal (+/−), or dyspedic (−/−). Data obtained from homozygous and heterozygous normal cultures were ultimately pooled together since no significant differences in macroscopic L-channel activity were observed between all normal myotubes (see Table). Expression of RyR1 in dyspedic myotubes was performed as previously described (Dirksen and Beam 1999). In brief, myotubes were microinjected (Tanabe et al. 1988) into a single nucleus with wild-type RyR1 cDNA (0.5 μg/μl) 6–8 d after the initial plating of myoblasts and were examined electrophysiologically 2–4 d later. Expressing myotubes were identified by observing contractions in response to external electrical stimulation (8.0 V, 10–30 ms). In some experiments, expressing myotubes were identified by the development of green fluorescence 2–4 d after coinjection with a mixture of RyR1 cDNA (0.5 μg/μl) and a cDNA expression plasmid encoding an enhanced green fluorescent protein (0.01 μg/μl) (Grabner et al. 1999).

Whole-cell patch-clamp experiments were carried out at room temperature (20–22°C), 7–11 d after the initial plating of myoblasts. For all experiments, the holding potential was −80 mV. T-type Ca²⁺ currents were eliminated by a conditioning prepulse consisting of a 1-s depolarization to −20 mV followed by a 25-ms repolarization to −50 mV before each test pulse (Adams et al. 1990; Dirksen and Beam 1999). Currents were recorded with either a Dagan 3900A (Dagan Corp.) or Axopatch 200A (Axon Instruments Inc.) amplifier and filtered at 2 kHz by a four-pole Bessel filter. Data were digitized at 10 kHz using a DigiData 1200 interface (Axon Instruments, Inc.) and analyzed using the pCLAMP (Axon Instruments, Inc.) and SigmaPlot (SPSS Inc.) software suites. Capacitative currents were minimized (>90%) using the capacitative transient cancellation feature of the amplifier. The remaining linear components were subtracted using a P/3 leak subtraction protocol. Cell capacitance (C_m) was determined by integration of the capacity transient resulting from a +10-mV pulse applied from the holding potential and was used to normalize ionic (pA/pF) and gating (nC/μF) currents obtained from different myotubes. Relatively large dyspedic myotubes were used to compensate for the relatively L-current density (∼1.0 pA/pF) found in these cells. C_m values were (pF): 322 ± 27 (n = 44), 607 ± 55 (n = 38), and 454 ± 33 (n = 82), for normal myotubes, dyspedic myotubes, and all experiments, respectively. The average series (access) resistance (R_s) after compensation was 1.1 ± 0.07 MΩ (n = 82), and the voltage error due to series resistance (V_e = R_s × I_Ca) was less than ∼5 mV (for these experiments, the average was 2.66 ± 0.19 mV, n = 82). The average time constant for charging the membrane capacitance (τ_m = R_s × C_m) was 0.37 ± 0.02 ms (n = 82) and was never larger than 1.21 ms. All data are presented as mean ± SEM.

The whole-cell variant of the patch clamp technique (Hamill et al. 1981) was used to compare the properties of macroscopic L-currents of normal myotubes, uninjected dyspedic myotubes, and dyspedic myotubes injected with cDNA encoding the wild-type rabbit RyR1. Patch pipettes were fabricated from borosilicate glass and had resistances of 1.5–2.0 MΩ when filled with internal solution (see below). Peak inward Ca²⁺ currents were assessed at the end of 200-ms test pulses of variable amplitude and plotted as a function of the membrane potential (I-V curves). I-V curves were subsequently fitted according to:

where V_rev is the extrapolated reversal potential of the calcium or barium current, V is the membrane potential during the test pulse, I is the peak current during the test pulse, G_max is the maximum L-channels conductance, V_G1/2 is the voltage for half activation of G_max, and k_G is the slope factor. The activation phase of macroscopic ionic currents was fitted using one of the following exponential functions ( and ):

where I(t) is the current at time t after the depolarization, A₀, A_fast, and A_slow are the steady state current amplitudes of each component with their respective time constants of activation (τ₀, τ_fast, and τ_slow), and C represents the steady state peak current. In all cases, the fitting procedure started at the zero current level, which corresponded to 5–7 ms after the initiation of the voltage pulse (>10 × τ_m). This approach limited artifacts introduced by the declining phase of Q_on, since the magnitude of Q_on reaches >90% of its maximal value before this time (see Fig. 1). In addition, L-currents were also recorded before and after ionic current blockade with 0.5 mM Cd²⁺ + 0.2 mM La³⁺ in a separate set of experiments. The Cd²⁺/La³⁺-sensitive currents lacked intramembrane charge movements and exhibited nearly identical activation kinetics as those obtained before gating current subtraction (data not shown). Thus, the fast component of L-current activation described in this study is not greatly influenced by the declining phase of the Q_on gating current transient.

