Potassium channels in basolateral membrane vesicles from
Necturus enterocytes: stretch and ATP sensitivity
William P.
Dubinsky,
Otilia
Mayorga-Wark, and
Stanley G.
Schultz
Department of Integrative Biology and Pharmacology, University of
Texas Medical School, Houston, Texas 77225
 |
ABSTRACT |
We have previously reported that ATP-inhibitable K+
channels, in vesicles derived from the basolateral membrane of
Necturus maculosus small intestinal cells, exhibit volume
regulatory responses that resemble those found in the intact tissue
after exposure to anisotonic solutions. We now report that increases in
K+ channel activity can also be elicited by exposure of
these vesicles to isotonic solutions containing glucose or alanine that
equilibrate across these membranes. We also demonstrate that swelling
after exposure to a hypotonic solution or an isotonic solution
containing alanine or glucose reduces inhibition of channel activity by
ATP and that this finding cannot be simply attributed to dilution of
intravesicular ATP. We conclude that ATP-sensitive, stretch-activated K+ channels may be responsible for the well-established
increase in basolateral membrane K+ conductance of
Necturus small intestinal cells after the addition of sugars
or amino acids to the solution perfusing the mucosal surface, and we
propose that increases in cell volume, resulting in membrane stretch,
decreases the sensitivity of these channels to ATP.
pump-leak parallelism; cross talk; volume regulation; epithelia; adenosine 5'-triphosphate
 |
INTRODUCTION |
EXPOSURE OF EPITHELIAL
CELLS to hypotonic solutions results in cell swelling and
concomitant increases in the K+ and, often, the
Cl
conductances of the basolateral membrane
(14, 20). These conductance changes permit
the efflux of KCl, accompanied by water, which either limits the degree
of swelling or, if swelling is marked, actually serves to restore the
swollen cell toward normal size; the latter is referred to as
"regulatory volume decrease" (14). Cell swelling can
also occur isotonically secondary to the intracellular accumulation of
osmotically active solutes accompanied by their osmotic equivalents of
water (14, 20). This phenomenon has long been
recognized for the case of Na+-coupled sugar and amino acid
transport, where the transported solutes are accumulated
intracellularly, in osmotically active forms, and this is also
accompanied by increases in basolateral membrane K+ and
Cl
conductances. The increase in K+
conductance not only prevents excessive accumulation of intracellular K+ and volume secondary to increased
Na+-K+ pump activity, but also serves to
restore the electrical driving force for the entry of these
Na+-coupled solutes across the apical membrane
(15, 21). The mechanism(s) responsible for
the increases in basolateral membrane K+ channel activity
in response to hypotonically or isotonically induced cell swelling are
not entirely resolved.
We have previously reported that K+ channels in vesicles
derived from the basolateral membranes of Necturus small
intestinal epithelial cells that are essentially devoid of soluble
intracellular contents, but possess cytoskeletal elements, respond to
exposure to anisotonic solutions, as do the intact cells
(8). Thus exposure to a solution that is hypotonic to the
intravesicular solution ("hypotonic shock") increases the activity
of these channels, whereas exposure to a hypertonic solution decreases,
and may abolish, this activity. Further, these "volume regulatory
responses" are dependent upon an intact cytoskeleton inasmuch as
treatment with cytochalasin D or depolymerization of actin without
employing pharmacological agents (8) abolishes these responses.
In the present study, we examine the effects of apparent vesicle
swelling on K+ channel activity under isotonic conditions
secondary to the accumulation of osmotically active solutes. We also
demonstrate that swelling of these vesicles reduces inhibition of
channel activity by ATP. The results suggest that the activity of these
basolateral membrane ATP-inhibitable, stretch-activated K+
channels is modulated by the effect of membrane stretch on ATP sensitivity and that these channels may be responsible for the increase
in K+ conductance associated with sugar and amino acid
absorption by the intact small intestine.
 |
METHODS |
The method for isolating a basolateral membrane fraction from
Necturus enterocytes has been described in detail
(6). Briefly, a membrane fraction enriched in
Na+-K+-ATPase activity was isolated from
mucosal scrapings of Necturus small intestine by
differential centrifugation without the use of enzymes. This method
results in a >20-fold enrichment of
Na+-K+-ATPase activity over that in the crude
homogenate with minimal contamination by enzyme markers for membranes
other than the basolateral membranes. The membranes were frozen, stored
in liquid N2, and thawed immediately before use.
