Passive water and urea permeability of a human Na+–glutamate cotransporter expressed in Xenopus oocytes

The human Na⁺-glutamate transporter (EAAT1) was expressed in Xenopus laevis oocytes. The passive water permeability, L _p, was derived from volume changes of the oocyte induced by changes in the external osmolarity. Oocytes were subjected to two-electrode voltage clamp. In the presence of Na⁺, the EAAT1-specific (defined in Discussion) L _p increased linearly with positive clamp potentials, the L _p being around 23 % larger at +50 mV than at –50 mV. l -Glutamate increased the EAAT1-specific L _p by up to 40 %. The K _0.5 for the glutamate-dependent increase was 20 ± 6 μ m , which is similar to the K _0.5 value for glutamate activation of transport. The specific inhibitor dl - threo -β-benzyloxyaspartate (TBOA) reduced the EAAT1-specific L _p to 72 %. EAAT1 supported passive fluxes of [¹⁴C]urea and [¹⁴C]glycerol. The [¹⁴C]urea flux was increased in the presence of glutamate. The data suggest that the permeability depends on the conformational equilibrium of the EAAT1. At positive potentials and in the presence of Na⁺ and glutamate, the pore is enlarged and water and urea penetrate more readily. The L _p was larger when measured with urea or glycerol as osmolytes as compared with mannitol. Apparently, the properties of the pore are not uniform along its length. The outer section may accommodate urea and glycerol in an osmotically active form, giving rise to larger water fluxes. The physiological role of EAAT1 for water homeostasis in the central nervous system is discussed.     

Activation of ferret erythrocyte Na+–K+–2Cl− cotransport by deoxygenation

Deoxygenation of ferret erythrocytes stimulates Na⁺–K⁺–2Cl⁻ cotransport by 111% ( s.d. , 46) compared to controls in air. Half-maximal activation occurs at a P _O2 of 24 mmHg ( s.d. , 2) indicating that physiological changes in oxygen tension can influence cotransport function. Approximately 25–35% of this stimulation can be attributed to the rise of intracellular free magnesium concentration that occurs on deoxygenation (from 0.82 ( s.d. , 0.07) to 1.40 m m ( s.d. , 0.17)). Most of the stimulation is probably caused by activation of a kinase which can be prevented or reversed by treating cells with the kinase inhibitors PP1 or staurosporine, or by reducing cell magnesium content to submicromolar levels. Stimulation by deoxygenation is comparable with that caused by calyculin A or sodium arsenite, compounds that cause a 2- to 3-fold increase in threonine phosphorylation of the cotransporter which can be detected with phospho-specific antibodies. However, the same approach failed to detect significant changes in threonine phosphorylation following deoxygenation. The results suggest that deoxygenation causes activation of a kinase that either phosphorylates the transporter, but probably not on threonine, or phosphorylates another protein that in turn influences cotransporter behaviour. They also indicate that more than one kinase and phosphatase are involved in cotransporter phosphorylation.     

Inhibition of rat Na+–HCO3– cotransporter (NBCn1) function and expression by the alternative splice domain

The Na⁺–HCO₃^– cotransporter NBCn1 (SLC4A7) has multiple variants depending upon splice domains in the cytoplasmic amino- and carboxy-termini of the protein. In this study, we examined the role of the amino-terminal splice domain containing 123 amino acids (cassette II) in the regulation of NBCn1 function and expression. Polymerase chain reaction detected NBCn1 mRNAs containing cassette II in a variety of tissues. Two variants, NBCn1-B containing cassette II and NBCn1-E lacking cassette II, were expressed in Xenopus oocytes and assessed by two-electrode voltage clamp to measure the ionic current mediated by the transporters. The two variants showed similar current–voltage ( I – V ) relations when measured 3–4 days after RNA injection. Replacment of Cl⁻ with gluconate did not affect the I – V relations. When exposed to solutions containing 20–50 m m Na⁺, the current produced by NBCn1-B was slightly more positive than that produced by NBCn1-E. The two currents were similar at 100 m m Na⁺. The slope conductances for the two variants were progressively increased at higher Na⁺ levels, and the increases were parallel and superimposed. Measured at different time points after RNA injection, NBCn1-B produced lower conductance than NBCn1-E at 24–48 h. Protein expression of NBCn1-B was also low at these time points as determined by immunoblot of oocyte membrane preparation. Expressed in opossum kidney (OK) cells, NBCn1-E caused a 1.5-fold increase in ouabain-sensitive production of p -nitrophenol from p -phenyl phosphate compared with control preparations, whereas NBCn1-B had negligible effect. We conclude that the primary function of cassette II is to reduce NBCn1 protein expression.     

Functional role of Na+–HCO3− cotransport in migration of transformed renal epithelial cells

Cell migration is crucial for immune defence, wound healing or formation of tumour metastases. It has been shown that the activity of the Na⁺–H⁺ exchanger (NHE1) plays an important role in cell migration. However, so far it is unknown whether Na⁺– HCO₃⁻ cotransport (NBC), which has similar functions in the regulation of intracellular pH (pH_i) as NHE1, is also involved in cell migration. We therefore isolated NHE-deficient Madin-Darby canine kidney (MDCK-F) cells and tested whether NBC compensates for NHE in pH_i and cell volume regulation as well as in migration. Intracellular pH was measured with the fluorescent pH indicator 2'7'-bis(carboxyethyl)-5-carboxyfluorescein (BCECF). The expression of NBC isoforms was determined with semiquantitative PCR. Migration was monitored with time-lapse video microscopy and quantified as the displacement of the cell centre. We found that MDCK-F cells express the isoform NBC1 ( SLCA4A gene product) at a much higher level than the isoform kNBC3 ( SLCA4A8 gene product). This difference is even more pronounced in NHE-deficient cells so that NBC1 is likely to be the major acid extruder in these cells and the major mediator of propionate-induced cell volume increase. NHE-deficient MDCK-F cells migrate more slowly than normal MDCK-F cells. NBC activity promotes migration during an acute intracellular acid load and increases migratory speed and displacement on a short timescale (< 30 min) whereas it has no effect on the long-term behaviour of migrating MDCK-F cells. Taken together, our results show that NBC actvity, despite many functional similarities, does not have the same importance for cell migration as NHE1 activity.