Water pumps

Abstract

The transport of water across epithelia has remained an enigma ever since it was discovered over 100 years ago that water was transported across the isolated small intestine in the absence of osmotic and hydrostatic pressure gradients. While it is accepted that water transport is linked to solute transport, the actual mechanisms are not well understood. Current dogma holds that active ion transport sets up local osmotic gradients in the spaces between epithelial cells, the lateral intercellular spaces, and this in turn drives water transport by local osmosis. In the case of the small intestine, which in humans absorbs about 8 l of water a day, there is no direct evidence for either local osmosis or aquaporin gene expression in enterocytes. Intestinal water absorption is greatly enhanced by glucose, and this is the basis for oral rehydration therapy in patients with secretory diarrhoea. In our studies of the intestinal brush border Na+-glucose cotransporter we have obtained evidence that there is a direct link between the transport of Na+, glucose and water transport, i.e. there is cotransport of water along with Na+ and sugar, that will account for about 50 % of the total water transport across the human intestinal brush border membrane. In this short review we summarize the evidence for water cotransport and propose how this occurs during the enzymatic turnover of the transporter. This is a general property of cotransporters and so we expect that this may have wider implications in the transport of water and other small polar molecules across cell membranes in animals and plants.

Figure 1. Sugar-coupled water flow can be studied in very fast solution changes in the water by SGLT1 expressed in oocytes

Water transport was measured optically and Na⁺-glucose cotransport electrically. At the time indicated, 10 mm sugar (α-methyl-d-glucopyranoside) replaced 10 mm mannitol in the saline superfusing the oocyte (mm: 90 NaCl, 20 mannitol, 2 KCl, 1 MgCl₂, 1 CaCl₂, 10 Hepes adjusted to pH 7.4). A, time course of the holding current. Membrane voltage was held at -50 mV. B, the noisy record is the oocyte volume and the smooth line is the Na⁺-glucose uptake reported as the charge influx (integral of the Na⁺-glucose inward current). The charge uptake superimposes on the volume uptake, assuming that 249 water molecules accompany two Na⁺ ions and one glucose molecule. C, the relationship between Na⁺ and water uptakes by an oocyte treated with 200 nm gramicidin. When the membrane potential was stepped from -45 to -100 mV by voltage clamping, a Na⁺ inward current of ∼1900 nA was recorded. Note that in this case there was no correlation between the integrated current and volume change of the oocyte. Modified from Meinild et al. (1998).

Figure 2. The two components of water transport by SGLT1

At the arrow labelled ‘ON’, sugar was abruptly added to the external solution. The data curve represented by J_v^total is the time course of the oocyte volume. There was an initial linear component J_vⁱⁿⁱ (continuous line, with slope 1.75 × 10⁻³ ± 3.85 × 10⁻¹⁹ cm s⁻¹). Subtracting this component from the total J_v^total yields the osmotic water flow J_v^osm. In the steady state, the slope was 4.41 × 10⁻³ ± 2.29 × 10⁻⁵ cm s⁻¹. The osmotic water flow occurred after a 40 s delay (the slope in the steady state was 2.66 × 10⁻³ ± 2.29 × 10⁻⁵ cm s⁻¹). The delay seen is similar to that observed for osmotic water flow caused by Na⁺ fluxes through ion channels such as Connexin 50 and ionophores gramicidin and nystatin. When the Na⁺-glucose cotransporter was turned off with a step jump of the membrane voltage to 0 mV (see Fig. 3B), there was an immediate reduction in the rate of water transport to that observed for osmotic water flow. Δ₁ and Δ₂ are the changes in slope of the fluid transport vs. time curve (i.e. the differences in the rates of fluid transport), which would be predicted if Na⁺-glucose cotransport was turned on and off. Modified from Zeuthen et al. (2001).

Figure 3. Sugar-coupled water flow can be turned rapidly on and off with voltage absence of an osmotic gradient

The figure shows that the volume change of the oocyte can be accounted for by a stoichiometric coupling between sugar transport and water flow. In A, a SGLT1-expressing oocyte was superfused with a solution containing 5 mm α-MDG with an osmotic gradient of 15 mosmol l⁻¹, and Na⁺-glucose cotransport was inactivated by clamping the membrane voltage to 0 mV. Na⁺-glucose cotransport was turned on by stepping membrane voltage to -100 mV. There was an immediate ∼4-fold increase in sugar-coupled current and water transport, from 280 to 950 nA and 9 to 40 pl s⁻¹. Modified from Loo et al. (1996). B, Na⁺-glucose-water cotransport is turned off instantaneously by membrane voltage. Initially membrane voltage was held at -80 mV and the rate of Na⁺-glucose cotransport was 1100 nA. The oocyte swelled at a rate of 48 ± 3 pl s⁻¹. When membrane voltage was jumped to 0 mV, the sugar-coupled Na⁺ current decreased to 350 nA, and the rate of water transport was reduced to 13 ± 1 pl s⁻¹. Modified from Zeuthen et al. (2001).

Figure 4. A kinetic model for Na⁺, glucose, water and urea transport by SGLT1 (see Parent et al. 1992b; Loo et al. 1998; Meinild et al. 2002)

The six-state alternating access model shows ordered binding of substrates. Two external Na⁺ ions bind first and this promotes a conformational change in the cotransporter that allows sugar, water and urea to ‘bind’. The fully-loaded complex (C3) then undergoes an isomerization step to expose the substrates to the internal surface of the membrane (C4) where sugar and Na⁺ dissociate. Na⁺ dissociates because of the low intracellular concentration. The dissociation of Na⁺ results in a relaxation of the protein conformation to a closed state (C6) resulting in the extrusion of water and urea. The catalytic cycle is completed with the isomerization of the protein from C6 to C1. Glucose is transported across the membrane as a result of the sodium gradient across the membrane, the membrane potential, which drives the C6 to C1 conformational change as well as increasing the rate of external sodium binding to the transporter, and the strict coupling between sodium and sugar transport. It is our hypothesis that the asymmetrical conformation changes in the protein during the catalytic cycle results in the cotransport of water and urea along with Na⁺ and glucose. Note that to accommodate 200-400 water molecules (6000-12 000 ų) in the sugar-binding pocket the volume of the pocket has to be only 6-12 % of the protein volume (100 000 ų).