Immobilization-resistant intramembrane charge movements were measured in whole-cell mode by a method described previously (Adams et al. 1990). Calcium currents were blocked by the addition of 0.5 mM CdCl₂ + 0.2 mM LaCl₃ to the extracellular recording solution. This combination of Cd²⁺ and La³⁺ effectively blocks ionic calcium currents carried through calcium channels in normal myotubes (Adams et al. 1990; Garcia and Beam 1994; Dirksen and Beam 1995). To prevent amplifier saturation, voltage clamp command pulses were exponentially rounded with a time constant of 240 μs. The amount of immobilization-resistant charge movement was estimated by integrating the transient of charge that moved outward after the onset of the test pulse (Q_on). The magnitude of the maximum immobilization-resistant charge movement (Q_max) was estimated by fitting the Q_on data according to:

where V_Q1/2 and k_Q have their usual meanings with regard to charge movement.

For measurements of macroscopic ionic and gating currents, the internal solution consisted of (mM): 140 Cs-aspartate, 10 Cs₂-EGTA, 5 MgCl₂, and 10 HEPES, pH 7.40 with CsOH. Macroscopic calcium currents were recorded in an external solution containing (mM): 145 TEA-Cl, 10 CaCl₂, 0.003 TTX (Alomone Laboratories), and 10 HEPES, pH 7.40 with TEA-OH. For measurements of macroscopic barium currents, 10 mM BaCl₂ was substituted for the 10 mM CaCl₂ in the external solution. The external calcium current recording solution was supplemented with 0.5 mM CdCl₂ + 0.2 mM LaCl₃ for measurements of intramembrane charge movement. For some experiments, the influence of RyR1 on the stimulatory action of (−) Bay K 8644 (0.005 mM), a pure DHP agonist, was evaluated after addition to the external calcium current recording solution. Except where noted, all chemical reagents were obtained from Sigma Chemical Co.

If the gating charge moved is similar for channels with different P_os, then the density of channel proteins can be estimated from the magnitude of the maximal immobilization-resistant intramembrane charge movement (Q_max) (Adams et al. 1990; Nakai et al. 1996). The values of Q_max reported for normal myotubes originating from the dysgenic line of mice (which lack a functional copy of the skeletal DHPR gene) have varied (nC/μF): 7.9 ± 1.4 (Adams et al. 1990), 7.2 ± 0.5 (Dirksen and Beam 1995), 5.6 ± 2.6 (Nakai et al. 1996), and 5.1 ± 0.9 (Garcia et al. 1994). This variability, coupled with the fact that the dyspedic mice were constructed from a separate mouse lineage, prompted us to compare the magnitude of Q_max in dyspedic myotubes with myotubes derived from their phenotypically normal littermates (normal myotubes). The voltage dependence of the charge movement obtained from these experiments is illustrated in Fig. 1. On average, the Q_max values of normal and dyspedic myotubes were 7.5 ± 0.8 (n = 17) and 4.5 ± 0.4 (n = 14) nC/μF, respectively. Thus, dyspedic myotubes exhibit a 40% reduction in Q_max compared with the magnitude of Q_max obtained from normal myotubes derived from their phenotypically normal littermates. This 40% reduction in the magnitude of Q_max in dyspedic myotubes is quantitatively similar to previous studies that reported a reduction in total DHP binding in dyspedic muscle (25%, Fleig et al. 1996; 50%, Buck et al. 1997). Interestingly, the V_Q1/2 of Q_on in dyspedic myotubes was ∼10 mV more hyperpolarized than that of normal myotubes (Fig. 1 C and Table). Thus, the RyR1 protein not only promotes the long-term functional expression of DHPRs, but also modifies the voltage sensitivity of DHPR's mediated gating currents. The difference in DHPR's gating current sensitivity to activation by voltage (i.e., V_Q1/2) was reversed 2–4 d after nuclear injection of dyspedic myotubes with cDNA encoding wild-type RyR1 (Fig. 1 C and Table). However, the restoration of Q_on voltage sensitivity by RyR1 expression in dyspedic myotubes occurred in the absence of an effect on Q_max (see also Nakai et al. 1996), suggesting that the restoration of a “normal” level of Q_max may reflect a slower process involving protein synthesis and/or membrane trafficking.