K+ channel activity of the vesicles was assayed using
86Rb+ as a tracer for K+, according
to the method of Garty et al. (10), as described previously (7). This method permits determination of the
time course of equilibration of the tracer with its electrochemical potential difference across the vesicle membrane over the course of
minutes, thereby obviating the need for rapid-sampling techniques. A
decrease in channel activity is reflected by a decrease in the initial
rate of uptake as well as in the steady-state level achieved (10). Vesicles were loaded by addition of 200 µl of
membranes (1.5-4 mg protein/ml) to 50 µl of 0.5 M
K2SO4 and 10 mM K-HEPES, pH 7.0, and other
reagents as indicated. The osmolarity of the loading solution was
adjusted with sucrose as indicated in the text. The mixture was frozen
in liquid N2 and thawed; during the freeze-thaw cycle, the
intravesicular compartment equilibrates with the loading solution, and
the cytoplasmic contents retained during the isolation procedure were
washed out. Columns were prepared from DOWEX 50W-X-8 (Tris form),
poured into glass Pasteur pipettes, and pretreated with three drops of
30% bovine serum albumin. The columns were washed with 4 ml of a
solution of sucrose, 10 mM Tris-HEPES, pH 7.6 adjusted to the
osmolarity of the loading solution. Two hundred microliters of the
vesicle suspension were pipetted onto the DOWEX column to remove
extravesicular K+ and is eluted with 2 ml of sucrose, 10 mM
Tris-HEPES, pH 7.0 buffer under mild vacuum; the sucrose wash is
adjusted to the test osmolarity and contains other reagents as
indicated. Thus the vesicles were eluted into a buffer that is
isotonic, hypertonic, or hypotonic relative to the intravesicular
solution, or isotonic but containing 40 mM glucose or alanine in place
of the osmotic equivalent of sucrose as specified in the text and/or
legends. After the vesicles were collected, a 10-µl aliquot of
86Rb+ (1-4 µCi) was added to initiate
uptake. At timed intervals, starting immediately (~5 s) after the
addition of tracer (nominally "zero time"), 200-µl aliquots were
withdrawn and placed on a second DOWEX column to remove all
extravesicular tracer. The vesicles were eluted from the column with 2 ml of the sucrose buffer directly into scintillation vials and assayed
for 86Rb+ content. Intravesicular
86Rb+ was expressed as the percent of total
radioactivity in a 200-µl aliquot of reaction mixture normalized to
the protein content of the vesicle suspension. As discussed previously
(7), because the intravesicular compartment is markedly
electrically negative with respect to the external compartment, only
channels oriented so that the intravesicular compartment corresponds to
the intracellular compartment will be active.
 |
RESULTS |
Effects of glucose or alanine.
The results of a series of experiments in which K+ channel
activity was determined in vesicles after exposure to an isotonc sucrose solution, a hypotonic sucrose solution, and isotonic solutions in which 40 mM D-glucose or L-alanine replaced
their isotonic equivalents of sucrose are shown in Fig.
1; the average measured osmolarities of these solutions are given in parentheses. The vesicles
that were exposed to isotonic solutions containing
D-glucose or L-alanine displayed increases in
86Rb+ uptake that were indistinguishable from
those that were exposed to the hypotonic sucrose solution.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 1.
86Rb+ uptake by vesicles
preloaded with a sucrose solution having an osmolarity of 426 mosM
after exposure to a solution isotonic with the loading solution
( ), a sucrose solution that is hypotonic to the loading
solution ( ), or isotonic solutions in which 40 mM
D-glucose ( ) or L-alanine
( ) replaced their osmotic equivalent of sucrose.
* Indicates that uptake by the vesicles suspended in the isotonic
sucrose solution was significantly lower than in the other 3 conditions
by P < 0.001.
|
|
Effect of stretch on ATP inhibition.
Van Wagoner (23) has reported that the KATP
channels found in rat atrial myocytes are mechanosensitive inasmuch as
inhibition by ATP can be overcome by cell swelling or suction applied
to an excised membrane patch. As shown in Figs.
2 and 3,
the same appears to be true for the K+ channel activity in
the basolateral membranes from Necturus enterocytes. Figure
2 shows that loading the vesicles with 1 mM ATP-
-S abolished 86Rb+ uptake by vesicles exposed to isotonic or
hypertonic sucrose solutions but had no significant effect on
86Rb+ uptake by vesicles swollen by exposure to
a hypotonic solution. The data shown in Fig. 3 indicate that the same
is true for isotonic swelling that resulted from the presence of 40 mM
alanine or glucose in the suspension. As shown in Fig.
