A malignant hyperthermia-inducing mutation in RYR1 (R163C): consequent alterations in the functional properties of DHPR channels

Abstract

Bidirectional communication between the 1,4-dihydropyridine receptor (DHPR) in the plasma membrane and the type 1 ryanodine receptor (RYR1) in the sarcoplasmic reticulum (SR) is responsible for both skeletal-type excitation-contraction coupling (voltage-gated Ca(2+) release from the SR) and increased amplitude of L-type Ca(2+) current via the DHPR. Because the DHPR and RYR1 are functionally coupled, mutations in RYR1 that are linked to malignant hyperthermia (MH) may affect DHPR activity. For this reason, we investigated whether cultured myotubes originating from mice carrying an MH-linked mutation in RYR1 (R163C) had altered voltage-gated Ca(2+) release from the SR, membrane-bound charge movement, and/or L-type Ca(2+) current. In myotubes homozygous (Hom) for the R163C mutation, voltage-gated Ca(2+) release from the SR was substantially reduced and shifted ( approximately 10 mV) to more hyperpolarizing potentials compared with wild-type (WT) myotubes. Intramembrane charge movements of both Hom and heterozygous (Het) myotubes displayed hyperpolarizing shifts similar to that observed in voltage-gated SR Ca(2+) release. The current-voltage relationships for L-type currents in both Hom and Het myotubes were also shifted to more hyperpolarizing potentials ( approximately 7 and 5 mV, respectively). Compared with WT myotubes, Het and Hom myotubes both displayed a greater sensitivity to the L-type channel agonist +/-Bay K 8644 (10 microM). In general, L-type currents in WT, Het, and Hom myotubes inactivated modestly after 30-s prepulses to -50, -10, 0, 10, 20, and 30 mV. However, L-type currents in Hom myotubes displayed a hyperpolarizing shift in inactivation relative to L-type currents in either WT or Het myotubes. Our present results indicate that mutations in RYR1 can alter DHPR activity and raise the possibility that this altered DHPR function may contribute to MH episodes.

Figure 1.

SR Ca²⁺ release is shifted to more hyperpolarized potentials in R163C MHS myotubes. Representative myoplasmic Ca²⁺ transients elicited by 50-ms depolarizations from −50 mV to test potentials of −30, −10, 10, and 30 mV are shown for WT (A), Hom (B), and Het (C) myotubes. Raw ΔF/F-V relationships are shown in D. The average ΔF/F-V relationships, obtained after normalizing to the [ΔF/F]_max in each individual experiment, are shown in E, with the smooth curves plotted according to Eq. 1 and best-fit parameters presented in Table I. Error bars represent ± SEM.

Figure 2.

Charge movements are shifted to more hyperpolarized potentials in R163C Het and Hom myotubes. Representative charge movements elicited by 20-ms depolarizations from −50 mV to test potentials of −30, −10, 10, and 30 mV are shown for WT (A), Het (B), and Hom (C) myotubes. Raw Q-V relationships are shown in D. The average Q-V relationships, obtained after normalizing to the Q_max in each individual experiment, are shown in E, with the smooth curves plotted according to Eq. 2 and best-fit parameters presented in Table II.

Figure 3.

L-type currents in R163C Het and Hom myotubes are shifted to slightly more hyperpolarized potentials. (A) Representative recordings of L-type Ca²⁺ currents elicited by 200-ms depolarizations from −50 mV to test potentials of 0, 10, 20, 30, and 40 mV are shown for WT (top), Het (middle), and Hom (bottom) myotubes. (B) Comparison of peak I-V relationships for WT (n = 25), Het (n = 22), and Hom (n = 28) cells. Currents were evoked at 0.1 Hz by test potentials ranging from −20 through +80 mV in 10-mV increments, following a prepulse protocol (Adams et al., 1990). Current amplitudes were normalized by linear cell capacitance (pA/pF). (C) I-V relationships for the same dataset shown in B, in which the current amplitudes have been normalized to the peak current in each cell. The smooth curves are plotted according to Eq. 3, with best-fit parameters presented in Table II.

Figure 4.

L-type currents in R163C Het and Hom myotubes are more sensitive to ±Bay K 8644. Representative recordings of L-type currents made in the presence of ±Bay K 8644 are shown for WT (A; left), Het (B; left), and Hom (C; left) myotubes. Currents were elicited by 200-ms depolarizations from −50 mV to test potentials of 0, 10, 20, and 30 mV. The average peak I-V relationships obtained in the absence (circles) and presence (diamonds) of 10 µM ±Bay K 8644 and are shown in A (right) for WT myotubes (◆, n = 9; •, n = 25), in B (right) for Het myotubes (◆, n = 6; •, n = 22), and in C (right) for Hom myotubes (◇, n = 9; ○, n = 28). Peak I-V data obtained in the absence of ±Bay K 8644 are replotted from Fig. 3, and the smooth curves are plotted according to Eq. 3, with best-fit parameters given in Table II.

Figure 5.

Steady-state inactivation of L-type current in WT and R163C MHS myotubes. (A) Voltage protocol. A 200-ms depolarization to +30 mV was applied just before (reference pulse) and immediately (test pulse) after 30-s prepulses to −50, −10, 0, 10, 20, and 30 mV. An interval of >1 min at the steady holding potential of −80 mV was used between each episode of the protocol. (B) Test currents elicited with this protocol for the indicated prepulse potentials (black traces) are shown for each genotype, with the corresponding control current (gray traces) evoked before the pulse run from −80 to +30 mV. The amplitudes of the currents shown in B have been normalized to the amplitude of the current elicited by the reference pulse. (C) Summary of results for WT (n = 12–19), Het (n = 6–12), and Hom (n = 8–23) myotubes at the indicated prepulse potentials. The average normalized inactivation values were fit by Eq. 5 (see Materials and methods), with the following respective parameters for WT, Het, and Hom myotubes: V_1/2inact = 6.1, 5.3, and −3.3 mV; k = −6.8, 4.3, and 6.6 mV.

Figure 6.

A model that can account for the effects on DHPR gating, which result from the R163C mutation in RYR1. In the model, the major component of DHPR gating charge (Q) moves rapidly (milliseconds) upon depolarization and results in a conformational change of a DHPR cytoplasmic domain(s) from “resting” to “ECC activating,” which in turn triggers a corresponding conformational change in the cytoplasmic (“foot”) domain of RYR1 that opens the RYR1 pore (black dotted cylinder). Although the movement of Q is necessary for the activation of L-type current via the DHPR, it is not sufficient. Opening of the L-type channel pore (white dotted cylinder) additionally depends on another DHPR charge (q), which moves more slowly (tens of milliseconds), is smaller than Q, and moves at more depolarized potentials than those causing ECC Ca²⁺ release (Dirksen and Beam, 1999). The R163C MHS mutation shifts the equilibrium of RYR1 toward the open state (Yang et al., 2003, 2006), and if this open state is linked to the ECC-activated conformation of the foot domain, it would retrogradely promote the ECC-activating conformation of the DHPR, with the result that less depolarization would be required to move Q (Fig. 2). The hyperpolarizing shift in the movement of Q also causes a hyperpolarizing shift in activation of the L-type channel (Fig. 3), but this shift is less pronounced because the movement of q is not directly affected by the transition of RYR1 to the ECC-activated state. During prolonged depolarization (tens of seconds), the DHPR shifts to a state that is inactivated for ECC (Beam and Horowicz, 2004). Entry into this ECC-inactivated state is slowed (Figs. 1 and 7 of Estève et al., 2010) because the ECC-activated states of RYR1 (and thus the DHPR) are stabilized by the R163C MHS mutation.