Novel regulators of RyR Ca2+ release channels: insight into molecular changes in genetically-linked myopathies.

There are many mutations in the ryanodine receptor (RyR) Ca2+ release channel that are implicated in skeletal muscle disorders and cardiac arrhythmias. More than 80 mutations in the skeletal RyR1 have been identified and linked to malignant hyperthermia, central core disease or multi-minicore disease, while more than 40 mutations in the cardiac RyR2 lead to ventricular arrhythmias and sudden cardiac death in patients with structurally normal hearts. These RyR mutations cause diverse changes in RyR activity which either excessively activate or block the channel in a manner that disrupts Ca2+ signalling in the muscle fibres. In a different myopathy, myotonic dystrophy (DM), a juvenile isoform of the skeletal RyR is preferentially expressed in adults. There are two regions of RyR1 that are variably spiced and developmentally regulated (ASI and ASII). The juvenile isoform (ASI(-)) is less active than the adult isoform (ASI(+)) and its over-expression in adults with DM may contribute to functional changes. Finally, mutations in an important regulator of the RyR, the Ca2+ binding protein calsequestrin (CSQ), have been linked to a disruption of Ca2+ homeostasis in cardiac myocytes that results in arrhythmias. We discuss evidence supporting the hypothesis that mutations in each of these situations alter protein/protein interactions within the RyR complex or between the RyR and its associated proteins. The disruption of these protein-protein interactions can lead either to excess Ca2+ release or reduced Ca2+ release and thus to abnormal Ca2+ homeostasis. Much of the evidence for disruption of protein-protein interactions has been provided by the actions of a group of novel RyR regulators, domain peptides with sequences that correspond to sequences within the RyR and which compete with the endogenous residues for their interaction sites.

Passive nitrate transport by root plasma membrane vesicles exhibits an acidic optimal pH like the H(+)-ATPase.

The net initial passive flux (J(Ni)) in reconstituted plasma membrane (PM) vesicles from maize (Zea mays) root cells was measured as recently described (P. Pouliquin, J.-P. Grouzis, R. Gibrat ¿1999 Biophys J 76: 360-373). J(Ni) in control liposomes responded to membrane potential or to NO(3)(-) as expected from the Goldman-Hodgkin-Katz diffusion theory. J(Ni) in reconstituted PM vesicles exhibited an additional component (J(Nif)), which was saturable (K(m) for NO(3)(-) approximately 3 mM, with J(Nifmax) corresponding to 60 x 10(-9) mol m(-2) s(-1) at the native PM level) and selective (NO(3)(-) = ClO(3)(-) > Br(-) > Cl(-) = NO(2)(-); relative fluxes at 5 mM: 1:0.34:0.19). J(Nif) was totally inhibited by La(3+) and the arginine reagent phenylglyoxal. J(Nif) was voltage dependent, with an optimum voltage at 105 mV at pH 6.5. The activation energy of J(Nif) was high (129 kJ mol(-1)), close to that of the H(+)-ATPase (155 kJ mol(-1)), and J(Nif) displayed the same acidic optimal pH (pH 6.5) as that of the H(+) pump. This is the first example, to our knowledge, of a secondary transport at the plant PM with such a feature. Several properties of the NO(3)(-) uniport seem poorly compatible with that reported for plant anion channels and to be attributable instead to a classical carrier. The physiological relevance of these findings is suggested.

Electrophysiological study with oxonol VI of passive NO3- transport by isolated plant root plasma membrane.

In contrast to animal cells, plant cells contain approximately 5-50 mM nitrate in cytosol and vacuole. The lack of specific spectroscopic probes, or suitable isotopes, impedes in vitro studies of NO3- transport. Reconstitution of root cell plasma membrane (PM) proteins in mixed soybean lipid:egg phosphatidylcholine allowed for the generation of large K+-valinomycin diffusion potentials (Em), monitored with the oxonol VI dye. Nevertheless, Em was restricted to approximately 130 mV by capacitor properties of biological membranes. This caused an increasing discrepancy at higher K+-Nernst potentials used for calibration. Therefore, Em was determined directly from the fluorescence of the dye free in buffer, bound at zero Em, and bound upon Em generation. Then, an electrophysiological analysis of the NO3--dependent dissipation rate of Em gave the net passive flux (JN) and the permeability coefficient to NO3- (PN). The plant root cell PM exhibited a strikingly large PN (higher than 10(-9) m s-1) at high Em (90-100 mV) and pH 6.5. At low Em (50-60 mV) and pH 7.4, PN decreased by 70-fold and became similar to that of the lipid bilayer. This agreed with the previous observation that 15 mM NO3- short-circuits the plant root PM H+-ATPase at its optimal pH of 6.5.

In vitro study of passive nitrate transport by native and reconstituted plasma membrane vesicles from corn root cells.

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.