Passive water and ion transport by cotransporters

1. The rabbit Na+-glucose (SGLT1) and the human Na+-Cl--GABA (GAT1) cotrans porters were expressed in Xenopus laevis oocytes, and passive Na+ and water transport were studied using electrical and optical techniques. Passive wa ter permeabilities (L-p) of the cotransporters were deter mined from the ch anges in oocyte volume in response to osmotic gradients. The specific SGLT1 and GAT1 L-p values were obtained by measuring L-p in the presence and abs ence of blockers (phlorizin and SKF89976A). In the presence of the blockers , the L-p values of oocytes expressing SGLT1 and GAT1 were indistinguishabl e from the L-p of control oocytes, 2. Passive Na+ transport (Na+ leak) was obtained from the blocker-sensitive Naf currents in the absence of substrates (glucose and GABA). Passive Naand water transport through SGLT1 were blocked by phlorizin with the same s ensitivity (inhibitory constant (K-i), 3-5 mu M). When Na+ was replaced wit h Li+, phlorizin also inhibited Li+ and water transport, but with a lower a ffinity (K-i, 100 mu M). When Na+ was replaced by choline, which is not tra nsported, the SGLT1 L-p was indistinguishable from that in Na+ or Li+, but in this case water transport was less sensitive to phlorizin. 3. The activation energies (E-a) for passive Na+ and water transport throug h SGLT1 were 21 and 5 kcal mol(-1), respectively. The high E-a for Na+ tran sport is comparable to that of Na+-glucose cotransport and indicates that t he process is dependent on conformational changes of the protein, while the low E-a for water transport is similar to that of water channels (aquapori ns). 4. GAT1 also behaved as an SKF89976A-sensitive water channel. We did not ob serve passive Na+ transport through GAT1. 5. We conclude that passive water and Na+ transport through cotransporters depend on different mechanisms: Na+ transport occurs by a saturable uniport mechanism, and water permeation is through a low conductance water channel . In the case of SGLT1, we suggest that both the water channel and water co transport could contribute to isotonic fluid transport across the intestina l brush border membrane.