FEMS Microbiology Letters 23 (1984) 293-297
Published by Elsevier
293
FEM 01818
Potassium transport in Escherichia coli: sodium is not a substrate
of the potassium uptake system TrkA
(Bacterial transport; K + uptake; Na + extrusion; H + extrusion; TrkA system)
E v e r t P. Bakker, R o h a n G. K r o l l * a n d I a n R. B o o t h
*
Fachgebiet Mikrobiologie, Fachbereich 5, Universiti~t Osnabruck, Postfach 4469, D -4500 Osnabr~ek, F.R.G., and * Department of
Microbiologp', Marischal College, University of Aberdeen, Aberdeen, AB91AS, U.K.
Received 30 March 1984
Accepted 11 April 1984
1. S U M M A R Y
In Escherichia coli cells depleted of both sodium
and potassium, the potassium uptake system TrkA
mediated a slow, electrogenic uptake of potassium.
Electroneutrality was maintained by the extrusion
of protons. Internal, but not external sodium
stimulated potassium uptake. This extra uptake
was coupled to a stoichiometric extrusion of
sodium. Triethanolamine also stimulated potassium uptake, presumably by increasing the cytoplasmic buffer capacity. These results are taken to
mean that sodium is not a substrate of the TrkA
system, but stimulates TrkA activity by facilitating
the reentry of protons through the sodium-proton
antiporter, and thereby preventing a prohibitive
increase in cytoplasmic pH.
2. I N T R O D U C T I O N
The constitutive K + transport system TrkA of
E. coli is responsible for K + uptake by the cells at
an external K + concentration of 200 /~M and
above. The system is genetically complex: net K +
transport may be affected by four unlinked genetic
loci (trkA, trkD, trkE, and trkG [1]). The mecha-
nism of K + uptake by the system is similarly
complex, in that it requires for activity both a high
transmembrane electrochemical proton gradient
a n d a high cytoplasmic ATP concentration [2].
Our recent work suggests that ATP is involved as a
regulator of TrkA function, and that the protonmotive force is the energy source for K + uptake
[3]. It has previously been determined, that the K +
gradient maintained by the TrkA system can exceed the membrane potential (Aq~) [4,5]. This appears to rule out the possibility that energy coupling occurs by K + uniport driven by A+. SOrensen and Rosen have proposed that TrkA may
function as a K + - N a + symporter [6], and is thus
driven by the sum of the membrane potential and
the electrochemical sodium gradient, which in its
turn is maintained by the N a + / H + antiporter of
the cells [7,8]. We have tested this model and
found that Na + is not a substrate of the TrkA
system. It is, therefore, concluded that the system
cannot function as a K + - N a + symporter.
3. M A T E R I A L S A N D M E T H O D S
3.1. Organism
E. coli TK1001 [9] ( F - , trkD, kdpABC5, lacZ,
0378-1097/84/$03.00 © 1984 Federation of European Microbiological Societies
294
rha, thi, gal) was used t h r o u g h o u t a n d was the
generous gift of Dr. W. Epstein ( U n i v e r s i t y of
Chicago, USA).
3.2. Preparation of cells
T h e cells were grown on m e d i u m K5 [10] suppl e m e n t e d with 1 / ~ g / m l thiamine a n d 10 m M
glucose. The cells were harvested in late exponential phase, treated with E D T A as previously described [11], washed twice with, a n d r e s u s p e n d e d
in the buffers of p H 6.8 described in the figure
legends.
i
i
u
4. R E S U L T S A N D D I S C U S S I O N
4.1. K +uptake induces H + extrusion
T h e d a t a from [6] indicate that K + - d e p l e t e d
cells s u s p e n d e d in a choline-chloride m e d i u m in
the absence of N a + ions take up K + relatively
slowly. Fig. 1A shows that u n d e r these c o n d i t i o n s
the a d d i t i o n of K + s t i m u l a t e d the rate with which
p r o t o n s a p p e a r e d in the m e d i u m . The extent of
this s t i m u l a t i o n of H + p r o d u c t i o n had an a p p r o x .
i
!
