Turtle Colon Research Articles

Epithelial localization of a reptilian Na+/H+ exchanger homologous to NHE-1.

Basolateral membranes of turtle (Pseudemys scripta) colon epithelial cells exhibit robust Na+/H+ exchange activity that can be activated by cell shrinkage and is blocked by amiloride [M. A. Post and D. C. Dawson. Am. J. Physiol. 262 Cell Physiol. 31):C1089-C1094, 1992]. The colonic epithelium actively absorbs Na+ and secretes K+ and HCO3-, but the role of basolateral Na+/H+ exchange, if any, in transepithelial transport is unknown. The current studies were undertaken to identify the gene product(s) responsible for the observed basolateral Na+/H+ exchange activity and to determine the cellular localization of the reptilian Na+/H+ exchange protein. We cloned and sequenced partial-length cDNAs that are likely to encode a reptilian homologue of the mammalian NHE-1 Na+/H+ exchanger isoform. The partial-length cDNAs were > 80% identical to mammalian NHE-1 homologues at the nucleotide level and recognized a transcript (approximately 5.8-6.0 kb) in RNA isolated from turtle colon, small intestine, stomach, kidney, urinary bladder, heart, and liver. In situ hybridization showed that mRNA encoding the reptile homologue of NHE-1 was expressed predominantly in the epithelial cells of these tissues. Immunofluorescent localization of the reptilian Na+/H+ exchanger protein using an antibody raised against a human NHE-1 fusion protein confirmed that protein expression paralleled abundant mRNA expression in epithelial cells of turtle stomach and colon, as well as in some nephron segments, and showed that the reptile NHE-1 homologue was localized exclusively to the basolateral membranes of these cells. The relatively high level of NHE-1 expression in epithelial cells, particularly those of the colon and stomach, suggests that NHE-1 function is important for the maintenance or regulation of ion transport processes that occur in these cell types.

The mammalian Na+/H+ antiporters NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent, but can couple to an H+ conductance.

Na+/H+ exchange in vertebrates is thought to be electroneutral and insensitive to the membrane voltage. This basic concept has been challenged by recent reports of antiport-associated currents in the turtle colon epithelium (Post and Dawson, 1992, 1994). To determine the electrogenicity of mammalian antiporters, we used the whole-cell patch clamp technique combined with microfluorimetric measurements of intracellular pH (pHi). In murine macrophages, which were found by RT-PCR to express the NHE-1 isoform of the antiporter, reverse (intracellular Na(+)-driven) Na+/H+ exchange caused a cytosolic acidification and activated an outward current, whereas forward (extracellular Na(+)-driven) exchange produced a cytosolic alkalinization and reduced a basal outward current. The currents mirrored the changes in pHi, were strictly dependent on the presence of a Na+ gradient and were reversibly blocked by amiloride. However, the currents were seemingly not carried by the Na+/H+ exchanger itself, but were instead due to a shift in the voltage dependence of a preexisting H+ conductance. This was supported by measurements of the reversal potential (Erev) of tail currents, which identified H+ (equivalents) as the charge carrier. During Na+/H+ exchange, Erev changed along with the measured changes in pHi (by 60-69 mV/pH). Moreover, the current and Na+/H+ exchange could be dissociated. Zn2+, which inhibits the H+ conductance, reversibly blocked the currents without altering Na+/H+ exchange. In Chinese hamster ovary (CHO) cells, which lack the H+ conductance, Na+/H+ exchange produced pHi changes that were not accompanied by transmembrane currents. Similar results were obtained in CHO cells transfected with either the NHE-1, NHE-2, or NHE-3 isoforms of the antiporter, indicating that exchange through these isoforms is electroneutral. In all the isoforms tested, the amplitude and time-course of the antiport-induced pHi changes were independent of the holding voltage. We conclude that mammalian NHE-1, NHE-2, and NHE-3 are electroneutral and voltage independent. In cells endowed with a pH-sensitive H+ conductance, such as macrophages, activation of Na(+)-H+ exchange can modulate a transmembrane H+ current. The currents reported in turtle colon might be due to a similar "cross-talk" between the antiporter and a H+ conductance.

Basolateral Na(+)-H+ antiporter. Mechanisms of electroneutral and conductive ion transport.

