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Three forms of muscular dystrophy-dystroglycanopathies are linked to the ribitol pathway. These include mutations in the isoprenoid synthase domain-containing protein (ISPD), fukutin-related protein (FKRP), and fukutin (FKTN) genes. The aforementioned enzymes are required for generation of the ribitol phosphate linkage in the O-glycan of alpha-dystroglycan. Mild cases of dystroglycanopathy present with slowly progressive muscle weakness, while in severe cases the eyes and brain are also involved. Previous research showed that ribose increased the intracellular concentrations of cytidine diphosphate-ribitol (CDP-ribitol) and had a therapeutic effect. Here, we report the safety and effects of oral ribose supplementation during 6 months in a patient with limb girdle muscular dystrophy type 2I (LGMD2I) due to a homozygous FKRP mutation. Ribose was well tolerated in doses of 9 g or 18 g/day. Supplementation with 18 g of ribose resulted in a decrease of creatine kinase levels of 70%. Moreover, metabolomics showed a significant increase in CDP-ribitol levels with 18 g of ribose supplementation (p < 0.001). Although objective improvement in clinical and patient-reported outcome measures was not observed, the patient reported subjective improvement of muscle strength, fatigue, and pain. This case study indicates that ribose supplementation in patients with dystroglycanopathy is safe and highlights the importance for future studies regarding its potential effects.

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We have determined the effects of coexpression of Kv2.1 with electrically silent Kv5.1 or Kv6.1 alpha-subunits in Xenopus oocytes on channel gating. Kv2.1/5.1 selectively accelerated the rate ofinactivation at intermediate potentials (-30 to 0 mV), without affecting the rate at strong depolarization (0 to +40 mV), and markedly accelerated the rate of cumulative inactivation evoked by high-frequency trains of short pulses. Kv5.1 coexpression alsoslowed deactivation of Kv2.1. In contrast, Kv6.1 was much less effective in speeding inactivation at intermediate potentials, had a slowing effect on inactivation at strong depolarizations, and had no effect on cumulative inactivation. Kv6.1, however, had profound effects on activation, including a negative shift of the steady-state activation curve and marked slowing of deactivation tail currents. Support for the notion that the Kv5.1's effects stem from coassembly of alpha-subunits into heteromeric channels was obtained from biochemical evidence of protein-protein interaction and single-channel measurements that showed heterogeneity in unitary conductance. Our results show that Kv5.1 and Kv6.1 function as regulatory alpha-subunits that when coassembled with Kv2.1 can modulate gating in a physiologically relevant manner.

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Heteromultimer formation between Kv potassium channel subfamilies with the production of a novel current is reported for the first time. Protein-protein interactions between Kv2.1 and electrically silent Kv6.1 alpha-subunits were detected using two microelectrode voltage clamp and yeast two-hybrid measurements. Amino terminal portions of Kv6.1 were unable to form homomultimers but interacted specifically with amino termini of Kv2.1. Xenopus oocytes co-injected with Kv6.1 and Kv2.1 cRNAs exhibited a novel current with decreased rates of deactivation, decreased sensitivity to TEA block, and a hyperpolarizing shift of the half maximal activation potential when compared to Kv2.1. Our results indicate that Kv channel subfamilies can form heteromultimeric channels and, for the first time, suggest a possible functional role for the Kv6 subfamily.

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Osmotic stress elicits hypertonic NaCl secretion and promotes structural and biochemical differentiation in avian salt glands. In addition to cholinergic control, Cl- secretion is stimulated by vasoactive intestinal peptide (VIP), suggesting that the cystic fibrosis transmembrane conductance regulator (CFTR) may be present and that its expression may be regulated by chronic salt stress. Anion efflux, assayed by 6-methoxy-N-(3-sulfopropyl)quinolinium fluorescence changes in single cells, was stimulated by VIP or 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate. Immunoblots with a COOH-terminal peptide antibody to human CFTR revealed approximately 170- and approximately 180-kDa bands in lysates from control and salt-stressed glands, respectively. Both variants reduced to approximately 140 kDa after N-glycanase digestion and gave identical tryptic phosphopeptide maps after immunoprecipitation and phosphorylation by protein kinase A. CFTR was localized to apical membranes by immunofluorescence and, additionally, to subapical vesicles by immunoelectron microscopy. Salt stress induced an approximately twofold increase in CFTR abundance/cell protein (approximately 5-fold/cell) and intensified apical membrane immunofluorescence. For comparison, Na+ pump expression increased approximately fourfold per cell protein with little change in actin. Thus differentiation induced by salt stress is accompanied by alteration in CFTR abundance and glycosylation. Upregulation of CFTR likely contributes to increased efficiency of Cl- secretion.