4, exposure of the vesicles to
increasingly hypotonic sucrose solutions resulted in a graded reversal
of the inhibitory effect of ATP on channel activity.

View larger version (32K):
[in this window]
[in a new window]
|
Fig. 2.
All vesicles were preloaded with a sucrose solution
having an osmolarity of 426 mosM with or without 1 mM ATP- -S.
86Rb+ uptake was determined after suspension in
isotonic, hypotonic (365 mosM), or hypertonic (460 mosM) sucrose
solutions as indicated. As indicated in the figure, ATP failed to
inhibit uptake by vesicles exposed to the hypotonic solution.
|
|

View larger version (26K):
[in this window]
[in a new window]
|
Fig. 3.
Vesicles were preloaded with isotonic sucrose solutions
with or without 1 mM ATP- -S. 86Rb+ was
determined after suspension in an isotonic sucrose solution or in
isotonic solutions containing 40 mM D-glucose or
L-alanine. ATP- -S did not inhibit
86Rb+ uptake by vesicles suspended in the
latter solutions. *Uptake by isotonic vesicles preloaded with ATP- -S
was significantly lower than in all other 3 conditions;
P < 0.01.
|
|

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 4.
86Rb+ uptake by vesicles
preloaded with an isotonic (426 mosM) sucrose solution containing 1 mM
ATP- -S after suspension in increasingly hypotonic solutions as
indicated in the figure.
|
|
To examine the possibility that the reversal of inhibition observed
after vesicle swelling is simply due to a dilution of intravesicular
ATP, a series of experiments was carried out to examine the effect of
varied ATP concentrations on K+ channel activity, and the
results of these studies are shown in Fig.
5. Although these data are admittedly
qualitative, comparison of the findings reported in Figs. 2-4 with
those reported in Fig. 5 leads to the conclusion that a decrease in
intravesicular ATP concentration cannot entirely account for the
reversal of inhibition that results from vesicle swelling. Thus the
inhibition by ATP is completely reversed by exposure to a solution that
is only 15-20% hypotonic to the control but is only partially
reversed by a 50% decrease in intravesicular ATP concentration.

View larger version (27K):
[in this window]
[in a new window]
|
Fig. 5.
86Rb+ uptake by vesicles
preloaded with an isotonic (426 mosM) sucrose solution containing
graded concentrations of ATP- -S as indicated in the figure.
|
|
 |
DISCUSSION |
The basolateral membranes of enterocytes possess carrier
mechanisms that "facilitate" the Na+-independent
equilibration of sugars and amino acids across that barrier. In the
present study, we have demonstrated that the suspension of basolateral
membrane vesicles derived from Necturus small intestinal cells, preloaded with a sucrose solution, in isotonic solutions containing 40 mM D-glucose or L-alanine,
results in increases in K+ channel activity (measured as
86Rb+ uptake) that closely resemble those
observed after exposure to hypotonic solutions of the same magnitude.
This is undoubtedly due to swelling under isotonic conditions secondary
to the equilibration of these solutes within the intravesicular space
accompanied by their isotonic equivalent of water. Breton et al.
(4) have demonstrated the same phenomenon after exposure
of the peritubular (basolateral) surface of collapsed, nontransporting
segments of rabbit proximal tubule to glucose and alanine. This
qualitatively resembles the events that accompany the absorption of
these solutes by intact small intestine and renal proximal tubule when
~5 mM glucose or alanine is present in the mucosal solution
(4, 15).
Na+-coupled absorption of sugars or amino acids by small
intestinal and renal proximal tubule epithelial cells is accompanied by: 1) an increase in Na+-K+ pump
activity at the basolateral membrane and hence a decrease in bulk
and/or local ATP activity, and 2) cell swelling. These effects have been best documented for rabbit proximal tubule. Thus Beck
et al. (3) have reported a decrease in cell ATP from 4.44 to 2.69 mM after stimulation of Na+ absorption by the
addition of 5.5 mM glucose and 6 mM alanine to the luminal perfusate
and a 9% increase in cell volume; a similar increase in cell volume
has been reported by Beck et al. (2) in a different study
under similar conditions. Both a decrease in cell ATP and an increase
in cell volume have been, individually, implicated in the increase in
basolateral membrane K+ conductance observed during the
absorptive process (20, 21, 25).