The basolateral Na-H antiporter of the turtle colon exhibits both conductive and electroneutral Na+ transport (Post and Dawson. 1992. American Journal of Physiology. 262:C1089-C1094). To explore the mechanism of antiporter-mediated current flow, we compared the conditions necessary to evoke conduction and exchange, and determined the kinetics of activation for both processes. Outward (cell to extracellular fluid) but not inward (extracellular fluid to cell) Na+ or Li+ gradients promoted antiporter-mediated Na+ or Li+ currents, whereas an outwardly directed proton gradient drove inward Na+ or Li+ currents. Proton gradient-driven, "counterflow" current is strong evidence for an exchange stoichiometry of > 1 Na+ or Li+ per proton. Consistent with this notion, outward Na+ and Li+ currents generated by outward Na+ or Li+ gradients displayed sigmoidal activation kinetics. Antiporter-mediated proton currents were never observed, suggesting that only a single proton was transported per turnover of the antiporter. In contrast to Na+ conduction, Na+ exchange was driven by either outwardly or inwardly directed Na+, Li+, or H+ gradients, and the activation of Na+/Na+ exchange was consistent with Michaelis-Menten kinetics (K1/2 = 5 mM). Raising the extracellular fluid Na+ or Li+ concentration, but not extracellular fluid proton concentration, inhibited antiporter-mediated conduction and activated Na+ exchange. These results are consistent with a model for the Na-H antiporter in which the binding of Na+ or Li+ to a high-affinity site gives rise to one-for-one cation exchange, but the binding of Na+ or Li+ ions to other, lower-affinity sites can give rise to a nonunity, cation exchange stoichiometry and, hence, the net translocation of charge. The relative proportion of conductive and nonconductive events is determined by the magnitude and orientation of the substrate gradient and by the serosal concentration of Na+ or Li+.

Tetraethylammonium-sensitive apical K+ channels mediating K+ secretion by turtle colon.

1. Apical membrane K+ channels in turtle colon were identified and characterized using current fluctuation analysis. 2. Under short-circuit conditions in NaCl-Ringer solution, the power density spectrum (PDS) of the short-circuit current (Isc) sometimes exhibited a clearly discernible Lorentzian component, indicating spontaneous fluctuations produced by a population of apical ion channels. The Lorentzian component had a characteristic corner frequency (fc) which averaged 10.2 +/- 0.9 Hz (mean +/- S.E.M., n = 20). 3. The power of the spontaneous fluctuations was enhanced (So increased) by manoeuvres that depolarize the apical membrane electrical potential (Va). Discernible fluctuations were enhanced or induced by raising the serosal K+ concentration ([K+]s = 50-115 mM, Na+ replacement), by clamping the transepithelial potential (Vt) to serosa-positive values, or by blocking basolateral K+ channels with Ba2+. 4. Mucosal amiloride (100 microM) attenuated the spontaneous fluctuations observed in NaCl-Ringer solution but had no effect in the presence of serosal high K+, indicating that amiloride did not block the K(+)-permeable channels but these channels resided in the same cells as the amiloride-sensitive Na+ channels. 5. Raising the mucosal K+ concentration attenuated spontaneous fluctuations. 6. In the presence of serosal high K+ and mucosal amiloride, the spontaneous fluctuations were often accompanied by a reversed Isc consistent with K+ secretion. These conditions were used to test the effects of putative channel blockers. 7. Mucosal Ba2+ and tetraethylammonium (TEA+) were effective inhibitors of the spontaneous fluctuations and the reversed Isc. At a concentration of 10 mM, TEA+ was maximally effective but the TEA+ analogues tetramethylammonium (TMA+) and tetrapropylammonium (TPrA+) were much less effective. Mucosal Rb+ or Cs+ did not inhibit at a concentration of 10 mM. 8. Mucosal lidocaine (200 microM), quinidine (200 microM), or diphenylamine-2-carboxylate (DPC, 1 mM) had little or no effect on the spontaneous fluctuations and reversed Isc. Quinine (100 microM), 4-aminopyridine (1 mM), and apamin (100 nM) were also without effect. 9. Mucosal TEA+ (10 mM) abolished the active secretory K+ flux measured in the presence of serosa-positive transepithelial potentials. 10. These experiments identified a population of TEA(+)-sensitive, apical K+ channels which mediate active K+ secretion in turtle colon. Sensitivity to external TEA+ distinguishes these channels from basolateral K+ channels in turtle colon and demonstrates similarity to apical K+ channels in mammalian colon.

Selective block of specific K(+)‐conducting channels by diphenylamine‐2‐carboxylate in turtle colon epithelial cells.

