274s
Biochemical SocietyTransactions (1 994) 22
Occlusion of Sodium by the NalGlucose Cotransporter.
ALLAN G. LOWE and BRUCE A. HIRAYAMA'.
Department of Biological Sciences, University of Manchester.
'Department of Physiology, UCLA School of Medicine, Los
Angeles, California, USA.
The detection of intermediates can make an important
contribution t o our understanding how enzymes or catalysed
transport systems operate. The (Na,K)ATPase is a prime
example of this since detection of (a) phosphorylated
intermediates and (b) tightly bound cations has provided a
useful insight into the way this transporter catalyses
transmembrane movements of sodium and potassium ions.
In particular, studies of binding indicate that one (outwardfacing) conformation the (Na,K)ATPase binds rubidium (used
as a congener of potassium) ions in a complex which is
stable enough t o pass carry the bound rubidium through a
cation exchange column (1). This stable complex has been
termed 'occluded rubidium', and addition of ATP has been
shown t o cause dissociation of rubidium (1), indicating that
binding of ATP t o the transporter induces a change in
conformation from the occluded E,(Rb+) complex t o a
loosely bound (inward-facing) E,(Rb+) complex from which
rubidium can rapidly dissociate. Thus evaluation of the
properties of the occluded intermediate has effectively
provided a mechanism for ATP-driven potassium transport by
the (Na,K)ATPase.
Recent studies of sodium-glucose cotransporters
(SGLT1) expressed in Xenopus oocytes (2) imply that
occlusion of sodium ions may occur in this cotransport
system. Two-electrode voltage clamp experiments have
shown that a transient transmembrane current attributable t o
reorientation of the negatively charged transporter between
outward- and inward-facing conformations is detectable after
changing the membrane potential from an inside-negative (eg
-100 mV) t o an inside-positive (eg 50 mV) voltage (3).
This transient current is dependent on having a relatively
high concentration of Na+ ions but no transported sugar
outside the oocyte before the voltage-change is imposed,
and this implies that dissociation of Na* ions from external
binding site(s) occurs before the negatively charged
transporter becomes free to re-orientate across the
membrane. The voltage-jump-induced transmembrane
current is inhibited by the presence of phlorizin, and since
phlorizin binding t o the transporter is Na +-dependent, it
would appear that phlorizin prevents the voltage-induced
dissociation of Na+ from the transporter. This abstract
reports experiments designed t o detect stable binding
(occlusion) of Na+ by the sodium-glucose cotransporter in
the presence of phlorizin.
Small intestinal brush border membranes (BBM) were
prepared from scrapes of rabbit small intestine and purified
by the calcium precipitation method (4). The method of
measuring occlusion of '?Na+ by the BBM vesicles was based
on the resin-column method used by Glynn & Richards (1
for n%b+-occIusion by the (Na,K)ATPase. Each column
consisted of a 1 ml plastic syringe loaded with 0.5 ml of a
suspension of Dowex 50W cation exchange resin (200-400
mesh) and the resin was covered with a ca. 2 m m depth of
acid-washed sand. The syringe was closed by a one-way
tap and connected t o needle fitted through a rubber bung
into a Buchner flask. The resin was pre-equilibrated with the
solution t o be used for suspension of the BMM, and before
use the syringe-space above the resinhand was rinsed twice
with distilled water t o remove any resin left there during
assembly.
22Na+-occIusionexperiments were carried out under
the following conditions with ice-chilled reagents.
+
Inside vesicles: 150 m M KCI/lO m M Hepesnris (pH 7.4).
Outside vesicles: 2 0 m M KCI, 26.7 m M HepeslK (pH 7.4).
2 2 0 m M mannitol, 1 uglml gramicidin, 10pM NaCI, 1 pCilml
22Na+. Gramicidin was present t o allow any 22Na+
transported into the vesicles t o diffuse out. When present
60 m M glucose replaced 6 0 m M mannitol, and phlorizin was
added at 1 mM. 0.5% (vlv) Ethanol was always present.
Procedure:
For each measurement of 22Na+-occIusiona resin-column
tap was fitted into a Buchner-suction set-up, then 0.5 ml
'2Na+-solution was mixed with 0.1 ml BBM-vesicle
suspension (containing about 600 p g protein) and
immediately loaded onto the column. 10 seconds after
mixing the column-tap was opened and the "Na +/vesicle
mixture was forced through the column into an Eppendorf
tube. 0.4 ml Column effluent was assayed by scintillation
counting and 2 0 pI was analyzed for protein.
The following table shows the result of a typical
experiment designed t o detect occlusion of 22Na+by BBM.
Measurements were made in quadruplicate with the same
preparation of BBM.
+
I1
I
Control BBM
(no phlorizin or
D-nlucose)
1 BBM + 1 m M phlorizin I
1 BBM + 6 0 m M D-glucose I
+
BBM
D-glucose
phlorizin
6.03
5.02
6.23
I
I
I
II
o.200
0.081
0.361
+
The increase in binding observed in the presence of phlorizin
was 1.01 pmol 22Na+per mg protein in the absence and
1.1 2 pmol 22Na+per mg protein in the presence of glucose
and in both cases the phlorizin-induced change was greater
than the sum of the standard deviations. These figures
compare with a SGLTl content of approximately 70
pmollmg protein assuming a purity of about 0.5% SGLTl in
the BBM. Additional experiments showed that increasing
Na+ t o 1 m M increased 22Na+occlusion t o a level similar to
the SGLT1-content of the BBM, but further experiments are
needed t o demonstrate saturation kinetics.
The results show that phlorizin does induce BBM t o
bind more 22Na+than is the case without the inhibitor, and
that this binding is of the right order of magnitude t o be
accounted for by SGLTl. More rigorous proof that the
occluded Na' is involved in transport of sodium and glucose
via SGLTl requires additional experiments t o demonstrate
saturation at high sodium concentrations, t o measure the
half time for dissociation of 22Na+with and without glucose,
and t o investigate possible competition between glucose and
phlorizin. Achievement of these ends should be possible
using BBM enriched in SGLTl, and combination of 22Na+occlusion data with results of voltage clamp studies of
SGLTl in Xenopus oocytes should give a valuable insight
into the mechanism of Nalglucose cotransport by SGLTl .
Visits t o UCLA by A.G. Lowe were supported by grants from
the Nuffield Foundation and SERC.
1.
2.
3.
4.
Glynn, I.M. & Richards, D.E. (1983) J. Physiol. 330,
17-43.
Umbach, J.. Coady, M.J. &Wright, E.M. (1990)
Biophys. J. 57, 1218-1 224.
Parent, L., Supplisson, S., Loo, D.F. & Wright, E.M.
(1992) J. Membr. Biol. 125, 49-62.
Stevens, B.R., Ross, H.J. &Wright, E.M. (1982) J.
Membr. Biol. 66, 213-225.
I
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