!
i
!
/
C
"_
. p~
,
"
I
H+ ...~jb
0
I
c
3.3. Cation transport
K + u p t a k e e x p e r i m e n t s were carried out at
20 o C with a cell suspension of 1 mg d r y w e i g h t / m l
that was shaken at 220 r e v . / m i n in a g y r o t o r y
shaker bath. G l u c o s e (10 m M ) was present as an
energy source. The K + and N a + c o n t e n t of the
cells was e s t i m a t e d b y c e n t r i f u g a t i o n - b a s e d assays,
as previously d e s c r i b e d [11]. In some e x p e r i m e n t s
the H + a n d K + c o n c e n t r a t i o n s in the suspension
were m o n i t o r e d s i m u l t a n e o u s l y with the aid of a
R a d i o m e t e r G K 2 3 2 1 C c o m b i n e d p H electrode
c o n n e c t e d to a K n i c k 644-1 p H meter, a n d with an
I n g o l d c o m b i n e d cation-sensitive glass electrode
c o n n e c t e d to a R a d i o m e t e r P H M 64 m V / p H meter. The two e l e c t r o d e signals were registered on a
t w o - c h a n n e l strip-chart r e c o r d e r ( L i n e a r Instruments, A b i m e d , Di~sseldorf, F.R.G.). T h e p H scale
was c a l i b r a t e d b y a d d i n g a k n o w n volume of
freshly p r e p a r e d 0.01 M HC1 to the suspension
(Fig. 1). T h e signal of the K + electrode (in mV)
was converted to the K + c o n c e n t r a t i o n in the
m e d i u m b y m e a n s of a c a l i b r a t i o n curve relating
log[K+]out with the electrode signal in mV.
i
A: Electrode signals
H÷
(500'nmol)
-..1
-
1/
I
"-
'
I
70
._~
/
~"
oop./
o -Olocoe
I
\\
I
I
I
I
I
5
6
B: Extents
60
H
pro* d ~
U m
-~50
~-Io~ 0
O•,--"~,
"630 ,-U--K+uptake
E
2cE
~-
20
-
/
~, I0>
I
-2
-1
0
I
I
I
I
2 3
Time {rain)
I
4
l
I
Fig. l. Stoichiometric relationship between K + uptake and H +
extrusion. EDTA-treated cells were incubated at 2.25 mg dry
weight/ml of a medium consisting of 5 mM bistrispropane
chloride, 150 mM choline chloride and 25 ~M KC1. Additions:
10 mM glucose at t = - 1.8 min; 800 btM KC1 at zero time. (A)
Electrode signals in mV; (B) extent of K ÷ uptake (O) or H ÷
production (O).
1 : 1 s t o i c h i o m e t r y with the extent of K + u p t a k e
(Fig. 1B). E x p e r i m e n t s with the fluorescent dye
3 , 3 ' - d i h e x y l o x a c a r b o c y a n i n e (diO-C6-(3)) ( n o t
shown) i n d i c a t e d that u n d e r these c o n d i t i o n s K +
u p t a k e i n d u c e d a r a p i d a n d extensive d e p o l a r i z a tion (see also [11,12]). Together, these results indi-
295
cate that the proton pumps of the cells respond to
the electrogenic uptake of K + with the electrogenic ejection of H + into the medium, and thereby
maintain electroneutrality.
