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) 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 Ca(2+) release occurred at approximately fivefold lower concentrations in nonexpressing and R1086H-expressing myotubes. Although maximal voltage-gated Ca(2+) release was similar in alpha(1S)- and R1086H-expressing myotubes, the voltage dependence of Ca(2+) release was shifted approximately 5 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 Ca(2+) 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 G(max) 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.