Null mutations of a single allele in a diploid organism could potentially result in a reduced probability of gene expression, and thus lead to haploinsufficiency. Therefore, we have investigated whether the presence of a single wild-type RyR1 allele is sufficient to supply the number of ryanodine receptors required to sustain normal L-channel activity. With this in mind, normal myotubes were genotyped and divided into two groups: homozygous normal (+/+) and heterozygous normal (+/−) for the wild-type RyR1 allele. However, no significant (P > 0.4) differences were found in the voltage-dependent parameters of either L-channel conductance or charge movement (Table) between these two genotypic groups. These data are in agreement with morphological studies that indicate that muscle fibers obtained from homozygous and heterozygous normal embryos are structurally indistinguishable (Takekura et al. 1995). Thus, data from homozygous and heterozygous normal myotubes were pooled together in all subsequent analyses.

Dyspedic myotubes exhibit a very modest amount of slowly activating L-current, which is greatly enhanced 2–4 d after nuclear injection of wild-type RyR1 cDNA (Nakai et al. 1996; Fig. 2A and Fig. B). In our experiments, the average peak L-currents were (at +30 mV) (pA/pF): 12.1 ± 0.6 (n = 27), 1.1 ± 0.4 (n = 9), and 8.4 ± 0.6 (n = 6) for normal, dyspedic, and RyR1-expressing dyspedic myotubes, respectively. Since normal myotubes exhibit a larger Q_max compared with uninjected and RyR1-expressing dyspedic myotubes (Fig. 1 and Table), we normalized peak L-current data by the Q_max value for each myotube (current-to-charge ratio). The normalized data (Fig. 2 C) demonstrates that expression of RyR1 in dyspedic myotubes restores the current-to-charge ratio at every potential and suggests that equivalent fractions of L-channels are upregulated in normal and RyR1-expressing dyspedic myotubes. The approximately fivefold difference in the current-to-charge (Fig. 2 C) and conductance-to-charge (G_max/Q_max) ratios between dyspedic myotubes and both normal and RyR1-expressing dyspedic myotubes (Table) supports the conclusion that RyR1 increases L-channel open probability and/or unitary conductance (Nakai et al. 1996).

The slow activation time course of skeletal L-current in cultured mouse myotubes has often been approximated by fitting the data to a single exponential function (Tanabe et al. 1990; Dirksen and Beam 1995; Strube et al. 1996; Beurg et al. 1997). However, L-current activation is apparently more complex. An adequate (χ² < 0.005) quantitative description of activation in BC3H1 cells requires two exponential components (Caffrey 1994). Fig. 3 A demonstrates that activation of L-currents in cultured skeletal myotubes also involves two kinetically distinct components. Single exponential fitting of normal L-currents failed to adequately describe current activation, particularly early during the depolarization (Fig. 3 A, left). However, fitting the data with the sum of two exponentials greatly improved (indicated by a >16-fold reduction in the value of χ²) the description of L-current activation (Fig. 3 A, right). The biexponential nature of the activation of the calcium current is best appreciated by replotting the normalized currents on a semilogarithmic scale (Fig. 3 A, bottom). The use of three exponential components did not significantly improve the fit of the data. The activation time course of the residual calcium current recorded from dyspedic myotubes was also best described by the sum of two exponential components (data not shown).

The second order exponential fitting procedure resulted in the extraction of the steady state amplitudes (A_fast and A_slow) and corresponding activation time constants (τ_fast and τ_slow) that comprise the macroscopic L-current. The influence of RyR1 on the amplitudes and time constants of macroscopic L-currents at +40 mV is summarized in Fig. 3B–D. Amplitudes and time constants were compared between normal (N), dyspedic (Y), and RyR1-expressing (R) dyspedic myotubes after 200-ms depolarizations to +40 mV, a potential at which L-current conductance is maximal (Adams et al. 1990; Garcia et al. 1994; Dirksen and Beam 1995). This analysis revealed that the marked reduction in dyspedic L-current arises from a parallel decrease in both A_fast (−2.20 ± 0.37 and −0.36 ± 0.03 for normal and dyspedic myotubes, respectively) and A_slow (−10.02 ± 1.04 and −1.14 ± 0.11 for normal and dyspedic myotubes, respectively). Thus, the fractional contribution of each component to the macroscopic L-current was similar in the presence and absence of RyR1 (Fig. 3 C). Interestingly, the faster overall activation of dyspedic L-currents (Nakai et al. 1996) arises from an approximately twofold reduction in both τ_fast and τ_slow (Fig. 3 D). Nuclear injection of dyspedic myotubes with wild-type RyR1 cDNA resulted in a marked increase in both A_fast and A_slow and also a corresponding increase in τ_fast and τ_slow (Fig. 3, B–D). Thus, the parallel dependence of both the amplitudes and time constants of the macroscopic L-current on the presence of RyR1 indicates that skeletal L-channels in cultured myotubes exhibit two distinct kinetic gating modes, each of which are modified by interaction with RyR1.