Thus in epithelia where KATP channels have been identified in the basolateral membranes, such as rabbit (22,
25) and Ambystoma (17) proximal
tubule, the local decrease in ATP activity is postulated to be directly
responsible for the increase in K+ conductance. In
addition, in all epithelia studied to date, cell swelling that results
from exposure to hypotonic solutions, and presumably unrelated to
increases in transepithelial transport, is followed by increases in
basolateral membrane K+ conductance; the mechanism(s)
responsible for this phenomenon remain unresolved.
The results of the present study suggest that in Necturus
small intestinal cells, the response to ATP and membrane stretch may be closely entwined. Thus an increase in Na+-coupled
solute entry into the cell across the apical membrane with subsequent
activation of the Na+-K+ basolateral membrane
pump will result in a decrease in local ATP activity. At the same time,
cell swelling, due to an accumulation of osmotically active solutes,
would be expected not only to activate stretch-activated K+
(SAK) channels, but also to dilute cell ATP and decrease its inhibitory effect on these channels. All of these mechanisms would act
synergistically to increase basolateral membrane K+
conductance. These results are consistent with the findings of Lau et
al. (16) that perfusion of Necturus small
intestine, with a galactose solution that is 20% hypertonic with
respect to control, prevents the increase in basolateral membrane
K+ conductance that is seen when the galactose-containing
solution is isotonic to the control perfusate.
Van Wagoner (23) has reported that the sensitivity of
KATP channels in atrial myocytes to ATP is reduced, and can
be abolished, by a stretch of patches of excised membrane resulting
from applied pressure or from hypotonic swelling of whole cells. Kim et
al. (13) have also presented evidence for
mechanosensitivity of atrial KATP channels. The
mechanism(s) underlying this interaction is obscure. It should be noted
that these KATP channels, which Ashcroft and Ashcroft
(1) classify as Type I, differ from those found in
Necturus enterocytes and other epithelia.
Finally, it is of interest that SAK channels have now been identified
in the basolateral membranes of Necturus proximal renal tubule cells (9, 18, 19),
Necturus gallbladder (24), and
Necturus small intestine (8). They have also
been identified by a number of investigators in the basolateral
membranes of frog proximal tubule (5, 11,
12) where evidence has been presented for their
involvement in the increase in the K+ conductance of that
barrier in response to swelling accompanying Na+-coupled
solute absorption (5).
 |
ACKNOWLEDGEMENTS |
We are grateful to Roxanne Ruiz for assistance with some of the
experiments reported in this paper.
 |
FOOTNOTES |
This research was supported by National Institute of Diabetes and
Digestive and Kidney Diseases Grant DK-45251.
Address for reprint requests and other correspondence: S. G. Schultz, Dept. of Integrative Biology and Pharmacology, Univ. of
Texas Medical School, PO Box 20708, Houston, TX 77225 (E-mail: sschultz{at}girch1.med.uth.tmc.edu).
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 4 February 2000; accepted in final form 20 March 2000.
 |
REFERENCES |
1.
Ashcroft, SJH,
and
Ashcroft FM.
Properties and functions of ATP-sensitive K-channels.
Cell Signal
2:
197-214,
1990[ISI][Medline].
2.
Beck, JS,
Breton S,
Laprade R,
and
Giebisch G.
Volume regulation and intracellular calcium in the rabbit proximal convoluted tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
260:
F861-F867,
1991[Abstract/Free Full Text].
3.
Beck, JS,
Breton S,
Mairböurl H,
Laprade R,
and
Giebisch G.
Relationship between sodium transport and intracellular ATP in isolated perfused rabbit proximal convoluted tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
261:
F634-F639,
1991[Abstract/Free Full Text].
4.
Breton, S,
Marsolais M,
Lapointe J-Y,
and
Laprade R.
Cell volume increases of physiological amplitude activate basolateral K and Cl conductances in the rabbit proximal convoluted tubule.
J Am Soc Nephrol
7:
2072-2087,
1996[Abstract].
5.
Cermerikic, S,
and
Sackin H.
Substrate activation of mechanosensitive whole cell currents in renal proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
264:
F697-F714,
1993[Abstract/Free Full Text].
6.
Costantin, J,
Alcalen S,
de Souza Otero A,
Dubinsky WP,
and
Schultz SG.
Reconstitution of an inwardly rectifying potassium channel from the basolateral membranes of Necturus enterocytes into planar lipid bilayers.
Proc Natl Acad Sci USA
86:
5252-5256,
1989[Abstract].
7.
Dubinsky, WP,
Mayorga-Wark O,
and
Schultz SG.
