Myotonic dystrophy type 1 (DM1) is a debilitating multisystemic disorder caused by a CTG repeat expansion in the DMPK gene. Aberrant splicing of several genes has been reported to contribute to some symptoms of DM1, but the cause of muscle weakness in DM1 and elevated Ca2+ concentrations in cultured DM muscle cells is unknown. Here, we investigated the alternative splicing of mRNAs of two major proteins of the sarcoplasmic reticulum, the ryanodine receptor 1 (RyR1) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) 1 or 2. The fetal variants, ASI(-) of RyR1 which lacks residue 3481-3485, and SERCA1b which differs at the C-terminal were significantly increased in skeletal muscles from DM1 patients and the transgenic mouse model of DM1 (HSA(LR)). In addition, a novel variant of SERCA2 was significantly decreased in DM1 patients. The total amount of mRNA for RyR1, SERCA1 and SERCA2 in DM1 and the expression levels of their proteins in HSA(LR) mice were not significantly different. However, heterologous expression of ASI(-) in cultured cells showed decreased affinity for [3H]ryanodine but similar Ca2+ dependency, and decreased channel activity in single-channel recording when compared with wild-type (WT) RyR1. In support of this, RyR1-knockout myotubes expressing ASI(-) exhibited a decreased incidence of Ca2+ oscillations during caffeine exposure compared with that observed for myotubes expressing WT-RyR1. We suggest that aberrant splicing of RyR1 and SERCA1 mRNAs might contribute to impaired Ca2+ homeostasis in DM1 muscle.

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The recently discovered CLIC-2 protein (where CLIC stands for chloride intracellular channel), which belongs to the ubiquitous glutathione transferase structural family and is expressed in the myocardium, is a regulator of native cardiac RyR2 (ryanodine receptor 2) channels. Here we show that recombinant CLIC-2 increases [3H]ryanodine binding to native and purified RyR channels, enhances substate activity in individual channels, increases the number of rare coupled gating events between associated RyRs, and reduces activation of the channels by their primary endogenous cytoplasmic ligands, ATP and Ca2+. CLIC-2 (0.2-10 microM) added to the cytoplasmic side of RyR2 channels in lipid bilayers depressed activity in a reversible, voltage-independent, manner in the presence of activating (10-100 microM) or sub-activating (100 nM) cytoplasmic Ca2+ concentrations. Although the number of channel openings to all levels was reduced, the fraction and duration of openings to substate levels were increased after exposure to CLIC-2. CLIC-2 reduced increases in activity induced by ATP or adenosine 5'-[beta,gamma-imido]triphosphate. Depression of channel activity by CLIC-2 was greater in the presence of 100 microM cytoplasmic Ca2+ than with 100 nM or 10 microM Ca2+. Further, CLIC-2 prevented the usual approximately 50-fold increase in activity when the cytoplasmic Ca2+ concentration was increased from 100 nM to 100 microM. The results show that CLIC-2 interacts with the RyR protein by a mechanism that does not require oxidation, but is influenced by a conserved Cys residue at position 30. CLIC-2 is one of only a few cytosolic inhibitors of cardiac RyR2 channels, and may suppress their activity during diastole and during stress. CLIC-2 provides a unique probe for substate activity, coupled gating and ligand-induced activation of cardiac RyR channels.

Proteins from phase-partitioned corn root plasma membrane were reconstituted into soybean lipids/egg PC (8:2, w:w) using deoxycholate and rapid gel filtration to eliminate the detergent. All (H+)ATPase molecules were inside-out reinserted and the initial activity was totally recovered in an homogeneous vesicle preparation. In addition, membrane tightness greatly increased, as shown by the size and stability of the response of the fluorescent membrane potential probe (oxonol VI) to an imposed K+ diffusion gradient. Consequently, the H(+)-pumping activity of the (H+)ATPase, monitored with the fluorescent pH probe (ACMA), increased 20-fold after reconstitution. A protein-mediated passive transport of nitrate was first demonstrated by the ability of NO3- to electrically short-circuit the (H+)ATPase in plasma membrane vesicles and not in liposomes containing only the purified enzyme. The passive transport was saturable (K(m) approximately 5 mM), thermolabile, inhibited by the arginine reagent phenylglyoxal, and selective (NO3- > I- approximately ClO3- approximately Br- > Cl- approximately NO2- > Iminodiacetate approximately SO4(2-)). Passive NO3- transport was also determined, independently of the (H+)ATPase, from the NO3(-)-dependent augmentation of the dissipation rate of imposed diffusion potentials. This second transport assay gave similar K(m) for NO3- and should be suitable to continue the functional and biochemical characterization of the NO3- transport system.