title: 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.
authors.affiliation: Department of Biochemical Pharmacology, Innsbruck Medical Univ., Peter-Mayr-Strasse 1, A-6020 Innsbruck, Austria.
authors.forename: Regina G
authors.initials: RG
authors.lastname: Weiss
authors.forename: Kristen M S
authors.initials: KM
authors.lastname: O'Connell
authors.forename: Bernhard E
authors.initials: BE
authors.lastname: Flucher
authors.forename: Paul D
authors.initials: PD
authors.lastname: Allen
authors.forename: Manfred
authors.initials: M
authors.lastname: Grabner
authors.forename: Robert T
authors.initials: RT
authors.lastname: Dirksen
mesh_terms: D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000187:drug effects / Q000235:genetics*; D002478:Cells, Cultured; D000697:Central Nervous System Stimulants / Q000494:pharmacology; D008305:Malignant Hyperthermia / Q000503:physiopathology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology*; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000187:drug effects / Q000378:metabolism; D018482:Muscle, Skeletal / Q000502:physiology*; D009154:Mutation; D019837:Ryanodine Receptor Calcium Release Channel / Q000187:drug effects / Q000378:metabolism; D012508:Sarcolemma / Q000502:physiology
chemical_list: D020746:Calcium Channels, L-Type; D000697:Central Nervous System Stimulants; D019837:Ryanodine Receptor Calcium Release Channel; D002110:Caffeine; D002118:Calcium