Pathogenic CaV1.1 Variants Cause Hyperpolarizing Shift of Depolarization-Induced Ca2+ Release in Malignant Hyperthermia Susceptibility

Figures

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  Fig. 1. DICR activity of reconstituted cells expressing CaV1.1 variants. a Schematic drawing of location of MH-associated variants (A560T, S879P, F1161L, and D1382V) in CaV1.1. Four voltage sensing domains (VSD) are indicated in Roman numerals (I–IV). b Schematic drawing of measurement of DICR in the reconstituted platform. HEK293 cells expressing RyR1 and R-CEPIA1er were infected with baculovirus carrying essential components (CaV1.1 WT or variants, β1a, Stac3, JP2) to reconstitute the DICR machinery. Kir2.1 was also transduced to hyperpolarize the membrane potential. Note that CaV1.1 carries a Ca2+ non-conducting mutation (N617D) in this experiment. DICR was triggered by depolarization of the membrane potential with high [K+]o solution and measured by a reduction in R-CEPIA1er fluorescence. c Typical results of time-lapse R-CEPIA1er fluorescence measurements at 5-s intervals for WT (left), A560T (center), and F1161L (right) CaV1.1 cells. High [K+]o solution ranging from 5 to 61 mM (symbols shown in WT) was applied 30 s after starting (arrowheads). d Resting [Ca2+]ER in cells expressing WT and CaV1.1 variants. Resting R-CEPIA1er fluorescence (F0) was normalized to Fmax, which was…

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  Fig. 2. Caffeine-induced Ca2+ release in reconstituted cells expressing CaV1.1 variants. a Schematic drawing of caffeine-induced Ca2+ release in the reconstituted platform. b Typical results of time-lapse R-CEPIA1er fluorescence measurement at 5-s intervals for WT (left), A560T (center), and F1161L (right) CaV1.1 cells. Caffeine solution ranging from 0 to 20 mM (symbols shown in WT) was applied 30 s after starting (arrows). c Caffeine dependence of R-CEPIA1er fluorescence in WT and Cav1.1-variant cells, corrected by resting [Ca2+]ER in WT cells (F/F0,WT). Data are means ± SD (n = 9). d EC50 values for caffeine. Data are means ± SD (n = 9). *p < 0.05 compared with WT. Note that the image in a was created by PowerPoint software.

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  Fig. 3. Effect of DICR activity on caffeine sensitivity. a Resting membrane potential of the reconstituted HEK293 cells in 5 mM and 1 mM [K+]o Krebs solution. Data are means ± SD (n = 8). b Resting [Ca2+]ER at 1 mM [K+]o solution in WT (black), S879P (red), and F1161L (blue) CaV1.1 cells. Data are means ± SD (n = 9). c [K+]o dependence of R-CEPIA1er fluorescence at 1 mM [K+]o solution in WT and Cav1.1-variant cells, corrected by resting [Ca2+]ER in WT cells (F/F0,WT). Data are means ± SD (n = 9). d EC50 values for [K+]o at 1 mM [K+]o solution. Data are means ± SD (n = 9). e Caffeine dependence of R-CEPIA1er fluorescence at 1 mM [K+]o solution in WT and CaV1.1-variant cells, corrected by resting [Ca2+]ER in WT cells (F/F0,WT). Data are means ± SD (n = 9). f EC50 values for caffeine at 1 mM [K+]o solution. Data are means ± SD (n = 9). g, h EC50 values for caffeine at 5 mM [K+]o (normal) Krebs solution in the reconstituted HEK293 cells without β1a (g) or Stac3 (h). *p < 0.05 compared with WT.

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  Fig. 4. Gating charge movement in CaV1.1 MH mutants. a Schematic illustration of gating current measurements using pore-dead N617D-mutant CaV1.1 channels. b Representative traces of test pulses and gating currents recorded from WT, S879P, and F1161L CaV1.1 channels. c Maximum gating charge (Qmax) obtained by fitting the Q–V relationship with a Boltzmann equation. d Voltage dependence of gating charge movement. Q was measured by integrating the ON-gating current over the first 2 ms of each test pulse, normalized to Qmax, and plotted as Q/Qmax against membrane potential. Curves represent Boltzmann fits. e, f Half-activation voltage (V0.5, e) and slope factor (k, f) derived from the Boltzmann fits shown in (d). Data are means ± SD (WT, n = 7; S879P, n = 9; F1161L, n = 7).

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  Fig. 5. Ionic currents and coupling efficiency in CaV1.1 MH mutants. a Schematic illustration of ionic current measurements using normal CaV1.1 channels. b Representative current traces evoked by test pulses (from −80 to 80 mV) in WT, S879P, and F1161L channels. Note that typical currents evoked by test pulses from −40 to +60 mV are shown. c Peak current density measured at +30 mV. d Current–voltage relationships fitted with a Boltzmann function. e Half-activation voltage (V0.5) derived from the fits in (d). f Activation time constants (τ) at +30 mV, obtained by single-exponential fitting. Data are means ± SD (WT, n = 5; S879P, n = 5; F1161L, n = 5). g Representative traces of test pulses and tail currents used to assess coupling efficiency. h Total gating charge movement determined by integrating ON-gating currents. i Peak tail current density upon repolarization to −50 mV. j Coupling efficiency calculated as the ratio of peak tail current to total gating charge (nA/pC). Data are means ± SD (WT, n = 10; S879P, n = 11; F1161L, n = 10).

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  Fig. 6. Proposed mechanism of pathogenesis of MH by CaV1.1 mutations. Left In WT controls, Ca2+ release from RyR1 is negligible under resting conditions (upper panel). Inhalational anesthetics, such as isoflurane, activate RyR1, but Ca2+ release is still low (lower panel). Center In patients carrying RyR1 MH variants, Ca2+ release activity is increased by gain-of-function of RyR1, but does not trigger regenerative Ca2+ release under resting conditions (upper panel). Inhalational anesthetics further activates RyR1 to trigger regenerative Ca2+ release (lower panel). Right In patients carrying CaV1.1 MH variants, DICR may occur under resting conditions, which increases Ca2+ in the junctional space (upper panel). Inhalational anesthetics activate RyR1, which can trigger regenerative Ca2+ release under such higher Ca2+ in the junctional space (lower panel).