The data illustrated in Table and Fig. 3 suggest that RyR1 influences the functional conformation of the skeletal L-channel. Since DHP modulation of the activity and voltage dependence L-channels is state dependent (Hille 1992), we set out to evaluate the effects of RyR1 on the DHP modulation of L-channels in cultured skeletal myotubes. We first compared whole-cell calcium currents in the absence (control) and presence of the pure DHP agonist, (−) Bay K 8644 (5 μM). Fig. 4 A shows representatives L-currents recorded from normal (left) and dyspedic (right) myotubes elicited by 200-ms depolarizations to +40 mV. To emphasize kinetic differences, control traces are shown after normalization to the peak current recorded in the presence of (−) Bay K 8644 (5 μM). In both myotubes, 5 μM (−) Bay K 8644 produced a 46% increase in peak calcium current (see also Fig. 5A and Fig. C) and a significant acceleration in the activation time course.

As demonstrated in Fig. 3 A, L-current activation was clearly best described by the sum of two exponential functions, and this becomes even more evident in the presence of DHP (where the second-order fit reduced χ² >43-fold, Fig. 4 A, left). This analysis revealed that at +40 mV the DHP-induced acceleration in L-channel activation kinetics in normal myotubes arises primarily from a preferential increase in the amplitude of the fast component (A_fast) of L-current (Fig. 4 B, left). Specifically, (−) Bay K 8644 caused a significant increase (83 ± 12%, n = 6) in the magnitude of A_fast, without significantly altering A_slow. However, the time constants of each component (τ_fast and τ_slow) were not significantly altered in the presence of the DHP agonist (Fig. 4 B, right). In dyspedic myotubes at +40 mV, (−) Bay K 8644 induced an increase in A_fast (174 ± 30%, n = 7) without significantly altering the magnitudes of A_slow, τ_fast, or τ_slow (Fig. 4 C). Consequently, in dyspedic myotubes, the DHP agonist increased the relative contribution of A_fast to the total L-current (at +40 mV, A_fast/[A_fast + A_slow] was 0.24 ± 0.02 in control and 0.49 ± 0.02 in 5 μM (−) Bay K 8644, P < 0.001). Thus, at +40 mV, (−) Bay K 8644 preferentially augmented the magnitude of A_fast in both normal and dyspedic myotubes without altering A_slow, τ_fast, or τ_slow.

The data described in Fig. 4 were obtained for test pulses to +40 mV, a potential at which L-channel conductance is maximal in both the presence and absence of DHP agonist. The effects of (−) Bay K 8644 (5 μM) on the voltage dependence of macroscopic skeletal L-currents and its component parts (A_fast and A_slow) are summarized in Fig. 5. The peak I-V relationships of normal (Fig. 5 A) and dyspedic (C) myotubes were obtained in the absence (•) and presence (○) of (−) Bay K 8644 (5 μM). In both types of myotubes, the DHP agonist produced both a similar increase in total L-current density and a hyperpolarizing shift (∼10 mV, Table) in the macroscopic I-V relation. However, these effects on the overall macroscopic currents arise from qualitatively distinct alterations in the two kinetic components of skeletal L-current. In Fig. 5, the magnitudes of A_fast and A_slow in normal (B) and dyspedic (D) myotubes were normalized by the average peak control value for each data set (A_slow and A_fast were: 11.89 ± 1.10 and 2.30 ± 0.40 pA/pF for normal myotubes, and 1.23 ± 0.16 and 0.47 ± 0.06 pA/pF for dyspedic myotubes). This analysis revealed that, even under control conditions, A_fast activates ∼10 mV more hyperpolarized than A_slow. In normal myotubes, (−) Bay K 8644 increased (up to approximately twofold) the normalized magnitude of both A_fast and A_slow. In addition, the DHP-induced hyperpolarizing shift in the voltage dependence of L-channel activation is reflected as a selective shift in the voltage dependence of A_slow. Interestingly, (−) Bay K 8644 produced an approximately threefold increase in A_fast in the absence of an alteration in the magnitude of A_slow in dyspedic myotubes. The remarkable DHP insensitivity of A_slow in dyspedic myotubes indicates that the RyR1 protein imparts a strong influence on the state-dependent action of DHP agonists on skeletal L-channels.

