Gene dose influences cellular and calcium channel dysregulation in heterozygous and homozygous T4826I-RYR1 malignant hyperthermia-susceptible muscle

Malignant hyperthermia susceptibility (MHS) is primarily conferred by mutations within ryanodine receptor type 1 (RYR1). Here we address how the MHS mutation T4826I within the S4-S5 linker influences excitation-contraction coupling and resting myoplasmi

Ryanodine Receptor-Mediated Calcium Release Has a Key Role in Hippocampal LTD Induction

The induction of both long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission entails pre- and postsynaptic Ca2+ signals, which represent transient increments in cytoplasmic free Ca2+ concentration. In diverse synapse types, Ca2+ release from intracellular stores contributes to amplify the Ca2+ signals initially generated by activation of neuronal Ca2+ entry pathways. Here, we used hippocampal slices from young male rats to evaluate whether pharmacological activation or inhibition of Ca2+ release from the endoplasmic reticulum (ER) mediated by ryanodine receptor (RyR) channels modifies LTD induction at Schaffer collateral-CA1 synapses. Pre-incubation of slices with ryanodine (1 μM, 1 h) or caffeine (1 mM, 30 min) to promote RyR-mediated Ca2+ release facilitated LTD induction by low frequency stimulation (LFS), but did not affect the amplitude of synaptic transmission, the profiles of field excitatory postsynaptic potentials (fEPSP) or the paired-pulse (PP) responses. Conversely, treatment with inhibitory ryanodine (20 μM, 1 h) to suppress RyR-mediated Ca2+ release prevented LTD induction, but did not affect baseline synaptic transmission or PP responses. Previous literature reports indicate that LTD induction requires presynaptic CaMKII activity. We found that 1 h after applying the LTD induction protocol, slices displayed a significant increase in CaMKII phosphorylation relative to the levels exhibited by un-stimulated (naïve) slices. In addition, LTD induction (1 h) enhanced the phosphorylation of the presynaptic protein Synapsin I at a CaMKII-dependent phosphorylation site, indicating that LTD induction stimulates presynaptic CaMKII activity. Pre-incubation of slices with 20 μM ryanodine abolished the increased CaMKII and Synapsin I phosphorylation induced by LTD, whereas naïve slices pre-incubated with inhibitory ryanodine displayed similar CaMKII and Synapsin I phosphorylation levels as naïve control slices. We posit that inhibitory ryanodine suppressed LTD-induced presynaptic CaMKII activity, as evidenced by the suppression of Synapsin I phosphorylation induced by LTD. Accordingly, we propose that presynaptic RyR-mediated Ca2+ signals contribute to LTD induction at Schaffer collateral-CA1 synapses

Functional and Biochemical Properties of Ryanodine Receptor Type 1 Channels from Heterozygous R163C Malignant Hyperthermia-Susceptible Mice

Mutations in ryanodine receptor type 1 (RyR1) confer malignant hyperthermia susceptibility. How inherent impairments in Ca2+ channel regulation affect skeletal muscle function in myotubes and adult fibers under basal (nontriggering) conditions are not understood. Myotubes, adult flexor digitorum brevis (FDB) fibers, and sarcoplasmic reticulum skeletal membranes were isolated from heterozygous knockin R163C and wild-type (WT) mice. Compared with WT myotubules, R163C myotubes have reduced Ca2+ transient amplitudes in response to electrical field pulses; however, R163C FDB fibers do not differ in their responses to electrical stimuli, despite heightened cellular cytoplasmic resting Ca2+ ([Ca2+]rest) and sensitivity to halothane. Immunoblotting of membranes from each genotype shows similar expression of RyR1, FK506 binding protein 12 kDa, and Ca2+-ATPase, but RyR1 2844Ser phosphorylation in R163C muscle is 31% higher than that of WT muscle (p < 0.001). RyR1 channels reconstituted in planar lipid bilayers reveal ∼65% of R163C channels exhibit ≥2-fold greater open probability (Po) than WT, with prolonged mean open dwell times and shortened closed dwell times. [3H]Ryanodine (Ry) binding and single-channel analyses show that R163C-RyR1 has altered regulation compared with WT: 1) 3-fold higher sensitivity to Ca2+ activation; 2) 2-fold greater [3H]Ry receptor occupancy; 3) comparatively higher channel activity, even in reducing glutathione buffer; 4) enhanced RyR1 activity both at 25 and 37°C; and 5) elevated cytoplasmic [Ca2+]rest. R163C channels are inherently more active than WT channels, a functional impairment that cannot be reversed by dephosphorylation with protein phosphatase. Dysregulated R163C channels produce a more overt phenotype in myotubes than in adult fibers in the absence of triggering agents, suggesting tighter negative regulation of R163C-RyR1 within the Ca2+ release unit of adult fibers