Functional analysis of the R1086H malignant hyperthermia mutation in the DHPR reveals an unexpected influence of the III-IV loop on skeletal muscle EC coupling

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Abstract
Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder caused by mutations in the skeletal muscle ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR) α1S-subunit. We characterized the effects of an MH mutation in the DHPR cytoplasmic III-IV loop of α1S (R1086H) on DHPR-RyR1 coupling after reconstitution in dysgenic (α1S null) myotubes. Compared with wild-type α1S, caffeine-activated Ca2+ release occurred at approximately fivefold lower concentrations in nonexpressing and R1086H-expressing myotubes. Although maximal voltage-gated Ca2+ release was similar in α1S- and R1086H-expressing myotubes, the voltage dependence of Ca2+ release was shifted ∼5 mV to more negative potentials in R1086H-expressing myotubes. Our results demonstrate that α1S functions as a negative allosteric modulator of release channel activation by caffeine/voltage and that the R1086H MH mutation in the intracellular III-IV linker disrupts this negative regulatory influence. Moreover, a low caffeine concentration (2 mM) caused a similar shift in voltage dependence of Ca2+ release in α1S- and R1086H-expressing myotubes. Compared with α1S-expressing myotubes, maximal L channel conductance ( Gmax) was reduced in R1086H-expressing myotubes (α1S 130 ± 10.2, R1086H 88 ± 6.8 nS/nF; P < 0.05). The decrease in Gmax did not result from a change in retrograde coupling with RyR1 as maximal conductance-charge movement ratio ( Gmax/Qmax) was similar in α1S- and R1086H-expressing myotubes and a similar decrease in Gmax was observed for an analogous mutation engineered into the cardiac L channel (R1217H). In addition, both R1086H and R1217H DHPRs targeted normally and colocalized with RyR1 in sarcoplasmic reticulum (SR)-sarcolemmal junctions. These results indicate that the R1086H MH mutation in α1S enhances RyR1 sensitivity to activation by both endogenous (voltage sensor) and exogenous (caffeine) activators.