Equimolar substitution of Ba²⁺ for extracellular Ca²⁺ produces an increase in peak L-current and a hyperpolarizing shift in the voltage dependence of channel activation in cardiac muscle (Kass and Sanguinetti 1984). Increased L-current amplitude has been attributed to a higher L-channel conductance to Ba²⁺ (Fox et al. 1987), while the shift in channel activation involves differences in the ability of Ba²⁺ and Ca²⁺ ions to screen external surface charges (Hille 1992). A similar substitution produces a smaller increase in L-current density and a similar hyperpolarizing shift in channel activation in skeletal muscle (Tanabe et al. 1990). We investigated the influence of the RyR1 protein on the effects of equimolar substitution of Ba²⁺ for extracellular Ca²⁺ on skeletal L-currents in Fig. 6. In both normal and dyspedic myotubes, L-channel activation was shifted ∼10 mV in the hyperpolarized direction upon replacement of extracellular Ca²⁺ with Ba²⁺ (Fig. 6; Table). In normal myotubes, peak L-current magnitude (Fig. 6 B, a) and conductance (Table) were increased (P < 0.01) nearly 40% with Ba²⁺ as the extracellular charge carrier. In contrast, peak L-current magnitude (Fig. 6 B, c) and conductance (Table) were unaltered upon the identical divalent substitution in dyspedic myotubes.

The relative contribution of A_fast and A_slow to the macroscopic Ca²⁺ and Ba²⁺ currents (Fig. 6 B, b and d) were extracted by fitting a second-order exponential function to the activation phase of the ionic currents (Fig. 6 A). Substitution of Ba²⁺ for extracellular Ca²⁺ increased both A_fast and A_slow in normal myotubes (Fig. 6 B, b) without greatly altering either τ_fast or τ_slow (data not shown). The identical divalent substitution failed to significantly alter the magnitude of either A_fast or A_slow in dyspedic myotubes (Fig. 6 B, d). Nevertheless, extracellular Ba²⁺ substitution caused a hyperpolarizing shift in the voltage dependence of both A_fast and A_slow in normal and dyspedic myotubes. Thus, the skeletal muscle ryanodine receptor appears to influence relative L-channel divalent conductance, but not the differential ability of Ca²⁺ and Ba²⁺ ions to modify external surface charge. These data suggest that the interaction of RyR1 with the skeletal muscle DHPR may exert long-range effects on the functional conformation of the pore of the skeletal L-channel. However, a direct evaluation of the effects of RyR1 on skeletal L-channel selectivity must await a systematic determination of the monovalent and divalent permeability sequence of L-channels in normal and dyspedic myotubes.

In skeletal muscle, the coupling of sarcolemmal depolarization to the release of SR calcium is thought to involve a direct physical interaction between the DHPR and the RyR1. If the interaction of RyR1 with the DHPR stabilizes certain L-channel conformational states by altering transition rates between states, then disruption of this interaction may result in altered L-channel function. However, no study has systematically characterized the influence of RyR1 on the biophysical and pharmacological properties of the skeletal L-current. Consequently, we have evaluated the influence of RyR1 on the expression level, voltage dependence, activation rate, DHP modulation, and divalent conductance of the skeletal L-channel. Our results indicate that the density of functional sarcolemmal L-channels is reduced by ∼40% in dyspedic myotubes compared with that of myotubes derived from their phenotypically normal littermates. We also demonstrated that dyspedic L-currents exhibit accelerated activation (τ_fast and τ_slow), a greater separation between Q-V and G-V relationships ([V_G1/2 − V_Q1/2] was 16.7, 33.7, and 13.5 mV for normal, dyspedic, and RyR1-expressing dyspedic myotubes, respectively), and similar macroscopic conductances to Ca²⁺ and Ba²⁺. The ability of RyR1 to enhance the coupling between charge movement and pore opening (i.e., reduce Q-V/G-V separation) and alter the relative conductance to divalent ions (Ca²⁺ versus Ba²⁺) suggests that RyR1 imparts long-range effects on the conformational state of the pore of the skeletal L-channel. In addition, (−) Bay K 8644 increased the magnitude of both the fast (A_fast) and slow (A_slow) components of the total L-current in normal myotubes, but only enhanced the fast component in dyspedic myotubes. Thus, our results indicate that the presence of RyR1 in skeletal muscle imparts a strong influence on several essential properties of the skeletal L-channel.