4.2. The effect o f external and internal N a + on K +
uptake
Fig. 2 shows that the simultaneous addition of
sodium phosphate (50 m M with respect to N a +)
and KC1 did not stimulate the rate of K + uptake
by K +- and Na+-depleted cells. By contrast, cells
preincubated with Na + did exhibit faster rates of
K + uptake. The extent of this stimulation correlated well with that of the extrusion of previously
acquired N a + from the cells (Figs. 2 and 3). Thus,
stimulation of K + uptake by Na + appears to be
transeffective, and occurs despite the Na + gradient being opposed to K + uptake by a putative
N a + symporter. We have previously shown that
200,
w
|
l
l
w
!
w
/
60
50
~ /..0
O
30
<1 20
10
0
0
10
20
30
/.,0
50
Z~ Na+ (nmollrog}
613
Fig. 3. Stoichiometric relationship between the extents of the
s t i m u l a t i o n of K ÷ u p t a k e a n d the extrusion of N a + ions.
E x p e r i m e n t a l c o n d i t i o n s were as in Fig. 2, except that s o d i u m
p h o s p h a t e was a d d e d at t = - 4 0 min (C)); t = - 2 5 min (z~);
t=-10
rain ( v , n ) or t = - 5
min (O). The difference at a
certain time in the a m o u n t of K + t a k e n up b y the cells
p r e i n c u b a t e d with N a ÷ and that of cells to which N a ÷ was
a d d e d at zero time is given as a function of the a m o u n t of N a +
e x t r u d e d between zero time a n d t = t. The d r a w n line represents the best fit of the d a t a points a c c o r d i n g to the linear
regression m e t h o d and has the e q u a t i o n of z~K + = 0.96 A N a +
- 0.76 (in n m o l / m g dry wt of cells).
I
(
~100
m
i
c
under such conditions an Na + symport is severely
inhibited [8].
._s
u
50
4.3. The role o f N a + in K + uptake
The trans effect of Na + on K + uptake can be
0
A
-5
--
0
"l'ime(mini
5
!
10
Fig. 2. S o d i u m s t i m u l a t e s K + u p t a k e from within. E D T A treated cells were i n c u b a t e d at 1.0 m g dry w t / m l of the
m e d i u m c o n s i s t i n g of 20 m M b i s t r i s p r o p a n e chloride a n d 150
m M choline chloride. A d d i t i o n s : at t = - 1 0
min, 10 m M
glucose; at zero time, 2 m M KCI. A, zx, C o n t r o l cells; o, O ,
ceils to which N a p h o s p h a t e (50 m M w i t h respect to N a + ) was
a d d e d at zero time; II, D, cells p r e i n c u b a t e d for 40 rain w i t h the
s a m e c o n c e n t r a t i o n of N a p h o s p h a t e . O p e n symbols, K + content; closed symbols, N a + content of the cells.
accounted for by the need to maintain overall
electroneutrality during K ÷ uptake. In Na+-free
cells K ÷ exchange against H ÷ is limited by the
requirement to maintain cytoplasmic p H constant.
Therefore, we tested the effect of increasing the
cytoplasmic buffering capacity during K ÷ uptake.
To this end, K+-depleted ceils were either preloaded with triethanolamine or with Na ÷. The
former compound is a weak base ( p K a 6.8), equilibrates across the membrane in its undissociated
form, and acts as a cytoplasmic buffer (unpublished observations). Addition of K ÷ to such cells
296
led to a rate of uptake of 160, 100 a n d 45 n m o l
K + . m i n 1. m g - i dry wt of cells in Na +-, trie t h a n o l a m i n e - a n d choline-loaded cells, respectively (Fig. 4). Thus, t r i e t h a n o l a m i n e can substitute for N a ÷ in stimulating K ÷ transport.
