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

Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder caused by mutations in the skeletal muscle ryanodine receptor (RyR1) and the dihydropyridine receptor ( DHPR) alpha(1S)-subunit. We characterized the effects of an MH mutation in the DHPR cytoplasmic III-IV loop of alpha(1S) (R1086H) on DHPR-RyR1 coupling after reconstitution in dysgenic (alpha(1S) null) myotubes. Compared with wild-type alpha(1S), 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 alpha(1S)- and R1086H-expressing myotubes, the voltage dependence of Ca2+ release was shifted similar to5 mV to more negative potentials in R1086H-expressing myotubes. Our results demonstrate that alpha(1S) 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 alpha(1S)- and R1086H-expressing myotubes. Compared with alpha(1S)-expressing myotubes, maximal L channel conductance (G(max)) was reduced in R1086H-expressing myotubes (alpha(1S) 130 +/- 10.2, R1086H 88 +/- 6.8 nS/nF; P < 0.05). The decrease in G(max) did not result from a change in retrograde coupling with RyR1 as maximal conductance-charge movement ratio (G(max)/Q(max)) was similar in alpha(1S)- 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 alpha(1S) enhances RyR1 sensitivity to activation by both endogenous ( voltage sensor) and exogenous ( caffeine) activators.