Previous studies have demonstrated that dyspedic muscle exhibits a 25–50% reduction in total DHP binding capacity compared with normal muscle (Fleig et al. 1996; Buck et al. 1997). Our finding that Q_max of dyspedic myotubes is reduced ∼40% compared with normal myotubes is in agreement with these reports and indicates that RyR1 promotes DHPR expression in skeletal muscle. However, this 40% reduction in DHPR expression cannot fully account for the ∼90% reduction in the value of G_max in dyspedic myotubes. Even after normalization of peak currents (Fig. 2) and G_max (Table) by Q_max, DHPRs in normal myotubes possess an approximately fivefold higher Ca²⁺-conducting activity compared with that of dyspedic myotubes. Thus, our results support the conclusion of Nakai et al. 1996 that RyR1 enhances either the open probability and/or the unitary conductance of skeletal L-channels. Interestingly, DHPR Ca²⁺-conducting activity (I_ca/Q_max and G_max/Q_max) was completely restored 2–4 d after nuclear injection of dyspedic myotubes with RyR1 cDNA without altering the magnitude of Q_max. Apparently, introduction of RyR1 proteins into dyspedic myotubes restores existing L-channel activity before increasing DHPR expression (as reflected in Q_max). It will be important for future experiments to determine whether or not long-term expression of RyR1 is able to restore Q_max to a value similar to that of normal myotubes and whether these changes are also associated with alterations in EC coupling. Interestingly, the preferential influx of Ca²⁺ through L-channels activates the calcium/calcineurin/NF-ATc transcription pathway and results in the induction of the type 1 1,4,5-inositol trisphosphate receptor in hippocampal neurons (Graef et al. 1999). Thus, it will be important to determine whether the enhancement of L-channel expression in skeletal myotubes also involves activation of a calcium-mediated transcription pathway (via Ca²⁺ influx and/or SR Ca²⁺ release).

The activation kinetics of skeletal L-currents in cultured myotubes has often been approximated by fitting the activation time course to a single exponential function. This approach has provided a convenient means for making qualitative comparisons in channel kinetics under different conditions (Tanabe et al. 1991; Dirksen and Beam 1995; Beurg et al. 1997). However, a precise quantitative description of skeletal L-current activation appears to require the sum of two exponential components (Fig. 3; and see Caffrey 1994). For example, Caffrey 1994 demonstrated that macroscopic L-current activation in BC3H1 myotubes was best described by the sum of two ascending exponential terms with time constants (τ₁ = 2–20 ms and τ₂ = 10–200 ms) similar to those reported here. Moreover, the voltage dependence and relative contribution of the faster component in BC3H1 cells (∼25% at +40 mV) is comparable with our results using primary myotube cultures. Our analyses indicate that while a single exponential often results in a reasonable approximation of L-channel activation at strong depolarizations (>20 mV), two activation terms are clearly required under conditions that enhance the relative contribution of the fast component of L-current activation. An increase in the relative contribution of A_fast results in a clear bi-exponential activation for L-currents activated at threshold potentials (e.g., −10 mV) and for L-currents in dyspedic myotubes treated with (−) Bay K 8644 (Fig. 4 A). In addition, we have recently reported that prolonged depolarization markedly increases the relative contribution of A_fast to the total L-current, resulting in a clearly bi-exponential L-channel activation time course (O'Connell and Dirksen 2000).

Apparent channel activation can be altered when a significant degree of inactivation occurs during the activation process. Thus, inactivation occurring during our 200-ms test pulses could influence the kinetic properties of L-channel activation (A_fast, τ_fast, A_slow, and τ_slow) reported here. Since we have not systematically characterized inactivation in normal and dyspedic myotubes in this study, we cannot rule out a possible contribution of inactivation to the effects of RyR1 on the kinetic properties of L-channel activation. However, any effects of inactivation on apparent L-channel activation would be anticipated to be minimal in our experiments since inactivation of L-currents is very slow (∼25× slower than τ_slow described here) in myotubes (Harasztosi et al. 1999).

Normal myotubes possess T-type Ca²⁺ channels (Adams et al. 1990; Dirksen and Beam 1995) that exhibit a similar rate of activation as that associated with A_fast. However, our data strongly suggest that A_fast does not arise from T-type channels. First of all, the sum of two exponentials is also required to fit L-current activation observed in normal myotubes that exhibit vanishingly small (or undetectable) T-type Ca²⁺ current (data not shown). Moreover, the magnitude of A_fast peaks at approximately +30 mV, ∼40 mV more depolarized than the peak of the current–voltage relationship of T-type Ca²⁺ channels (Garcia and Beam 1994). In addition, A_fast is potentiated by (−) Bay K 8644 (Fig. 4 and Fig. 5) and exhibits a greater conductance for Ba²⁺ than Ca²⁺ (Fig. 6 B). These observations clearly contrast with the classic profile of T-type Ca²⁺ channels (Hille 1992), and are most consistent with the notion that A_fast originates from L-type Ca²⁺ channels.