Colocalization of glycolytic enzyme activity and KATP channels in basolateral membrane of Necturus enterocytes.
Am J Physiol Cell Physiol
275:
C1653-C1659,
1998[Abstract/Free Full Text].
8.
Dubinsky, WP,
Mayorga-Wark O,
and
Schultz SG.
Volume regulatory responses of basolateral membrane vesicles from Necturus enterocytes: role of the cytoskeleton.
Proc Natl Acad Sci USA
96:
9421-9426,
1999[Abstract/Free Full Text].
9.
Filipovic, D,
and
Sackin H.
Stretch- and volume-activated channels in isolated proximal tubule cells.
Am J Physiol Renal Fluid Electrolyte Physiol
262:
F857-F870,
1992[Abstract/Free Full Text].
10.
Garty, H,
Rudy B,
and
Karlish SJD
A simple and sensitive procedure for measuring isotope fluxes through ion-specific channels in heterogeneous populations of membrane vesicles.
J Biol Chem
258:
13094-13099,
1983[Abstract/Free Full Text].
11.
Hunter, M.
Stretch-activated channels in the basolateral membrane of single proximal cells of frog kidney.
Pflügers Arch
416:
448-453,
1983.
12.
Kawahara, K.
A stretch-activated channel in the basolateral membrane of Xenopus kidney proximal tubule cells.
Pflügers Arch
415:
624-629,
1990[ISI][Medline].
13.
Kim, SH,
Cho KW,
Chang SH,
Kim SZ,
and
Chae SW.
Glibenclamide suppresses stretch-activated ANP secretion: involvements of K+ATP channels and L-type Ca2+ channel modulation.
Pflügers Arch
434:
362-372,
1997[ISI][Medline].
14.
Lang, F,
Busch GL,
Ritter M,
Völkl H,
Waldegger S,
Gulbins E,
and
Höussinger D.
Functional significance of cell volume regulation.
Physiol Rev
78:
247-306,
1998[Abstract/Free Full Text].
15.
Lapointe, J-Y,
Hudson RL,
and
Schultz SG.
Current-voltage relations of Na-coupled sugar transport across the apical membrane of Necturus small intestine.
J Membr Biol
93:
205-219,
1986[ISI][Medline].
16.
Lau, KR,
Hudson RL,
and
Schultz SG.
Effect of hypertonicity on the increase in basolateral conductance of the Necturus small intestine in response to Na-sugar cotransport.
Biochim Biophys Acta
855:
193-196,
1986[ISI][Medline].
17.
Mauerer, UR,
Boulpaep EL,
and
Segal AS.
Properties of an inwardly rectifying ATP-sensitive K+ channel on the basolateral membrane of renal proximal tubule.
J Gen Physiol
111:
139-160,
1997[Abstract/Free Full Text].
18.
Sackin, H.
Stretch-activated potassium channels in renal proximal tubule.
Am J Physiol Renal Fluid Electrolyte Physiol
253:
F1253-F1262,
1987[Abstract/Free Full Text].
19.
Sackin, H.
A stretch-activated K+ channel sensitive to cell volume.
Proc Natl Acad Sci USA
86:
1731-1735,
1989[Abstract].
20.
Schultz, SG,
Dubinsky WP,
and
Lapointe J-Y.
Cell Volume Regulation, edited by Lang F.. Basel: Karger, 1998, p. 205-259.
21.
Schultz, SG,
and
Hudson RL.
How do sodium absorbing cells do their job and survive?
News Physiol Sci
1:
185-189,
1986[Abstract/Free Full Text].
22.
Tsuchiya, K,
Wang W,
Giebisch G,
and
Welling PA.
ATP is a coupling modulator of parallel Na, K-ATPase-K channel activity in the renal proximal tubule.
Proc Natl Acad Sci USA
89:
6418-6422,
1992[Abstract].
23.
Van Wagoner, DR.
Mechanosensitive gating of atrial ATP-sensitive potassium channels.
Circ Res
72:
973-983,
1993[Abstract].
24.
Vanoye, GV,
and
Reuss L.
Stretch-activated single K+ channels account for whole-cell currents elicited by swelling.
Proc Natl Acad Sci USA
96:
6511-6516,
1999[Abstract/Free Full Text].
25.
Welling, PA.
Cross-talk and the role of KATP channels in the proximal tubule.
Kidney Int
48:
1017-1023,
1995[ISI][Medline].
Am J Physiol Cell Physiol 279(3):C634-C638
0363-6143/00 $5.00
Copyright © 2000 the American Physiological Society