Fig. 2: TRPV1 variations from patients who suffer from malignant hyperthermia.

(a) Segregation ofTRPV1 variations. Individuals tested MHEh are depicted with hatched symbols. Black symbols refer to patients diagnosed with a congenital myopathy and MH sensitivity. Arrows indicate probands referred to the laboratory for genetic studies according to initial diagnosis: malignant hyperthermia crisis for family 1 and myopathy for family 2. Family 1: the proband was referred to genetic investigation for postoperative hyperthermia after anesthesia. No significant variant was found in the RYR1 gene sequence. The c.1836C>T; pThr612Met variation was found in targeted analysis of the gene. Family 2: the proband was referred to genetic investigation for muscle weakness. He responded positively to the in vitro contracture test (MHS) and further familial studies were undertaken for MH sensitivity. Two mutations in the RYR1 gene (c.1205T>C;p.Met402Thr and c.11653C>T;p.Arg3885X) were found inherited from each of the parents in the proband and his symptomatic sister’s DNA. The haplo insufficiency that was revealed during sequencing of the father RYR1 messenger RNA (mRNA) cannot account for its MH sensitivity to halothane in the in vitro contracture test (MHSh). The c.1180-82delAAC; pAsn394del variant was found in a targeted analysis of theTRPV1 gene. The variation was also found in his daughter’s DNA. (b) Electropherograms from DNA sequencing showingTRPV1 variation c.1835C>T; pThr612Met and c.1180-82delAAC; pAsn394del respectively for family 1 and 2. (c–h) Ca²⁺ imaging experiments were performed using the cytosolic Ca²⁺ probe fura-2 (at 37 °C). Representative time course of cytosolic Ca²⁺ concentration following capsaicin (10 µM) or isoflurane (0.5 mM) exposure in HEK-293 cells transfected with the wild-type TRPV1 or the TRPV1 mutants. Each experiment was repeated three times (field of 35–45 cells) and representative experiments are presented (mean ± SE). (i) Cell-surface biotinylation analysis of TRPV1 wild-type and mutants transfected cells. The empty vector was used as a control (mock). TRPV1 expression was analyzed by immunoblotting for the biotinylated plasma membrane fraction (TRPV1PM) or total cell lysates (TRPV1TL) and calnexin was used as a loading control. (j) Endoplasmic reticulum (ER) enrichment of WT HEK or transiently transfected with TRPV1 N394del or T612M and total fraction. GRP78 is used as an ER loading control, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for the cytosol compartment, and B-actin as a general loading control. (k,l) Patterns of expression of human or mutant (T612M and N394del) TRPV1 transfected in flexor digitorum brevis (FDB) muscle fibers of trpv1^−/− mice. Representative confocal images of immunofluorescence labeling of (1) Trpv1, (2) human TRPV1-mcherry, (3) TRPV1 T612M-mcherry, and (4) TRPV1 N394del-mcherry. (l) Average intensity profiles superposed: native Trpv1 (black line), hTRPV1-mcherry (gray line), TRPV1 T612M- mcherry (blue line), and TRPV1 N394del-mcherry (green line)