Dysgenic myotubes, which lack an intact gene for the skeletal muscle DHPR, exhibit a rapidly activating, DHP-sensitive L-current (I_dys; Adams and Beam 1989) that may be attributable to the cardiac α_1C subunit. It is also likely that the channels that account for I_dys make a significant contribution to the residual immobilization-resistant intramembrane charge movement found in dysgenic myotubes (Q_dys; Adams et al. 1990). The fast component of L-current activation (A_fast) described in this study resembles I_dys in that both currents peak at approximately +20 mV, activate rapidly (τ_act ∼ 5 ms), and are augmented two- to threefold by (−) Bay K 8466. However, several findings suggest that the fast component of skeletal L-current activation is distinct from I_dys. For example, I_dys is more strongly stimulated by (−) Bay K 8644 than L-current in normal myotubes (Adams and Beam 1989; Strube et al. 1998). However, the data in Fig. 5 shows that (−) Bay K 8644 stimulates both kinetic components of L-current activation (A_fast and A_slow) to a roughly similar degree. In addition, expression of RyR1 in dyspedic myotubes causes a marked increase in A_fast without altering the magnitude of Q_max (Fig. 3), an observation clearly inconsistent with I_dys as the identity of A_fast. Finally, substitution of Ba²⁺ for extracellular Ca²⁺ doubles the size of I_dys, but only enhanced A_fast in normal, and not dyspedic, myotubes (Fig. 6B and Fig. D). Thus, our data are inconsistent with I_dys as the identity of the fast component of skeletal L-channel activation, but rather support the notion that A_fast represents an intrinsic gating property of the skeletal L-channel.

It is uncertain whether the two components of L-channel activation in mouse skeletal myotubes reflect the gating of separate ion channels or two gating modes of a single Ca²⁺ channel protein. However, our data support the latter possibility since RyR1 regulates several properties of these two components in a quantitatively similar manner. For example, A_fast and A_slow are each reduced approximately sevenfold in the absence of RyR1, resulting in a constant relative contribution of fast and slow components to the total L-current in both normal and dyspedic myotubes. Interestingly, RyR1 expression increased A_fast to a value comparable with that of normal myotubes and only partially restored A_slow, resulting in a moderately significant (P < 0.05) increase in the relative contribution of A_fast. In addition, both τ_fast and τ_slow are approximately twofold faster in dyspedic myotubes, and expression of RyR1 in dyspedic myotubes restores both τ_fast and τ_slow to values similar to those of normal myotubes. Based on single channel recordings (Dirksen and Beam 1996) and gating current measurements (Dirksen and Beam 1999), slow skeletal L-channel activation has been accounted for by a linear reaction scheme in which the rate-limiting transition exhibits an asymmetric voltage dependence. According to this scheme, faster L-channel activation observed in the absence of RyR1 (dyspedic myotubes) could arise from an increase in the forward rate constant (“δ” in the model of Dirksen and Beam 1996) governing this rate-limiting transition. In this way, RyR1 may act like a tether stabilizing one or more of the closed states traversed during channel activation. According to this hypothesis, single L-channels recorded from dyspedic myotubes would be anticipated to exhibit a briefer time to first opening after depolarization and possibly a decrease in channel closed times.

Skeletal muscle dihydropyridine receptors and ryanodine receptors colocalize in clusters that are randomly distributed in punctate foci throughout the muscle cell (Flucher et al. 1993; Franzini-Armstrong and Jorgensen 1994). These clusters occur at junctions between terminal SR and both the surface and transverse-tubule (t-tubules) membranes and presumably represent functional sites of skeletal muscle EC coupling (Franzini-Armstrong et al. 1991; Franzini-Armstrong and Jorgensen 1994). Within these junctions, SR Ca²⁺ release channels are packed in highly ordered arrays. Moreover, clusters of four evenly spaced membrane particles (tetrads), apparently representing DHPRs, are positioned in the sarcolemma such that each particle is located immediately above each of the four RyR1 subunits of the release channel homotetramere (Block et al. 1988). In dyspedic myotubes, junctions containing large clusters of DHPRs are present periodically throughout the sarcolemma (though in a limited number; Takekura et al. 1995). Thus, RyR1 proteins are not required for the targeting of DHPRs to the junctional domains. However, the junctional clusters of DHPRs in dyspedic myotubes are not organized into tetrads, indicating that RyR1 proteins dictate the positioning of DHPRs into tetrads (Takekura and Franzini-Armstrong 1999).

