doi: 10.1073/pnas.93.23.13367.
Cotransport of water by the Na+/glucose cotransporter
Affiliations
- PMID: 8917597
- PMCID: PMC24099
- DOI: 10.1073/pnas.93.23.13367
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Cotransport of water by the Na+/glucose cotransporter
Proc Natl Acad Sci U S A.
.
Abstract
Water 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 flow 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+/CI-/gamma-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.
Figures
Volume changes in oocytes expressing SGLT1. The
relative change in volume
V/Vo
(Vo initial volume), was plotted as a
function of time. The oocyte was injected with rabbit SGLT1 cRNA, and
the plasma membrane capacitance (Cm), SGLT1
transient charge movements (Q), and steady-state
sugar-induced currents were recorded simultaneously (11). The oocyte
was equilibrated in a buffer containing 90 mM NaCl, 2 mM KCl, 1 mM
CaCl2, 1 mM MgCl2, 10 mM Hepes·Tris (pH
7.4), and 20 mM mannitol at 22°C. After volume equilibration, the
oocyte was continuously superfused (arrow) with a test solution at
22°C: In A the test solution was identical to the
equilibration buffer and the volume change was less than 0.03% per
minute. In B the 20 mM mannitol was absent producing an
inward osmotic gradient of 20 milliosmoles/liter. In C
the 20 mM mannitol was replaced with 5 mM α-methyl
d -glucopyranoside (αMDG), a saturating concentration of
transported sugar. In D the test solution was identical
to that in C except that 100 μM phlorizin, a specific
competitive inhibitor of SGLT1, was included. In E the
test solution was identical to that in C, but the
experiment was at 30°C. The membrane potential was clamped at −100
mV for C–E. The plasma membrane area was 0.33
cm2, as estimated from the capacitance (325 nF) (11).
Qmax, the maximal charge transfer, was 90
nC, and given a valence of 3.5 (12) this is equivalent to 1.6 ×
1011 SGLT1 transporters in the plasma membrane. The
sugar-induced currents were 1900 nA in C and 5000 nA in
E. The increase in current (transport) was proportional
to the increase in sugar-dependent water flow.
Arrhenius plots of Na+/glucose
cotransport and glucose-dependent water flow. Sugar transport
(•) is given as the current produced by a saturating
concentration of αMDG (5 mM) in oocytes expressing rabbit SGLT1 and
clamped at −100 mV. Water transport (○) is estimated from
the rate of volume change of the oocyte in the presence of αMDG in
the same oocytes clamped at −100 mV. The line was drawn by
linear regression with slope −13 ± 2 and corresponds to an
activation energy Ea
(Ea = −R·slope,
R is the gas constant) of 26 ± 4 kcal/mol. The
experiment was performed on five oocytes with
Qmax = 65 ± 9 nC and
Cm = 430 ± 34 nF. At 22°C, the
sugar-dependent current was 2063 ± 420 nA. Data without error
bars indicate individual experiments, and n was 5
oocytes at 22°C and 4 at 12°C.
Relationship between water transport and
Na+/glucose cotransport. The data were obtained at
different clamp voltages and temperatures: 31°C(⋄), 30°C
(○), 27°C (▵), 22°C (•), and
12°C (□). At 0 nA, the data represented by •
were obtained on control noninjected oocytes (n =
4) and ♦ were obtained in the presence of 5 mM αMDG and
100 μM phlorizin (n = 5). The slope 24 ±
2 × 10−3 pl per sec per nA corresponds to a turnover
of 130 ± 9 mol of water per mol of inward positive charge. There
was also a linear relationship between Jw
and Qmax (slope, 4.9 ± 0.7 ×
10−1 pl per sec per nC) and between
Imax and Qmax
(slope, 23 ± 2/sec).
Dependence of water transport on membrane
voltage. During the experiment, the oocyte expressing SGLT1 was
superfused with a test solution containing 5 mM αMDG with an inward
osmotic gradient of 15 milliosmoles/liter (Fig. 1). Membrane
voltage was initially ≈0 mV, and after 150 sec, it was stepped to
−100 mV. The sugar-coupled current and water transport both increased
≈4-fold, from 280 to 950 nA and 9 to 40 pl/sec. The increase in
water transport was immediate (<10 sec) with the voltage jump and the
increase in current.
Relationship between volume flow and osmotic
gradient in oocytes in the presence of sugar (αMDG) and the presence
of sugar and phlorizin. The experiment was performed on five oocytes by
dilution of mannitol in the bathing medium (50 mM NaCl/100 mM
mannitol/2 mM KCl/1 mM CaCl2/1 mM MgCl2/10 mM
Hepes·Tris at pH 7.4) (at 22°C) to 80, 60, and 0 mM in the presence
of 5 mM αMDG. The lines were obtained by linear regression
with slopes 1.2 ± 0.2 pl per sec per milliosmole per liter in the
presence (▪) and 1.0 ± 0.1 pl per sec per milliosmole
per liter in the absence (•) of phlorizin. These slopes
correspond to an Lp of 1.3 ± 0.1
× 10−4 cm/sec, indistinguishable from that for control
noninjected oocytes. Symbols without error bars indicate that the error
is smaller than the size of the symbol. We did not observed any
sustained cell swelling when sugar was added to a clamped oocyte in the
absence of an external osmotic gradient (stippled line), and this is
probably due to mechanical properties of the oocyte (16).
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