Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder caused by mutations in the skeletal muscle ryanodine receptor (RyR1) and the dihydropyridine receptor (DHPR) α_1S-subunit. We characterized the effects of an MH mutation in the DHPR cytoplasmic III-IV loop of α_1S (R1086H) on DHPR-RyR1 coupling after reconstitution in dysgenic (α_1S null) myotubes. Compared with wild-type α_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 α_1S- 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 α_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 α_1S- and R1086H-expressing myotubes. Compared with α_1S-expressing myotubes, maximal L channel conductance (G_max) was reduced in R1086H-expressing myotubes (α_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 α_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 α_1S enhances RyR1 sensitivity to activation by both endogenous (voltage sensor) and exogenous (caffeine) activators.