The Xenopus laevis oocyte is widely used to express exogenous channels and transporters and is well suited for functional measurements including currents, electrolyte and nonelectrolyte fluxes, water permeability and even enzymatic activity. It is difficult, however, to transform functional measurements recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here a method of estimating the number of exogenously expressed channels and transporters inserted in the plasma membrane of oocytes. The method is based on the facts that the P (protoplasmic) face in water-injected control oocytes exhibit an extremely low density of endogenous particles (212 +/- 48 particles/microns2, mean, SD) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/microns2) only on the P face. The utility and generality of the method were demonstrated by estimating the "gating charge" per particle of the Na+/glucose cotransporter (SGLT1) and a nonconducting mutant of the Shaker K+ channel proteins, and the single molecule water permeability of CHIP (Channel-like In-tramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a "gating charge" of approximately 3.5 electronic charges for SGLT1 and approximately 9 for the mutant Shaker K+ channel from the ratio of Qmax to density of particles measured on the same oocytes. The "gating charges" were 3-fold larger than the "effective valences" calculated by fitting a Boltzmann equation to the same charge transfer data suggesting that the charge movement in the channel and cotransporter occur in several steps. Single molecule water permeabilities (pfs) of 1.4 x 10(-14) cm3/sec for CHIP and of 1.5 x 10(-16) cm3/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (Pf) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit approximately 100-fold less effective than CHIP.

Communicated by Jared M. Diamond, University of California, Los Angeles, CA, August 28, 1996 (received for review June 24, 1996 ABSTRACTWater is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water f low is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na ؉ ͞glucose cotransporter. The Na ؉ ͞glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na ؉ ͞Cl ؊ ͞␥-aminobutyric acid, Na ؉ ͞iodide and H ؉ ͞amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.The human small intestine absorbs 10 liters of water a day, and perturbations may result in severe health problems. Worldwide, 10,000 children under 5 years of age die every day from dehydration caused by cholera. Another 3000 children suffering from dehydration are saved each day by oral rehydration therapy (1). Oral rehydration therapy is based on the fact that glucose stimulates salt and water transport across the small intestine. Fluid transport occurs in the absence or even against significant external osmotic or hydrostatic gradients, but it is secondary to active solute transport. Water movement has been postulated to be driven by local osmotic gradients within the tissue (2, 3). It is difficult to explain how local osmotic coupling takes place because (i) osmotic gradients have not been found within epithelial tissues (4, 5), (ii) no water channel proteins have been found in the small intestine (6-8), and (iii) the water permeability of brush border and basolateral membranes is extremely low (4, 9).One alternative explanation is that water transport is directly coupled to the movement of solutes by cotransporters (4, 5), and we have tested the hypothesis that water is transported across the apical membrane of the intestine by the Na ϩ ͞glucose cotransporter (SGLT1). Our results suggest that water is cotransported with sodium and sugar. The system chosen was the cloned SGLT1 expressed in Xenopus oocytes (10), and the advantages are (i) the cloned SGLT1 protein is expressed at high levels in the plasma membrane of oocytes, greater than 10 11 copies per cell (11, 12); (ii) the coupled transport of Na ϩ and glucose and the number of transporters expressed in the plasma membrane can be monitored by electrophysiological methods (11, 12); (iii) Na ϩ ͞glucose cotransport may be rapidly blocked...