1. The conduction and gating properties of K(+)-conducting channels were studied in isolated turtle colon cells in an attempt to identify the single channels responsible for specific components of the macroscopic conductance of the basolateral membrane. Three types of Ca(2+)-activated channel were identified, two of which were selective for K+ over Na+ and a third which was selective for monovalent cations over anions, but did not discriminate between K+ and Na+. 2. One of the K(+)-selective channels was a large-conductance 'maxi' K+ channel. A second was characterized by a lower conductance and pronounced inward rectification. 3. The inward-rectifying K+ channel was selectively blocked by diphenylamine-2-carboxylate (DPC). Neither the maxi K+ channel nor a previously identified K(+)-selective channel thought to be activated by cell swelling was affected by this compound. DPC also blocked the non-selective cation channel. 4. An inward-rectifying, DPC-sensitive current was prominent in whole cell-recordings, and DPC blocked basolateral K+ currents in colonic cell layers apically permeabilized with amphotericin-B. In addition, the compound blocked active Na+ absorption. 5. The selective block of a class of epithelial K+ channels by DPC may be a useful tool for determining the contribution of this specific subpopulation to macroscopic conductance and transepithelial salt transport.

Inactivation of a volume-sensitive basolateral potassium conductance in turtle colon: effect of metabolic inhibitors

Previous work has shown that the basolateral membrane of turtle colon epithelium contains a quinidine-sensitive potassium conductance which can be activated by osmotic cell swelling. In this work and in the present study, potassium flow across the basolateral membrane was measured as a short-circuit current across intact pieces of epithelial tissue in which amphotericin B was used to permeabilize the apical membrane. Quinidine-sensitive currents were generated when the mucosal bath contained chloride, a permeant anion. Replacement of chloride by sulfate or addition of sucrose to the bathing solutions abolished 75–90% of the current and caused the quinidine-inhibitable fraction of the current to go from over 90% to around 6% — suggesting that decreases in cell volume had brought about inactivation of the quinidine-sensitive conductance. When metabolic inhibitors were present, inactivation of the conductance by these maneuvers was prevented. Activation of the conductance by replacement of mucosal SO 4 by Cl, however, was not affected.

Cholinergic modulation of apical Na+ channels in turtle colon: analysis of CDPC-induced fluctuations

Current fluctuation analysis was used to investigate the properties of apical Na+ channels during muscarinic inhibition of active Na+ absorption. A reversible Na+ channel blocker, 6-chloro-3,5-diaminopyrazine-2-carboxamide (CDPC), was used to induce fluctuations in the short-circuit current (I(sc)). Power density spectra of the CDPC-induced fluctuations exhibited a clearly discernible Lorentzian component, characterized by a corner frequency that was linearly related to CDPC concentration between 20 and 100 microM. The on (k'on) and off (k(off)) rate coefficients for the CDPC blocking reaction were k'on = 11.1 +/- 0.8 rad.s-1.microM-1 and k(off) = 744 +/- 53 rad/s, and the microscopic inhibition constant was 67 microM (n = 11). CDPC blocking kinetics were not significantly different after inhibition of Isc by 5 microM serosal carbachol. Single-channel Na+ current (iNa) and the density of open and blocked Na+ channels (N(ob)) were estimated from the fluctuations induced by 40 microM CDPC. Under control conditions, iNa was 0.43 +/- 0.05 pA and N(ob) was 251 +/- 42 X 10(6)/cm2 (n = 10). After exposure to serosal carbachol (2-10 microM) for 60 min, Na+ current and N(ob) were reduced by approximately 50%, but iNa was not changed significantly. These results indicate that muscarinic inhibition of electrogenic Na+ absorption was associated with a reduction in the number of open Na+ channels in the apical membrane. They also suggest that this downregulation of transport involved a coordinated decrease in both apical and basolateral membrane conductances.

Digitonin-permeabilized colonic cell layers. Demonstration of calcium-activated basolateral K+ and Cl- conductances.

