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. 2012 Mar 1;590(5):1139-54.
doi: 10.1113/jphysiol.2011.226316. Epub 2012 Jan 16.

Cotransport of water by Na⁺-K⁺-2Cl⁻ cotransporters expressed in Xenopus oocytes: NKCC1 versus NKCC2

Thomas Zeuthen  1 , Nanna Macaulay
Affiliations

Cotransport of water by Na⁺-K⁺-2Cl⁻ cotransporters expressed in Xenopus oocytes: NKCC1 versus NKCC2

Thomas Zeuthen et al. J Physiol. .

Abstract

The NKCC1 and NKCC2 isoforms of the mammalian Na⁺–K⁺–2Cl⁻ cotransporter were expressed in Xenopus oocytes and the relation between external ion concentration and water fluxes determined.Water fluxes were determined from changes in the oocytes volume and ion fluxes from 86Rb+ uptake. Isotonic increases in external K⁺ concentration elicited abrupt inward water fluxes in NKCC1; the K⁺ dependence obeyed one-site kinetics with a K₀.₅ of 7.5 mM. The water fluxes were blocked by bumetanide, had steep temperature dependence and could proceed uphill against an osmotic gradient of 20 mosmol l⁻¹. A comparison between ion and water fluxes indicates that 460 water molecules are cotransported for each turnover of the protein. In contrast, NKCC2 did not support water fluxes.Water transport in NKCC1 induced by increases in the external osmolarity had high activation energy and was blocked by bumetanide. The osmotic effects of NaCl were smaller than those of urea and mannitol. This supports the notion of interaction between ions and water in NKCC1 and allows for an estimate of around 600 water molecules transported per turnover of the protein. Osmotic gradients did not induce water transport in NKCC2. We conclude that NKCC1 plays a direct role for water balance in most cell types, while NKCC2 fulfils its role in the kidney of transporting ions but not water. The different behaviour of NKCC1 and NKCC2 is discussed on the basis of recent molecular models based on studies of structural and molecular dynamics.

