Impaired gating of an L-Type Ca(2+) channel carrying a mutation linked to malignant hyperthermia

Abstract

Recently, we characterized the functional properties of a mutant skeletal muscle L-type Ca(2+) channel (CaV1.1 R174W) linked to the pharmacogenetic disorder malignant hyperthermia. Although the R174W mutation neutralizes the innermost basic amino acid in the voltage-sensing S4 helix of the first conserved membrane repeat of CaV1.1, the ability of the mutant channel to engage excitation-contraction coupling was largely unaffected by the introduction of the bulky tryptophan residue. In stark contrast, the mutation ablated the ability of CaV1.1 to produce L-type current under our standard recording conditions. In this study, we have investigated the mechanism of channel dysfunction more extensively. We found that CaV1.1 R174W will open and conduct Ca(2+) in response to strong or prolonged depolarizations in the presence of the 1,4-dihydropyridine receptor agonist ±Bay K 8644. From these results, we have concluded that the R174W mutation impedes entry into both mode 1(low Po) and mode 2 (high Po) gating states and that these gating impairments can be partially overcome by maneuvers that promote entry into mode 2.

Figure 1

Potentiation of wild-type Ca_V1.1 tail currents by Bay K 8644. Representative currents evoked by the illustrated voltage protocol are shown for dysgenic myotubes expressing YFP-Ca_V1.1 in the absence (A) and the presence (B) of ±Bay K 8644 (10 μM). The currents elicited by depolarization to +40 or +90 mV are indicated in green and red, respectively, with the tail currents upon repolarization to −50 mV shown on an expanded time base in the insets in A and B. (C) Summary of amplitudes of YFP-Ca_V1.1 tail currents recorded in the absence (♦; n = 18) and presence (⋄; n = 10) of ±Bay K 8644. (D) Summary of half-times of YFP-Ca_V1.1 tail current decay recorded in the absence (left panel) and presence (right panel) of ±Bay K 8644. Asterisks indicate significant differences (^* denotes p < 0.05, ^** denotes p < 0.005, ^*** denotes p < 0.001, unpaired t-test in C and ANOVA for both panels in D). Bars represent mean ± SE throughout.

Figure 2

Potentiated Ca_V1.1 R174W conducts inward Ca²⁺ tail current. Representative currents evoked by the illustrated voltage protocol are shown for dysgenic myotubes expressing YFP-Cav1.1 R174W in the absence (A) and the presence (B) of ±Bay K 8644 (10 μM). The currents elicited by depolarization to +40 or +90 mV are indicated in green and red, respectively, with the tail currents upon repolarization to −50 mV shown on an expanded time base in the insets in A and B. (C) Summary of amplitudes of YFP-Ca_V1.1 R174W tail currents recorded in the absence (●; n = 9) and presence (○; n = 10) of ±Bay K 8644. Also shown in panel C are the corresponding control data for naive dysgenic myotubes in the presence of ±Bay K 8644 (●; n = 6). (D) Summary of half-times of YFP-Ca_V1.1 R174W tail current decay recorded in the absence (black bars) and presence (white bars) of ±Bay K 8644. Asterisks indicate significant differences (^* denotes p < 0.05; ^** denotes p < 0.005; ^*** denotes p < 0.001, ANOVA in both C and D).

Figure 3

After strong depolarization in the presence of ±Bay K 8644, Ca_V1.1 R174W produces long-lasting inward Ca²⁺ current. Currents from a YFP-Ca_V1.1 R174W-expressing dysgenic myotubes were elicited by an initial 200 ms step depolarization to either +60 mV (black trace) or +90 mV (red trace), followed by 100 ms at +60 mV, before final repolarization to −20 mV (illustrated at top). Note the substantial, sustained inward current recorded at +60 mV following the step to +90 mV and the enhanced tail current upon repolarization to −20 mV. Similar behavior was observed in a total of six cells (see text).

Figure 4

Ca_V1.1 R174W opens in response to long depolarizations in the presence of ±Bay K 8644. Recordings of Ca²⁺ currents elicited by 2 s depolarizations from −50 mV to the indicated test potentials are shown for dysgenic myotubes expressing either YFP-Ca_V1.1 (A) or YFP-Ca_V1.1 R174W (B).

Figure 5

Model for Ca_V1.1 R174W dysfunction. Mode 0 represents closed states of Ca_V1.1. Upon sufficient depolarization, the voltage sensors of both wild-type Ca_V1.1 and Ca_V1.1 R174W translocate within the membrane field producing the movement of charge (Q) and engage EC coupling, although the channel remains in mode 0. Stronger depolarization of sufficient duration causes the slow movement of additional charge (q) and entry into mode 1, in which the pore opens with low P_o. Our present work indicates that the malignant hyperthermia-linked R174W mutation impairs the transitions necessary for the channel to enter mode 1. However, strong or prolonged depolarization in the presence of ±Bay K 8644 sufficiently accelerates the transition (big arrow) into higher P_o mode 2 to overcome the gating impediment caused by the R174W mutation.