Identification and functional characterization of malignant hyperthermia mutation T1354S in the outer pore of the Cavalpha1S-subunit

Abstract

To identify the genetic locus responsible for malignant hyperthermia susceptibility (MHS) in an Italian family, we performed linkage analysis to recognized MHS loci. All MHS individuals showed cosegregation of informative markers close to the voltage-dependent Ca(2+) channel (Ca(V)) α(1S)-subunit gene (CACNA1S) with logarithm of odds (LOD)-score values that matched or approached the maximal possible value for this family. This is particularly interesting, because so far MHS was mapped to >178 different positions on the ryanodine receptor (RYR1) gene but only to two on CACNA1S. Sequence analysis of CACNA1S revealed a c.4060A>T transversion resulting in amino acid exchange T1354S in the IVS5-S6 extracellular pore-loop region of Ca(V)α(1S) in all MHS subjects of the family but not in 268 control subjects. To investigate the impact of mutation T1354S on the assembly and function of the excitation-contraction coupling apparatus, we expressed GFP-tagged α(1S)T1354S in dysgenic (α(1S)-null) myotubes. Whole cell patch-clamp analysis revealed that α(1S)T1354S produced significantly faster activation of L-type Ca(2+) currents upon 200-ms depolarizing test pulses compared with wild-type GFP-α(1S) (α(1S)WT). In addition, α(1S)T1354S-expressing myotubes showed a tendency to increased sensitivity for caffeine-induced Ca(2+) release and to larger action-potential-induced intracellular Ca(2+) transients under low (≤ 2 mM) caffeine concentrations compared with α(1S)WT. Thus our data suggest that an additional influx of Ca(2+) due to faster activation of the α(1S)T1354S L-type Ca(2+) current, in concert with higher caffeine sensitivity of Ca(2+) release, leads to elevated muscle contraction under pharmacological trigger, which might be sufficient to explain the MHS phenotype.

Fig. 1.

Segregation of MHS5 microsatellite markers and of the c.4060A>T malignant hyperthermia susceptibility (MHS) mutation within the pedigree of family NA-6. I, II, and III indicate the 3 generations investigated; squares symbolize male and circles female family members. Black symbols denote individuals identified as MHS by invasive in vitro contracture test; white symbols marked with N denote those identified as MH normal (MHN); black/white symbols denote those identified as MH equivocal (MHE); and symbols with a question mark denote untested family members (disease status unknown). The index patient (II:1; indicated by an arrow) developed an MH crisis during anesthesia. All identical haplotypes are symbolized by bars in identical patterns. The haplotype 4–6-T-9 (black bar) is shared by all of the affected individuals (MHS and MHE) and by none of the unaffected individuals (MHN).

Fig. 2.

Thr1354, 3 amino acids before segment IVS6, is highly conserved in all α_1S-subunits but is replaced by Ser in the cardiac/neuronal Ca_Vα_1C- and Ca_Vα_1D-subunits. A: location of the mutation T1354S in a transmembrane domain model of GFP-α_1S. I-IV indicate the 4 homologous repeats, and pore loop domains are indicated in dark gray. Double line indicates the sarcolemmal membrane. B: sequence alignments of the C-terminal half of the pore-forming IVS5-S6 loop of Ca_Vα_1S, Ca_Vα_1C, and Ca_Vα_1D from different species and their positions in the domain model. For each isoform, residues that are identical in all species are boxed in black. The selectivity filter Glu of pore loop domain IV (dark gray cylinder) is indicated by an asterisk. Thr, 3 amino acids before transmembrane segment IVS6 (Thr1354 in human and rabbit), is indicated by a light-gray double arrowhead. As indicated by a black double arrowhead, a conserved Ser is found at the same position in Ca_Vα_1C and Ca_Vα_1D, similar to the MH mutation T1354S. Sequences for cat and horse Ca_Vα_1C and Ca_Vα_1D, for chicken Ca_Vα_1S, and for frog Ca_Vα_1S and Ca_Vα_1C were extracted from genomic assemblies at http://www.ensembl.org using a BLAST search with rabbit Ca_Vα_1S, Ca_Vα_1C, or Ca_Vα_1D cDNA sequences. GeneBank Accession Nos. for Ca_Vα_1S, Ca_Vα_1C, or Ca_Vα_1D, respectively, of the different species are as follows: human (Homo sapiens) NM_000069 (α_1S), NM_199460 (α_1C), NM_000720 (α_1D); rabbit (Oryctolagus cuniculus) NM_001101720 (α_1S), NM_001136522 (α_1C); rat (Rattus norvegicus) NM_017298 (α_1D), mouse (Mus musculus) NM_014193 (α_1S), NM_009781 (α_1C), NM_028981 (α_1D); dog (Canis lupus familiaris) XM_843592 (α_1S), XM_534932 (α_1C), XM_844338 (α_1D); cat (Felis catus) NM_001038605 (α_1S); horse (Equus caballus) XM_001916128 (α_1S); chicken (Gallus gallus) XM_416388 (α_1C), NM_205034 (α_1D); and frog (Xenopus tropicalis) NM_001079461 (α_1D).