Sheets of isolated turtle colon were exposed to digitonin on the mucosal side to chemically remove the apical membrane as a permeability barrier. Increases in the mucosal uptake of 86Rb, [3H]mannitol, and 45Ca-EGTA, and the appearance of the cytosolic marker enzyme lactate dehydrogenase in the mucosal bath confirmed the permeabilizing effect of the detergent. Basolateral K+ and Cl- currents were generated by imposing transmural ion gradients, and cytosolic free Ca2+ was manipulated by means of a Ca2+-EGTA buffer system in the mucosal bathing solution. Raising the cytosolic free Ca2+ concentration from the nanomolar to the micromolar range activated basolateral conductances for K+ and Cl-. Differences in ion selectivity, blocker specificity, calcium activation kinetics, and divalent cation activation selectivity indicated that the Ca2+-induced increases in the K+ and Cl- conductances were due to separate populations of channels. The results are consistent with the notion that the apical membranes of turtle colon epithelial cells can be functionally removed under conditions that preserve some of the conductive properties of the basolateral membrane, specifically Ca2+-activated conductive pathways for K+ and Cl-. This permeabilized preparation should offer a means for the identification of macroscopic currents that are due to presumed Ca2+-activated channels, and may also provide a model system for the functional reconstitution of channel regulatory mechanisms.

Osmotically induced basolateral K+ conductance in turtle colon: lidocaine-induced K+ channel noise

The basolateral membrane of amphotericin-treated turtle colon can exhibit two distinct types of K+ conductance, one of which is associated with cell swelling and is blocked by quinidine or lidocaine. Fluctuations in basolateral K+ currents were analyzed under swelling (mucosal KCl) and nonswelling (mucosal K gluconate) conditions. Under nonswelling conditions, it was not possible to detect a spontaneous Lorentzian component in the power density spectrum (PDS) and the addition of lidocaine neither inhibited the macroscopic current nor induced a Lorentzian component in the PDS. Under swelling conditions, however, lidocaine induced a Lorentzian component in the PDS and the corner frequency increased linearly with blocker concentration as expected for reversible blockade of the channel. The gating and conductance properties of osmotically induced channels estimated from a two-state model were similar to those determined recently in single-channel recordings from isolated colonic cells.

Cholinergic regulation of Na absorption by turtle colon: role of basolateral K conductance.

The mechanism underlying the muscarinic inhibition of colonic Na absorption is unknown. In this study the effects of carbachol on active Na transport and basolateral K conductance were compared in the isolated turtle colon. Carbachol produced a biphasic response in both Na transport and basolateral K conductance. The response consisted of a transient activation followed by a sustained inhibition and was blocked by atropine. Submucosal cholinergic neurons were implicated in the regulation of colonic transport by employing depolarizing agents to release endogenous acetylcholine. Depolarizing agents produced a carbachol-like response that was atropine-sensitive. Finally, experiments with the Ca ionophores, A23187 and ionomycin, suggested that the muscarinic response may be mediated, at least in part, by changes in cellular Ca. These experiments provide evidence that cholinergic neurons are present in the turtle colon submucosa, muscarinic agonists cause a change in basolateral K conductance that may be an important event in the regulation of colonic Na absorption, and a Ca second messenger system may be involved in mediating the response.

Resting and osmotically induced basolateral K conductances in turtle colon.

Two types of K conductance can be distinguished in the basolateral membranes of polyene-treated colonic epithelial cells (see Germann, W. J., M. E. Lowy, S. A. Ernst, and D. C. Dawson, 1986, Journal of General Physiology, 88:237-251). The significance of these two types of K conductance was investigated by measuring the properties of the basolateral membrane under conditions that we presumed would lead to marked swelling of the epithelial cells. We compared the basolateral conductance under these conditions of osmotic stress with those observed under other conditions where changes in cell volume would be expected to be less dramatic. In the presence of a permeant salt (KCl) or nonelectrolyte (urea), amphotericin-treated colonic cell layers exhibited a quinidine-sensitive conductance. Light microscopy revealed that these conditions were also associated with pronounced swelling of the epithelial cells. Incubation of tissues in solutions containing the organic anion benzene sulfonate led to the activation of the quinidine-sensitive gK and was also associated with dramatic cell swelling. In contrast, tissues incubated with an impermeant salt (K-gluconate) or nonelectrolyte (sucrose) did not exhibit a quinidine-sensitive basolateral conductance in the presence of the polyene. Although such conditions were also associated with changes in cell volume, they did not lead to the extreme cell swelling detected under conditions that activated the quinidine-sensitive gK. The quinidine-sensitive basolateral conductance that was activated under conditions of osmotic stress was also highly selective for K over Rb, in contrast to the behavior of normal Na transport by the tissue, which was supported equally well by K or Rb and was relatively insensitive to quinidine. The results are consistent with the notion that the basolateral K conductance measured in the amphotericin-treated epithelium bathed by mucosal K-gluconate solutions or in the presence of sucrose was due to the same channels that are responsible for the basolateral K conductance under conditions of normal transport. Conditions of extreme osmotic stress, however, which led to pronounced swelling of the epithelial cells, were associated with the activation of a new conductance, which was highly selective for K over Rb and was blocked by quinidine or lidocaine.

