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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


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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 (black-triangle) or L-alanine (black-down-triangle ) 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-gamma -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.


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Fig. 2.   All vesicles were preloaded with a sucrose solution having an osmolarity of 426 mosM with or without 1 mM ATP-gamma -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.



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Fig. 3.   Vesicles were preloaded with isotonic sucrose solutions with or without 1 mM ATP-gamma -S. 86Rb+ was determined after suspension in an isotonic sucrose solution or in isotonic solutions containing 40 mM D-glucose or L-alanine. ATP-gamma -S did not inhibit 86Rb+ uptake by vesicles suspended in the latter solutions. *Uptake by isotonic vesicles preloaded with ATP-gamma -S was significantly lower than in all other 3 conditions; P < 0.01.



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Fig. 4.   86Rb+ uptake by vesicles preloaded with an isotonic (426 mosM) sucrose solution containing 1 mM ATP-gamma -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.


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Fig. 5.   86Rb+ uptake by vesicles preloaded with an isotonic (426 mosM) sucrose solution containing graded concentrations of ATP-gamma -S as indicated in the figure.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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




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