Fig. 3.

L-type Ca²⁺ currents are accelerated in myotubes expressing α_1ST1354S. A: representative traces of intracellular Ca²⁺ release (top) and L-type Ca²⁺ currents (bottom) recorded in response to 200-ms test pulses from dysgenic myotubes expressing wild-type α_1S (α_1SWT) and α_1ST1354S. B: mean voltage dependences and amplitudes of peak Ca²⁺ transients (ΔF/F; α_1SWT, n = 40, and α_1ST1354S, n = 27) and of L-type Ca²⁺ currents (α_1SWT, n = 31, and α_1ST1354S, n = 26) are indistinguishable in α_1SWT- and α_1ST1354S-expressing myotubes. I-V, current-voltage relationship. C: time to peak of Ca²⁺ currents is significantly (**P < 0.01) reduced in myotubes expressing α_1ST1354S (α_1SWT, n = 31, and α_1ST1354S, n = 26).

Fig. 4.

Interaction between Ca_Vα_1S and α₂δ-1 is not affected by the T1354S mutation. A: double immunofluorescence labeling of GFP-tagged α_1S (a and b, clusters indicated by arrows) and of α₂δ-1 (c and d, clusters indicated by arrows) in myotubes expressing α_1SWT (left) and α_1ST1354S (right). Merged images (e and f) show proper colocalization of both α_1S-constructs with the endogenous α₂δ-1. B and C: activating phases of the Ca²⁺ currents from Fig. 3A were fitted either by double or monoexponential functions and corresponding contributions to the total current amplitudes (B) and time constants of activation (C) were analyzed. B: relative contribution of fast and slow fractions to total current amplitude was similar for α_1SWT (white background, black hatched bars) and α_1ST1354S (black background, white hatched bars). C: time constants for all slow and fast components (τ_slow, τ_fast, α_1SWT, n = 18, and α_1ST1354S, n = 13) were significantly reduced (*P < 0.05) in α_1ST1354S-expressing (black background, white hatched bars) compared with α_1SWT-expressing (white background, black hatched bars) myotubes. A similar tendency was observed for τ_mono (α_1SWT, n = 13, and α_1ST1354S, n = 13) (C).

Fig. 5.

Caffeine sensitivity of RyR1 in α_1ST1354S-expressing myotubes. A: representative caffeine concentration-response curves recorded from Indo-1-loaded myotubes transfected with α_1SWT (top), α_1ST1354S (middle), and untransfected dysgenic myotubes (bottom). At the beginning of each trace, a train of 3 extracellular electrical stimuli was applied to elicit voltage-gated Ca²⁺ release (black arrows). No electrically induced Ca²⁺ release was observed in untransfected dysgenic myotubes. Subsequently, increasing concentrations of caffeine were applied for 60 s to each myotube separated by 60-s washing steps. B: number of cells (%) responding to caffeine concentrations of 3 and 5 mM. C: concentration dependence of caffeine-induced Ca²⁺ release for α_1SWT (●, n = 19), α_1ST1354S (○, n = 22), and dysgenic myotubes (▾, n = 21).

Fig. 6.

Ca²⁺ release induced by extracellular electrical stimuli in α_1SWT- and α_1ST1354S-expressing myotubes. A and C: Ca²⁺ transients from myotubes were elicited by 3 extracellular electrical pulses during sequential 30-s application of 0, 1, and 2 mM caffeine (indicated by gray bars of different width) in extracellular solutions containing 2 mM Ca²⁺ (A) and 0 mM Ca²⁺ (C, bottom lines). Between the pulse trains myotubes were incubated for 2 min with extracellular caffeine-free solution containing 10 mM Ca²⁺ for SR reloading (A). B: integral of Ca²⁺ transients in 2 mM extracellular Ca²⁺ as a measure for total Ca²⁺ release in response to one action potential. A trend towards higher Ca²⁺ release with constructs α_1ST1354S (n = 23) and α_1SΔ29 (n = 29) compared with α_1SWT (n = 19) is observable under 1 mM caffeine and is significant under 2 mM caffeine (*P, #P < 0.05, respectively). D: integral of Ca²⁺ transients in 0 mM extracellular Ca²⁺ shows no difference between α_1SWT (n = 13), α_1ST1354S (n = 6), and α_1SΔ29 (n = 14) in relation to different caffeine exposures.