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Results: 5

1.
Fig. 4.

Fig. 4. From: Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor.

 SR Ca2+ store is partially depleted in CaV1.1 R174W-expressing dysgenic myotubes. (A) Representative ionomycin (5 μM)-induced Ca2+ transients in dysgenic myotubes stably expressing either wild-type CaV1.1 (shaded trace) or CaV1.1 R174W (solid trace). (B) Quantification of peak transient amplitude (Left) and integrated transient area (Right) for ionomycin-induced Ca2+ transients in dysgenic myotubes expressing CaV1.1 (n = 33) and CaV1.1 R174W (n = 39). Asterisks indicate significant differences (**P < 0.01; ***P < 0.001; unpaired t test).

Jose Miguel Eltit, et al. Proc Natl Acad Sci U S A. 2012 May 15;109(20):7923-7928.
2.
Fig. 3.

Fig. 3. From: Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor.

Dysgenic myotubes expressing CaV1.1 R174W (Right) display elevated resting myoplasmic Ca2+ and hypersensitivity to MH triggers compared with dysgenic myotubes expressing wild-type CaV1.1 (Left). Myoplasmic Ca2+ levels measured with Ca2+-sensitive microelectrodes are shown before (control) and after exposure to either isoflurane (0.1% vol/vol; Right, hatched,bar) or halothane (0.1% vol/vol; Left, hatched bar). &&&, significant difference (P < 0.001, ANOVA) in resting Ca2+ between wild-type CaV1.1 and CaV1.1 R174W control groups. ***, significant (P < 0.001, ANOVA) difference between control and anesthetic-treated CaV1.1 R174W-expressing myotubes. In dysgenic myotubes expressing wild-type CaV1.1, no significant changes in myoplasmic Ca2+ were observed following exposure to the volatile anesthetics relative to control.

Jose Miguel Eltit, et al. Proc Natl Acad Sci U S A. 2012 May 15;109(20):7923-7928.
3.
Fig. 5.

Fig. 5. From: Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor.

The R174W mutation in CaV1.1 disrupts the resting interaction between CaV1.1 and RyR1 that supresses Ca2+ leak from the SR. (A) Our previous work in dysgenic myotubes has demonstrated that the absence of CaV1.1 reveals a leak state of RyR1 (Left) that increases resting Ca2+ (22). Additionally, the absence of the CaV1.1 voltage sensor renders dysgenic myotubes incapable of EC coupling or generating L-type Ca2+ current during plasma membrane depolarization (Right) (42). (B) When present, CaV1.1 inhibits Ca2+ leak from the SR via RyR1 at rest (Left) and responds to depolarization by engaging EC coupling in Ca2+ release and producing L-type current (Right) (6). (C) The R174W mutation alters the conformation of CaV1.1 such that it does not suppress resting SR Ca2+ leak, leading to an increase in resting Ca2+ and enhanced sensitivity to MH triggers (Left; Fig. 3); the R174W mutation also ablates the ability of CaV1.1 to produce L-type current but has little, if any, effect on the ability of CaV1.1 to engage EC coupling (Right).

Jose Miguel Eltit, et al. Proc Natl Acad Sci U S A. 2012 May 15;109(20):7923-7928.
4.
Fig. 2.

Fig. 2. From: Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor.

The R174W mutation has little effect on the ability of CaV1.1 to function as the voltage sensor for EC coupling. (A and B) Recordings of myoplasmic Ca2+ transients elicited by 50-ms depolarizations from −50 mV to −20, −10, 0, 10, 20, and 30 mV are shown for dysgenic myotubes expressing either YFP-CaV1.1 (A) or YFP-CaV1.1 R174W (B). (C) Average ΔF/F–V relationships for dysgenic myotubes expressing either YFP-CaV1.1 (●; n = 11) or YFP-CaV1.1 R174W (○; n = 7) fitted by Eq. 3 with the parameters presented in Table S1. (D and E) Representative elevated K+-induced Ca2+ transients in dysgenic myotubes stably expressing wild-type CaV1.1 (D) or CaV1.1 R174W (E). (F) K+ dose–response relationships for dysgenic myotubes stably expressing either wild-type CaV1.1 (n = 19) or CaV1.1 R174W (n = 32) fitted by Eq. 4. (G and H) Representative caffeine-induced Ca2+ transients in dysgenic myotubes stably expressing CaV1.1 (G) or CaV1.1 R174W (H). At the beginning of each experiment, myotubes were exposed to K+ (60 mM) for 10 s to confirm either wild-type or mutant CaV1.1 expression. (I) Caffeine dose–response relationships for dysgenic myotubes stably expressing either wild-type CaV1.1 (n = 24) or CaV1.1 R174W (n = 28) fitted by Eq. 4.

Jose Miguel Eltit, et al. Proc Natl Acad Sci U S A. 2012 May 15;109(20):7923-7928.
5.
Fig. 1.

Fig. 1. From: Malignant hyperthermia susceptibility arising from altered resting coupling between the skeletal muscle L-type Ca2+ channel and the type 1 ryanodine receptor.

Ca2+ currents and charge movements recorded from primary dysgenic myotubes expressing either YFP-CaV1.1 or YFP-CaV1.1 R174W. (A and B) Representative currents evoked from −50 mV to −10, 10, 30, and 50 mV are shown for dysgenic myotubes expressing either YFP-CaV1.1 R174W (A) or YFP-CaV1.1 (B). (C) Peak I–V relationships for naive dysgenic myotubes (●; n = 8) or dysgenic myotubes expressing either YFP-CaV1.1 (●; n = 32) or YFP-CaV1.1 R174W (○; n = 7). In the case of YFP-CaV1.1 R174W, I–V data were collected only from the 7 myotubes where channel expression was confirmed by charge movement; no inward current was observed in 10 additional dysgenic myotubes displaying yellow fluorescence. Currents were evoked at 0.1 Hz by test potentials ranging from −20 mV through +90 mV in 10-mV increments, following a prepulse protocol (41). Current amplitudes were normalized by linear cell capacitance (pA/pF). The smooth curve for YFP-CaV1.1 is plotted according to Eq. 1, with best-fit parameters presented in Table S1. (D and E) Representative charge movements evoked from −50 mV to −30, −10, 10, and 30 mV are shown for dysgenic myotubes expressing either YFP-CaV1.1 R174W (D) or YFP-CaV1.1 (E). Charge movements were evoked at 0.1 Hz by test potentials ranging from −40 mV through +50 mV in 10-mV increments following a prepulse protocol. (F) Q–V relationships for naïve dysgenic myotubes (n = 6) or dysgenic myotubes expressing either YFP-CaV1.1 (n = 12) or YFP-Cav1.1 R174W (n = 7). The smooth curves are plotted according to Eq. 2, with respective best-fit parameters presented in Table S1.

Jose Miguel Eltit, et al. Proc Natl Acad Sci U S A. 2012 May 15;109(20):7923-7928.

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