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Figures

Figure 1
Figure 1. Hypothesis and experimental set-up
A, previous experiments have shown that Na+, Cl− and possibly K+ are linked to water transport in NKCC1 by a molecular mechanism closely associated with the protein itself (Hamann et al. 2005, 2010). In the present paper we express NKCC1 (and NKCC2) in Xenopus oocytes in order to study the effects of abrupt changes of external ion concentrations on oocyte volume. Most of our experiments are explained if NKCC1 cotransports 460 water molecules into the oocyte for each 1Na+, 1K+ and 2 Cl− ions. In contrast, NKCC2 cotransports no water. B, Xenopus oocytes were placed in a small chamber and held by two microelectrodes that also served to monitor the membrane potential or to voltage-clamp the oocyte. The oocyte was illuminated from above, and its cross-section recorded via an inverted microscope. The oocyte volume could then be recorded online with a time resolution of 1 s and a volume sensitivity of 20 pl (Zeuthen et al. 1997, 2006).
Figure 3
Figure 3. Rapid onset of water transport in NKCC1 induced by isosmotic increase in K+ concentration
A, at t = 0, the extracellular K+ concentration was increased by 10 mm; the Na+ concentration was reduced concurrently to maintain osmolarity. This initiated an increase in the volume of a NKCC1-expressing oocyte (ΔV full) as well as a depolarization of the membrane potential (ΔEm). The time course of this depolarization is determined by the increase in K+ concentration at the oocyte surface. If water is cotransported with the ions in NKCC1 in a fixed ratio, the increase in oocyte volume can be calculated to follow the dashed curve; see text. B shows the onset of the K+- induced volume increase at high resolution (magnified from the trace in A). The noise of the baseline is characterized by the upper and lower 95% confidence levels (dashed lines CL). When the K+ concentration is increased at t = 0, the oocyte volume begins to increase and exceeds the upper CL after about 1 s. Bath temperature 25 °C.
Figure 7
Figure 7. Uphill water transport by NKCC1
A, inward water transport (oocyte swelling, ΔV) was initiated by an isosmotic increase in external K+ concentration of 15 mm. In B, the K+ concentration was increased simultaneously with an increase in osmolarity of 10 mosmol l−1 implemented by urea (10 U); this did not prevent inward water transport. C, if K+ was added together with 20 mosmol l−1 of urea, there was no net influx of water. D, increase in urea alone (20 mosmol l−1, no change in K+ concentration) caused efflux of water. E, increases in K+ concentrations gave rise to influx of water despite opposing osmotic gradients (Δπ) of 10 or 20 mosmol l−1 (filled symbols). Osmotic challenges alone gave rise to effluxes of water (open symbols). The K+-induced component of water transport (the vertical distance between the two lines) was independent of the osmotic gradient imposed (number of oocytes in parentheses). The experiments were performed at 25°C; similar results were obtained at temperatures of 35°C (not shown).
Figure 4
Figure 4. Influx of water by NKCC1 as a function of the external K+ concentration
A, changes in oocyte volume (ΔV) in response to isosmotic increases in external K+ concentration (ΔK+). Values of ΔK+ between 1.25 and 30 mm were tested for durations of 40 s. The recordings are from the same oocyte; experiments as in Figs 2 and 3; the control bathing solution contained 2 mm K+. B, the rates of change in oocyte volume (dV/dt) obtained in the 40 s test period in A were plotted as a function of ΔK+ and fitted to a one-site Michaelis–Menten curve. For this oocyte we obtained K0.5 = 4.5 ± 0.4 mm, and the maximal influx of water (Bmax) of 50.5 ± 0.4 pl s−1 (R2 = 0.99). Experiments at 25 °C.
Figure 2
Figure 2. Cotransport of water by NKCC1 but not by NKCC2
At t = 0 the external K+ concentration was increased isosmotically by 15 mm (black bar), i.e. the Na+ concentration was reduced concurrently. This caused immediate swelling in a NKCC1-expressing oocyte (first vertical dashed line) but not in a NKCC2-expressing oocyte which only began to swell about 28 s later as indicated by the second dashed line. The NKCC1-expressing oocyte was from a batch with an average Rb+ uptake of 5.7 ± 0.6 pmol s−1 (n = 5). The NKCC2-expressing oocyte is from a batch with a 40% higher uptake, 9.5 ± 0.7 pmol s−1 (n = 4). Experiments at 35 °C.
Figure 5
Figure 5. Effects of bumetanide on steady state volume and K+-induced volume change in an NKCC1-expressing oocyte
Addition of 100 μm bumetanide (+ bum) to the control bathing solution induced an increase in oocyte volume at a rate of about 2 pl s−1. In the presence of bumetanide, an increase in K+ concentration of 22 mm did not induce any volume changes. Experiment at 25°C.
Figure 6
Figure 6. Rb+ fluxes induced by increases in bathing solution Rb+ concentration
The influx of Rb+ (formula image) was obtained from 150 or 300 s exposures to 7.5 mm Rb+ with tracer amounts of 86Rb+ added. A, the intracellular concentration of Rb+ (formula image) was a linear function of time, which supports the assumption that the rate of uptake formula image obtained at 300 s is similar to the initial rate of uptake. B, to measure effluxes, NKCC1-expressing oocytes were preloaded for 3 days in Kulori medium that contained tracer amounts of 86Rb+. The preloaded oocytes were exposed (for 300 s) to a bathing solution in which 7.5 mm of Na+ was replaced by 7.5 mm of non-labelled Rb+. formula image (the efflux of Rb+ and K+) was obtained from the efflux of 86Rb+ and the amount of 86Rb+ remaining in the oocyte (see Methods). Bumetanide (bum) reduced the fluxes (filled bars). Number of batches in parenthesis; bath temperature 35 °C.
Figure 8
Figure 8. Water transport induced by osmotic gradients. Effects of temperature and osmolyte
A, at t = 0, an NKCC1-expressing oocyte was exposed to an extracellular hyperosmolarity of 20 mosmol l−1 by adding urea (20U) to the bathing solution. After about 20 s, bumetanide (100 μm) was added; this caused an abrupt reduction in shrinkage rate. B, the oocyte was initially shrinking in response to 20 mosmol l−1 of urea. After about 16 s the urea was replaced by 10 mm NaCl (equivalent to 20 mosmol l−1), which caused an abrupt reduction in rate of shrinkage. Experiments in A and B were performed at 35°C. C, rates of shrinkage can be expressed as water permeability, Lp; see Methods. Lp increases with temperature; 25, 30 and 35°C were tested. This applies in the absence of bumetanide (open bars) and to the bumetanide-poisoned oocytes (filled bars), as well as to the difference, i.e. the bumetanide sensitive component. The Lp obtained with NaCl as osmoticum (hatched bar) was similar to that obtained in the presence of bumetanide (compare A and B). The Lp of NKCC2 expressing oocytes was tested at 35°C and equalled that of native oocytes; in neither were there any effects of bumetanide. Numbers of oocytes are in parentheses. All control bathing solutions contained 1 mm of K+.
Figure 9
Figure 9. Coupling ratio (n) estimated from the balance between chemical and osmotic driving forces
The driving force of NKCC1 was changed by adding NaCl (ΔNaCl) to the outside solution. This has two effects: the increased chemical potentials of the ions tend to increase the driving force in the inward direction, while the reduction of the chemical potential of water (i.e. increased outside osmolarity) tends to decrease the driving force. The resulting change in driving force can be calculated for different values of the coupling ratio (n); see text. For values of n of about 600, the effect of increased NaCl concentrations is more or less balanced by the concomitant change in external osmolarity. This applies for ΔNaCl in the range 0–50 mm. For low values of the coupling ratio (n = 200), the ionic concentration terms dominate, and the driving force increases in the inward direction for increasing ΔNaCl, i.e. oocyte swelling. At high values of coupling (n = 1000) the increase in external osmolarity (reduced water chemical potential) dominates and the driving force decreases, i.e. there is oocyte shrinkage. The calculation is related to a control situation with bathing solution concentrations for Na+, K+, Cl− of 100 mm, 1 mm, 106 mm, and an osmolarity of 208 mosmol l−1.
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