Upon infection of soybean roots, nitrogen-fixing bacteria become enclosed in a specific organelle known as the symbiosome. The symbiosome membrane (SM) is a selectively permeable barrier that controls metabolite flux between the plant cytosol and the symbiotic bacterium inside. Nodulin 26 (NOD 26), a member of the aquaporin (AQP) water channel family, is a major protein component of the SM. Expression of NOD 26 in Xenopus oocytes gave a mercury-sensitive increase in osmotic water permeability (P f ). To define the biophysical properties of NOD 26 water channels in their native membranes, symbiosomes were isolated from soybean root nodules and the SM separated as vesicles from the bacteria. Permeabilities were measured using stopped-flow fluorimetry in SM vesicles with entrapped carboxyfluorescein. Osmotic water permeability (P f ) of SM was high, with a value of 0.05 ؎ 0.003 cm/s observed at 20°C (mean ؎ S.E.; n ‫؍‬ 15). Water flow exhibited a low activation energy, was inhibited by HgCl 2 (0.1 mM), and exhibited a unit conductance of 3.2 ؎ 1.3 ؋ 10 ؊15 cm 3 /s, a value 30-fold lower than that of AQP 1, the red blood cell water channel. Diffusive water permeability (P d ) was 0.0024 ؎ 0.0002 cm/s, and the resulting P f to P d ratio was 18.3, indicating that water crosses the SM in single file fashion via the NOD 26 water channel. In addition to high water permeability, SM vesicles also show high mercury-sensitive permeability to glycerol and formamide, but not urea, suggesting that NOD 26 also fluxes these solutes. Overall, we conclude that NOD 26 acts as a water channel with a single channel conductance that is 30-fold lower than AQP 1. Because the solutes that permeate NOD 26 are far larger than water, and water appears to cross the channel via a single file pathway, solute flux across NOD 26 appears to occur by a pathway that is distinct from that for water.Soil bacteria of the Rhizobium and Bradyrhizobium genera invade the roots of specific leguminous plants and establish a nitrogen fixing symbiosis. During this process, a developmental program is triggered resulting in the formation of root nodules. The bacterium becomes enclosed in a specific organelle, the symbiosome (1), within the infected cells of the nodule. This organelle is delimited by a membrane of plant origin, known as the symbiosome membrane (SM).1 This membrane regulates the efflux of fixed nitrogen from the endosymbiont to the plant cytosol as well as the influx of dicarboxylates (e.g. malate) from the plant, providing energy for nitrogen fixation (2-4).During nodule development, the expression of a number of nodule-specific proteins, known as nodulins, is induced. One of these proteins, nodulin 26 (NOD 26), is a major protein component of the SM (5). NOD 26 is a member of an ancient family of membrane channel proteins that include bacterial glycerol transporters, aquaporin (AQP) water channels, and various other intrinsic membrane proteins (6, 7). Structural similarities shared by family members include the presence of six putative transmemb...

In this paper we compare the water-transport properties of Aquaporin (AQP1), a known water channel, and those of the 28 kD Major Intrinsic Protein of Lens (MIP), a protein with an undefined physiological role. To make the comparison as direct as possible we measured functional properties in Xenopus laevis oocytes injected with cRNAs coding for the appropriate protein. We measured the osmotic permeability, Pf, (using rate of swelling) and the surface density of plasma membrane proteins (using freeze-fracture electron microscopy) in the same oocytes. Knowing both Pf and the number of exogenously expressed proteins in the membrane, we estimated the single-molecule permeability to be 2.8 x 10(-16) cm3/sec for MIP and 1.2 x 10(-14) cm3/sec for AQP1. As a negative control, a mutant MIP, truncated at the carboxyl-terminal, was shown by western blotting to be expressed, but this protein resulted in no increase in either water permeability or particle density. (Interestingly, the truncated protein was glycosylated, while the complete MIP transcript was not.) Water transport by MIP had a higher activation energy (approximately 7 Kcal/ mole) than water transport by AQP1 (approximately 2.5 Kcal/Mole) but a substantially lower activation energy than water flux across bare oolemma (approximately 20 Kcal/mole). Though the water-transport properties of MIP and AQP1 differ quantitatively, they are qualitatively quite similar. We conclude that MIP, like AQP1, forms water channels when expressed in oocytes. Thus water transport in the lens seems a plausible physiological role for MIP.

Cotransporters are membrane proteins which couple the downhill transport of cations (Na¤ or H¤) to the uphill transport of substrates (sugars, amino acids, neurotransmitters, vitamins and ions) into cells. In addition to the secondary active transport of solutes, many cotransporters exhibit a cationic leak current (uniporter mode), which is the passive transport of Na¤ (or H¤) in the absence of substrates (see Wright et al. 1996). We have previously suggested that cotransporters can function as passive (osmotic) and secondary active water transporters. For example, the Na¤glucose cotransporter SGLT1 exhibits an osmotic water permeability that is blocked by the inhibitor phlorizin (Zampighi et al. 1995;Loo et al. 1996;Loike et al. 1996;Meinild et al. 1998a). Under sugar-transporting conditions, SGLT1 transports 200-260 HµO molecules along with two sodium ions and one glucose molecule for every transport cycle. This solute-coupled water transport occurs in the absence of, and even against, an osmotic gradient (Loo et al. 1996;Zeuthen et al. 1997;Meinild et al. 1998a;Wright et al. 1998). Similar observations on the K¤-Cl¦, H¤lactate, Na¤-Cl¦-GABA, Na¤-iodide and H¤-amino acid transporters indicate that water transport may be a general phenomenon in this class of membrane transport proteins with coupling coefficients varying from 50 to 500 water molecules per substrate molecule (