Rhoads a n d Epstein [13] have suggested that
N a + ions directly activate the T r k A system from
the inside. This effect is, however, very small or
non-existing, since even at high intracellular
s o d i u m c o n c e n t r a t i o n s the major part of the enhanced rate of K + uptake was due to simultaneously occurring N a ÷ extrusion (Fig. 4 a n d
[14,151).
a/'
I
I
i
i
500
We conclude that the K + uptake via T r k A is
not coupled directly to the electrochemical gradient of N a +. Rather, N a + stimulates indirectly by
allowing the reentry of protons, which are initially
ejected to electrically balance the K + ions taken
up (Fig. 1). In earlier reports of N a + stimulation
of T r k A the intracellular N a ÷ c o n c e n t r a t i o n was
not m o n i t o r e d [6]. Since in these experiments the
cells were p r e i n c u b a t e d with N a + [6], it is likely
that the effects observed were due to intracellular,
rather than extracellular N a + . Similarly, it is likely
that the molecular basis of the stimulatory effects
of N a + on K + uptake in Streptococcusfaecalis [16]
a n d in Mycoplasma mycoides var. capri [17,18] are
the same as shown here for the E, coli T r k A
system.
ACKNOWLEDGEMENTS
4OO
We thank W e r n e r Mangerich for technical
assistance a n d Drs. K. A l t e n d o r f a n d W. Epstein
for helpful discussion. This work was supported by
a grant from the Deutsche Forschungsgemeinschaft (Ba 7 1 3 / 2 ) to E.P. Bakker and by travelling
grants from the S.E.R.C. a n d Royal Society to Ian
R. Booth.
E
o
E
.-£c300
,
5. C O N C L U S I O N
.~-200
REFERENCES
100
:f
,AL~
~
Ab~aL
3
4
Time (rain)
5
6
Fig. 4. K + uptake and Na + extrusion of cells incubated in
various media. EDTA-treated cells were washed with and incubated in either one of the following buffers: 20 mM bistrispropane chloride plus 150 mM choline chloride (O, O); 200
mM Na N-(2-acetamido)-2-aminoethanesulphonicacid (Aces)
(ll, rn); 200 mM triethanolamine Aces (A, zx); 150 mM triethanolamine chloride ( . , ~). Additions: 10 mM glucose at
t = -10 rain, and 2 mM KCI at zero time. Open symbols, K +
content; closed symbols, Na + content.
[1] Epstein, W. and Laimins, L. (1980) Trends Biochem. Sci.
5, 21-23.
[2] Rhoads, D.B. and Epstein, W. (1977) J. Biol. Chem, 252,
1394-1401.
[3] Stewart, L.M.D., Bakker, E.P. and Booth, I.R. (1984)
Biochem. Soc. Trans., in press.
[4] Bakker, E.P. (1982) Biochim. Biophys. Acta 681,474-483.
[5] Bakker, E.P. (1983) FEMS Microbiol. Lett. 16, 229-233.
[6] SSrensen, E.M. and Rosen, B.P. (1980) Biochemistry 19,
1458-1462.
[7] Schuldiner, S. and Fishkes, H. (1978) Biochemistry 17,
706-711.
[8] Stewart, L.M.D. and Booth, I.R. (1983) FEMS Microbiol.
Lett. 19, 161-164.
[9] Rhoads, D.B., Waters, F.B. and Epstein, W. (1976) J. Gen.
Physiol. 67, 325-341.
[10] Epstein, W. and Kim, B.S. (1971) J. Bacteriol. 108,
639-644.
297
[11] Bakker, E.P. and Mangerich, W.E. (1981) J. Bacteriol. 147,
820-826.
[12] Kroll, R.G. and Booth, I.R. (1981) Biochem. J. 198,
691-698.
[13] Rhoads, D.B. and Epstein, W. (1978) J. Gen. Physiol. 78,
283-295.
[14] Schulz, S.G., Epstein, W. and Solomon, A.K. (1963) J.
Gen. Physiol. 47, 329-346.
[15] Bakker, E.P. and Mangerich, W.E. (1983) Biochim. Biophys. Acta 730, 379-386.
[16] Harold, F.M. and Papineau, D. (1972) J. Membrane Biol.
8, 45-62.
[17] Benyoucef, M., Regaud, J.L. and Leblanc, G. (1982) Biochem. J. 208, 529-538.
[18] Benyoucef, M., Regaud, J.L. and Leblanc, G. (1982) Biochem. J. 208, 539-547.