Our results, in which injection with RyR1 cDNA restores both the I_Ca/Q_max (Fig. 2) and G_max/Q_max (Table) ratios of dyspedic myotubes to values comparable with those of normal myotubes, indicate that the vast majority of dyspedic sarcolemmal L-channels are functionally “recoupled” upon RyR1 expression. Thus, it is possible that the expressed RyR1 proteins are efficiently targeted to the majority of junctions throughout the injected dyspedic myotubes. However, Lorenzon et al. 1999 demonstrated that the distribution of GFP-tagged RyR1 proteins expressed in dyspedic myotubes are restricted to the region surrounding the site of injection. Since our experiments were performed on morphologically compact myotubes and we have not monitored RyR1 localization after nuclear injection, the spatial restrictions of the expressed RyR1 proteins in our experiments cannot be easily inferred. However, it will be interesting to determine whether the periodic distribution of DHPRs throughout the dyspedic sarcolemma are reorganized in such a way as to permit interaction with a restricted distribution of RyR1 proteins.

The functional properties of voltage-dependent Ca²⁺ channels are markedly influenced by direct interactions with auxiliary Ca²⁺ channel subunits and intracellular signaling proteins. With regard to L-channels, β-subunits augment peak Ca²⁺ current by increasing the number of channels in the surface membrane (Lacerda et al. 1991; Singer et al. 1991; Wei, et al. 1991), increasing channel open probability (Neely et al. 1993; Shistik et al. 1995), and facilitating channel pore opening (Neely et al. 1993). In addition, β-subunits also accelerate the kinetics of L-channel activation and inactivation (Singer et al. 1991; Wei et al. 1991) and shift activation to more hyperpolarized voltages (Neely et al. 1993). In skeletal muscle, the β₁-subunit accelerates L-channel activation (Lacerda et al. 1991; Varadi et al. 1991), augments total DHP binding (Lacerda et al. 1991), and promotes the targeting of α₁-subunits to the plasma membrane (Strube et al. 1996; Beurg et al. 1997). In addition, the β_1a-subunit, and not the β_2a-subunit, restores both L-current and EC coupling in myotubes derived from mice carrying a null mutation in the β₁ gene (Beurg et al. 1999). The α₂-δ-subunit generally acts to potentiate the effects of β subunits on L-current amplitude and kinetics (Singer et al. 1991) and both the α₂-δ- and β-subunits enhance DHP binding to L-channels (Singer et al. 1991).

Several important intracellular signaling molecules are also known to interact and modify the functional properties of voltage-dependent Ca²⁺ channels. For example, the properties of neuronal N- and P/Q-type Ca²⁺ channels are modulated by interaction with synaptic membrane proteins (e.g., syntaxin and the 25-kD synaptosome-associated protein, SNAP25) that control vesicle docking and membrane fusion during neurotransmitter release (for review, see Catterall 1999). G protein β/γ-subunits directly inhibit neuronal N-, P/Q-, and R-type Ca²⁺ channels by reducing current amplitude, slowing channel activation, and shifting channel activation to more depolarized potentials (for review, see Dolphin 1998). Additionally, Ca²⁺-dependent inactivation of cardiac L-channels has recently been suggested to involve a constitutive interaction between calmodulin and the Ca²⁺-channel complex (Peterson et al. 1999). The interaction of ryanodine receptors with L-channels in skeletal muscle (Nakai et al. 1996) and neurons (Chavis et al. 1996) causes an enhancement of Ca²⁺ flux through these channels. Our results demonstrate that the skeletal muscle ryanodine receptor also modifies other important channel properties, including L-channel expression level, voltage dependence and kinetics of activation, modulation by DHPs, and the relative conductance to Ca²⁺ and Ba²⁺. These results support the notion that RyR1 is an important allosteric modulator of the skeletal L-channel, analogous to that of a conventional Ca²⁺ channel accessory subunit.

We thank Drs. Kurt G. Beam and Paul D. Allen for providing us access to the dyspedic mice used in this study, as well as for their advice and continued support. We also thank Dr. Ted Begenisich for helpful discussions and comments on the manuscript and Linda Groom for excellent technical assistance.

This work was supported by National Institutes of Health grant AR44657 (R.T. Dirksen), a Neuromuscular Disease Research grant (R.T. Dirksen), and CONACYT postdoctoral fellowship 990236 (G. Avila).