Differentiation of two distinct K conductances in the basolateral membrane of turtle colon.

The K conductance of the basolateral membrane of turtle colon was measured in amphotericin-treated cell layers under a variety of ionic conditions. Changing the composition of the bathing solutions changed not only the magnitude but also the physical properties of the basolateral K conductance. The results are consistent with the notion that altered ionic environments can lead to changes in the relative abundance of two different populations of K channels in the basolateral membrane, which can be differentiated on the basis of pharmacological specificity, ion selectivity, and tracer kinetics. In the following article (Germann, W. J., S. A. Ernst, and D. C. Dawson, 1986, Journal of General Physiology, 88:253-274), we present evidence consistent with the hypothesis that one of these conductances was due to the same channels that give rise to the normal resting basolateral K conductance of the transporting cells, while the other was associated with experimental maneuvers that led to extreme swelling of the epithelial cells.

Single potassium channels blocked by lidocaine and quinidine in isolated turtle colon epithelial cells

The patch-clamp technique for recording single-channel currents across cell membranes was applied to single turtle colon epithelial cells isolated with hyaluronidase. With electrodes fabricated from Corning #7052 glass, high-resistance seals were consistently formed to these cells. In on-cell patches with low K (2.5 mM) in the pipette and high K (114.5 mM) in the bath, outward K currents were recorded that had a slope conductance of 17 pS and a reversal potential greater than -70 mV. Currents through this K channel were blocked by lidocaine, quinidine, and barium. These agents also block a cell swelling-induced K conductance identified by macroscopic current measurements in the basolateral membranes of the intact colonic epithelium, suggesting that the 17 pS K channel identified by single-channel recording in isolated turtle colon cells may be responsible for this macroscopically defined K conductance.

Aldosterone does not stimulate the Na:K pump in isolated turtle colon.

The study of the mechanisms by which mineralocorticoids stimulate sodium absorption across distal epithelia has focused on three possible sites of action: apical sodium permeability, the basolateral Na:K pump, and the production of high-energy substrates. Recently we developed a method for direct measurement of the current generated by the basolateral Na:K pump of the turtle colon [15]. In the presence of mucosal amphotericin-B and serosal barium the short-circuit current across the colon can be equated with the current produced by active electrogenic exchange of sodium for potassium across the basolateral membrane. This pump current is a measure of the transport capacity of the epithelial Na:K pump that is uncomplicated by changes in apical membrane sodium permeability. Pump currents, thus defined, were compared in control tissues and tissues treated with aldosterone in vitro. After 9 h Na absorption was increased 4-fold in the aldosterone-treated tissues but the values of the pump current were identical in the two groups. This result indicates that acute stimulation of sodium absorption by aldosterone does not occur by stimulating the Na:K pump directly.

Passive cation permeability of turtle colon: Evidence for a negative interaction between intracellular sodium and apical sodium permeability

The role of intracellular sodium in the regulation of apical sodium permeability was investigated in an electrically "tight" epithelium, the turtle colon. In the presence of low mucosal sodium (3 mM) and serosal ouabain, an inhibitor of the basolateral sodium pump, the apical membrane retained a substantial amiloride-sensitive, sodium conductance and the basolateral membrane exhibited a barium-sensitive potassium conductance in parallel with a significant sodium (and lithium) conductance. In the presence of a high mucosal sodium concentration (114 mM), however, inhibition of active sodium absorption by ouabain led to a disappearance of the amiloride-sensitive, transepithelial conductance that was due, at least in part, to a virtual abolition of the apical sodium permeability. Two lines of evidence indicate that this permeability decrease was dependent upon an increase in intracellular sodium content. First, raising the mucosal sodium concentration from 3-114 mM in the presence of ouabain reversibly inhibited the amiloride-sensitive conductance. The time course of the decline in conductance paralleled the apparent intracellular accumulation of sodium in exchange for potassium, which was monitored as a transient deflection in the amiloride-sensitive, short-circuit current. Second, the inhibitory effect of mucosal sodium-addition was markedly attenuated by serosal barium, which prevented the accumulation of sodium by blocking the electrically coupled, basolateral potassium exit. These results support the notion of a "negative feedback" effect of intracellular sodium on the apical sodium permeability.

