Purinergic inhibition of the epithelial Na+ transport via hydrolysis of PIP2.

Stimulation of purinergic receptors inhibits amiloride-sensitive Na+ transport in epithelial tissues by an unknown mechanism. Because previous studies excluded the role of intracellular Ca2+ or protein kinase C, we examined whether purinergic regulation of Na+ absorption occurs via hydrolysis of phospholipid such as phosphatidylinositol-bisphosphates (PIP2). Inhibition of amiloride-sensitive short-circuit currents (Isc-Amil) by adenine 5'-triphosphate (ATP) in native tracheal epithelia and M1 collecting duct cells was suppressed by binding neomycin to PIP2, and recovery from ATP inhibition was abolished by blocking phosphatidylinositol-4-kinase or diacylglycerol kinase. Stimulation by ATP depleted PIP2 from apical membranes, and PIP2 co-immunoprecipitated the beta subunit of ENaC. ENaC was inhibited by ATP stimulation of P2Y2 receptors in Xenopus oocytes. Mutations in the PIP2 binding domain of betaENaC but not gammaENaC reduced ENaC currents without affecting surface expression. Collectively, these data supply evidence for a novel and physiologically relevant regulation of ENaC in epithelial tissues. Although surface expression is controlled by its C terminus, N-terminal binding of betaENaC to PIP2 determines channel activity.

Expression of membrane transporters in cane toad Bufo marinus oocytes.

Membrane transport proteins (transporters and ion channels) have been extensively expressed in amphibian oocytes. The aims of this study were to determine whether oocytes from the cane toad Bufo marinus could be used as an alternative expression system to the broadly used Xenopus laevis oocytes. mRNAs encoding plasma membrane transporters NaSi-1 and sat-1 (sulphate transporters), NaDC-1 (dicarboxylate transporter), SGLT-1 (Na(+)/glucose cotransporter) and rBAT and 4F2 hc (amino acid transporters) were injected into B. marinus oocytes. All led to significant induction of their respective transport activities. Uptake rates were comparable with those in X. laevis oocytes, with the exception of rBAT, which was able to induce amino acid uptake only in X. laevis oocytes, suggesting that rBAT may require an endogenous X. laevis oocyte protein that is absent from B. marinus oocytes. Transport kinetics were determined for the NaSi-1 cotransporter in B. marinus oocytes, with identical results to those obtained in X. laevis oocytes. NaSi-1 specificity for the Na(+) cation was determined, and the anions selenate, molybdate, tungstate, oxalate and thiosulphate could all inhibit NaSi-1-induced sulphate transport. This study demonstrates that cane toad oocytes can be used successfully to express plasma membrane proteins, making this a viable heterologous system for the expression of proteins.