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

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 (α1Snull) 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 α1Sfunctions 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 (α1S130 ± 10.2, R1086H 88 ± 6.8 nS/nF; P < 0.05). The decrease in Gmaxdid 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 Gmaxwas 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 α1Senhances RyR1 sensitivity to activation by both endogenous (voltage sensor) and exogenous (caffeine) activators.