Control of potassium transport by turtle colon: role of membrane potential.

To more clearly define the role of the transepithelial electrical potential difference (V m----s), potassium permeability, and sodium-potassium pump rate in transcellular potassium transport by isolated turtle colon, we measured transmural potassium fluxes under open-circuit conditions in the presence and absence of putative blockers of potassium transport: amiloride and barium. The results were consistent with the notion that V m----s is a major determinant of cellular potassium secretion, whereas active potassium absorption is insensitive to changes in V m----s. These observations suggest that "coupling" between colonic sodium absorption and potassium secretion in vivo could be due primarily to the effect of the lumen negative V m----s on transcellular secretory potassium flow. Amiloride-induced inhibition of potassium secretion appeared to be due to the reductions in V m----s and sodium-potassium pump rate that accompanied the inhibition of active sodium absorption.

Mechanism of epithelial lithium transport. Evidence for basolateral Na:Na and Na:Li exchange.

Measurement of transmural sodium fluxes across isolated, ouabain-inhibited turtle colon in the presence of a serosal-to-mucosal sodium gradient shows that in the absence of active transport the amiloride-sensitive cellular path contains at least two routes for the transmural movement of sodium and lithium, one a conductive path and the other a nonconductive, cation-exchange mechanism. The latter transport element can exchange lithium for sodium, and the countertransport of these two cations provides a mechanistic basis for the ability of tight epithelia to actively absorb lithium despite the low affinity of the basolateral Na/K-ATPase for this cation.

Cation Activation of the Basolateral Sodium-Potassium Pump in Turtle Colon

The current generated by electrogenic sodium-potassium exchange at the basolateral membrane of the turtle colon can be measured directly in tissues that have been treated with serosal barium (to block the basolateral potassium conductance) and mucosal amphotericin B (to reduce the cation selectivity of the apical membrane). We studied the activation of this pump current by mucosal sodium and serosal potassium, rubidium, cesium, and ammonium. The kinetics of sodium activation were consistent with binding to three independent sites on the cytoplasmic side of the pump. The pump was not activated by cellular lithium ions. The kinetics of serosal cation activation were consistent with binding to two independent sites with the selectivity Rb > K > Cs > NH4. The properties and kinetics of the basolateral Na/K pump in the turtle colon are at least qualitatively similar to those ofthe well-characterized Na/K-ATPase of the human red blood cell .

Basolateral potassium channel in turtle colon. Evidence for single-file ion flow.

Treatment of the apical surface of the isolated, ouabain-inhibited turtle colon with the polyene antibiotic amphotericin B permitted the properties of a barium-sensitive potassium conductance in the basolateral membrane to be discerned from the measurements of transepithelial fluxes and electrical currents. Simultaneous measurements of potassium currents and 42K fluxes showed that the movement of potassium was not in accord with simple diffusion. Two other cations, thallium and rubidium, were also permeable and, in addition, exhibited strong interactions with the potassium tracer fluxes. The results indicate that permeant cations exhibit positive coupling, which is consistent with a single-file mechanism of ion translocation through a membrane channel.

Active sodium transport by turtle colon via an electrogenic Na-K exchange pump.

Active sodium absorption by a variety of epithelia is abolished by ouabain, but the obligatory coupling between the movement of sodium and potassium expected from a basolateral (Na+ + K+) ATPase has not been convincingly demonstrated. According to the model of Koefoed-Johnsen and Ussing, the asymmetric cation selectivities of the apical and basolateral membranes prevent basolateral Na-K exchange from being expressed as opposing transmural ion flows. An additional consequence of this asymmetry is that the short-circuit current (Isc) cannot be identified with the current through the sodium-potassium pump. We used the polyene antibiotic, amphotericin-B, to reduce the resistance and the cation selectivity of the apical membrane of isolated turtle colon so that the basolateral membrane current could be dissected into two components: one through a barium-sensitive potassium channel and another which represents the current associated with ouabain-sensitive, electrogenic, Na-K exchange. Comparison of cation fluxes and short circuit current indicates that in these conditions active sodium absorption is entirely attributable to an electrogenic Na-K pump with a stoichiometry of approximately 3Na:2K.