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The basolateral Na-H antiporter of the turtle colon exhibits both conductive and electroneutral Na+ transport (Post and Dawson. 1992. American Journal of Physiology. 262:C1089-C1094). To explore the mechanism of antiporter-mediated current flow, we compared the conditions necessary to evoke conduction and exchange, and determined the kinetics of activation for both processes. Outward (cell to extracellular fluid) but not inward (extracellular fluid to cell) Na+ or Li+ gradients promoted antiporter-mediated Na+ or Li+ currents, whereas an outwardly directed proton gradient drove inward Na+ or Li+ currents. Proton gradient-driven, "counterflow" current is strong evidence for an exchange stoichiometry of > 1 Na+ or Li+ per proton. Consistent with this notion, outward Na+ and Li+ currents generated by outward Na+ or Li+ gradients displayed sigmoidal activation kinetics. Antiporter-mediated proton currents were never observed, suggesting that only a single proton was transported per turnover of the antiporter. In contrast to Na+ conduction, Na+ exchange was driven by either outwardly or inwardly directed Na+, Li+, or H+ gradients, and the activation of Na+/Na+ exchange was consistent with Michaelis-Menten kinetics (K1/2 = 5 mM). Raising the extracellular fluid Na+ or Li+ concentration, but not extracellular fluid proton concentration, inhibited antiporter-mediated conduction and activated Na+ exchange. These results are consistent with a model for the Na-H antiporter in which the binding of Na+ or Li+ to a high-affinity site gives rise to one-for-one cation exchange, but the binding of Na+ or Li+ ions to other, lower-affinity sites can give rise to a nonunity, cation exchange stoichiometry and, hence, the net translocation of charge. The relative proportion of conductive and nonconductive events is determined by the magnitude and orientation of the substrate gradient and by the serosal concentration of Na+ or Li+.

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The effects of HgCl2 on ion transport were investigated using isolated sheets of flounder urinary bladder, a model epithelium that is capable of electrically silent NaCl absorption and electrogenic K secretion. Exposure of the mucosal surface of the bladder to submicromolar doses of HgCl2 reduced K secretion, but the effect was not due to blockade of apical K channels. Rather, the effects of HgCl2 were virtually identical to those seen with experimental maneuvers that blocked the thiazide-sensitive NaCl cotransporter in the apical membrane, e.g., hydrochlorothiazide, Cl-free solutions, and Na-free solutions. Mucosal HgCl2 also blocked 22Na absorption, suggesting that the effect of the metal was mediated by blockade of NaCl entry. The effects of HgCl2 had a rapid onset and were readily reversed by washing, suggesting a noncovalent binding reaction. The abundance of polyanionic Hg complexes in salt solutions prompts the speculation that one of these may bind to the Cl-binding site on the cotransporter, thereby blocking it. The results provide the first evidence that the thiazide-sensitive NaCl cotransporter is a specific site of action for inorganic mercury.

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An amiloride-inhibitable, Na(+)-H+ antiporter was identified in the basolateral membrane of turtle colon by measuring 22Na+ fluxes across isolated tissues apically permeabilized with the pore-forming antibiotic amphotericin B. In cells shrunken by exposure to Cl(-)-free (gluconate) solutions and treated with ouabain to block the Na-K-ATPase, Na+ movement across the basolateral membrane was due entirely to the antiporter. Elevation of cytosolic Na+ was associated with an amiloride-inhibitable outward current across the basolateral membrane. The sensitivity of the current to various amiloride analogues paralleled that of Na+ exchange rather than that of the apical Na+ channel. Furthermore, cell volume changes altered basolateral Na+ exchange and basolateral Na+ conductance in a parallel fashion. We propose that this amiloride-sensitive basolateral Na+ conductance represents an altered operating mode of a basolateral Na(+)-H+ exchanger.

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