In skeletal muscle, the interaction between two proteins, the dihydropyridine receptor (DHPR) in the plasma membrane/transverse-tubules and the type 1 ryanodine receptor (RyR1) in the sarcoplasmic reticulum (SR), is essential for linking electrical excitation to contraction (excitation-contraction coupling, EC coupling). In particular, it is thought that conformational changes of the DHPR (containing Cav1.1 as its principal subunit) in response to depolarization cause RyR1 to open and release calcium from the SR, and that this signaling depends on physical links between the two proteins. Significantly, mutations of the DHPR or RyR1 in humans can result in the inherited disorder of malignant hyperthermia susceptibility (MHS), whereby volatile anesthetics cause dysregulation of calcium release that can lead to a fatal rise in body temperature unless there is rapid intervention. With the long-term objective of understanding the interactions between the DHPR and RyR1, and the mechanisms of MH, the specific aims of Project 3 are:
Aim 1. 1: To use whole cell patch clamping to measure L-type Ca2+ current and charge movement (which arise directly from the DHPR), together with Ca release from the SR, to determine how MHS mutations (RyR1-R2435H and -T4826I;Cav1.1-R174W) in mouse and human myotubes affect bi-directional signaling.
Aim 1. 2: To determine the effect of dantrolene on bi-directional signaling in MHS myotubes.
Aim 1. 3: In collaboration with Core D, to determine if MHS mutations alter the frequency or disposition of DHPR tetrads in myotubes.
Aim 2 : To measure membrane currents and myoplasmic Ca2+ transients in dissociated FDB fibers from adult (3-6 mo) and aged (12-18 mo) male and female MHS mice (Cav1.1-R174W and RyR1-R163C,-R2435H, and -T4826I) to determine the heterogeneity that results from differences in gender, age and locus.
Aim 3 : To measure membrane currents and Ca2+ transients in FDB fibers from 3-6 month old male Het RyR1-T4826I MHS mice that have been crossed with mice over-expressing SERCA1 (enhanced SR Ca2+ filling) or dnTPRC6 (reduced SOCE), or which were administered 4-OH-BDE49 (reduced RyR1 leak) or salicylamine (?KA scavenger) to determine if modification of one of the 4 key elements associated with MHS can mitigate or abrogate alterations in RyR-DHPR bi-directional signaling.
Aim 4. 1: To determine whether Ca2+ currents, charge movements or voltage-gated Ca2+ transients are differentially affected by volatile anesthetics in WT or MHS mutant (RyR1-R1630, -R2435H, -T4826I;Cav1.1-R174W) FDBs.
Aim 4. 2: To determine whether effects of volatile anesthetics on Ca2+ channel function are prevented by treatment with dantrolene.
Aim 5 : To use expression in myotubes of proteins harboring MHS mutations newly discovered by Core C in order to determine their effects on bi-directional signaling and hypersensitivity to volatile anesthetics.
Voluntary movements depend on excitation-contraction (EC) coupling, in which an electrical signal causes calcium to be released inside skeletal muscle cells, which in turn causes contraction. Inherited mutations can cause this system to malfunction during general anesthesia and result in the potentially fatal condition termed malignant hyperthermia (MH). The proposed experiments will investigate why these mutations cause MH susceptibility.
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