Rapid stimulation of human renal ENaC by cAMP in Xenopus laevis oocytes

Among the compensatory mechanisms restoring circulating blood volume after severe haemorrhage, increased vasopressin secretion enhances water permeability of distal nephron segments and stimulates Na+ reabsorption in cortical collecting tubules via epithelial sodium channels (ENaC). The ability of vasopressin to upregulate ENaC via a cAMP-dependent mechanism in the medium to long term is well established. This study addressed the acute regulatory effect of cAMP on human ENaC (hENaC) and thus the potential role of vasopressin in the initial compensatory responses to haemorrhagic shock. The effects of raising intracellular cAMP (using 5 mmol/L isobutylmethylxanthine (IBMX) and 50 ìmol/L forskolin) on wild-type and Liddle-mutated hENaC activity expressed in Xenopus oocytes and hENaC localisation in oocyte membranes were evaluated by dual-electrode voltage clamping and immunohistochemistry, respectively. After 30 min, IBMX + forskolin had stimulated amiloride-sensitive Na+ current by 52 % and increased the membrane density of Na+ channels in oocytes expressing wild-type hENaC. These responses were prevented by 5 ìmol/L brefeldin A, which blocks antegrade vesicular transport. By contrast, IBMX + forskolin had no effects in oocytes expressing Liddle-mutated hENaC. cAMP stimulated rapid, exocytotic recruitment of wild-type hENaC into Xenopus oocyte membranes, but had no effect on constitutively over-expressed Liddle-mutated hENaC. Extrapolating these findings to the early cAMP-mediated effect of vasopressin on cortical collecting tubule cells, they suggest that vasopressin rapidly mobilises ENaC to the apical membrane of cortical collecting tubule cells, but does not enhance ENaC activity once inserted into the membrane. We speculate that this stimulatory effect on Na+ reabsorption (and hence water absorption) may contribute to the early restoration of extracellular fluid volume following severe haemorrhage (AU)

Rapid stimulation of human renal ENaC by cAMP in Xenopus laevis oocytes.

Among the compensatory mechanisms restoring circulating blood volume after severe haemorrhage, increased vasopressin secretion enhances water permeability of distal nephron segments and stimulates Na(+) reabsorption in cortical collecting tubules via epithelial sodium channels (ENaC). The ability of vasopressin to upregulate ENaC via a cAMP-dependent mechanism in the medium to long term is well established. This study addressed the acute regulatory effect of cAMP on human ENaC (hENaC) and thus the potential role of vasopressin in the initial compensatory responses to haemorrhagic shock. The effects of raising intracellular cAMP (using 5 mmol/L isobutylmethylxanthine (IBMX) and 50 µmol/L forskolin) on wild-type and Liddle-mutated hENaC activity expressed in Xenopus oocytes and hENaC localisation in oocyte membranes were evaluated by dual-electrode voltage clamping and immunohistochemistry, respectively. After 30 min, IBMX + forskolin had stimulated amiloride-sensitive Na(+) current by 52% and increased the membrane density of Na(+) channels in oocytes expressing wild-type hENaC. These responses were prevented by 5 µmol/L brefeldin A, which blocks antegrade vesicular transport. By contrast, IBMX + forskolin had no effects in oocytes expressing Liddle-mutated hENaC. cAMP stimulated rapid, exocytotic recruitment of wild-type hENaC into Xenopus oocyte membranes, but had no effect on constitutively over-expressed Liddle-mutated hENaC. Extrapolating these findings to the early cAMP-mediated effect of vasopressin on cortical collecting tubule cells, they suggest that vasopressin rapidly mobilises ENaC to the apical membrane of cortical collecting tubule cells, but does not enhance ENaC activity once inserted into the membrane. We speculate that this stimulatory effect on Na(+) reabsorption (and hence water absorption) may contribute to the early restoration of extracellular fluid volume following severe haemorrhage.

Basolateral potassium (IKCa) channel inhibition prevents increased colonic permeability induced by chemical hypoxia.

Major liver resection is associated with impaired intestinal perfusion and intestinal ischemia, resulting in decreased mucosal integrity, increased bacterial translocation, and an increased risk of postoperative sepsis. However, the mechanism by which ischemia impairs intestinal mucosal integrity is unclear. We therefore evaluated the role of Ca(2+)-sensitive, intermediate-conductance (IK(Ca)) basolateral potassium channels in enhanced intestinal permeability secondary to chemical hypoxia. The effects of chemical hypoxia induced by 100 µM dinitrophenol (DNP) and 5 mM deoxyglucose (DG) on basolateral IK(Ca) channel activity and whole cell conductance in intact human colonic crypts, and paracellular permeability (G(S)) in isolated colonic sheets, were determined by patch-clamp recording and transepithelial electrical measurements, respectively. DNP and DG rapidly stimulated IK(Ca) channels in cell-attached basolateral membrane patches and elicited a twofold increase (P = 0.004) in whole cell conductance in amphotericin B-permeabilized membrane patches, changes that were inhibited by the specific IK(Ca) channel blockers TRAM-34 (100 nM) and clotrimazole (CLT; 10 µM). In colonic sheets apically permeabilized with nystatin, DNP elicited a twofold increase (P = 0.005) in G(S), which was largely inhibited by the serosal addition of 50 µM CLT. We conclude that, in intestinal epithelia, chemical hypoxia increases G(S) through a mechanism involving basolateral IK(Ca) channel activation. Basolateral IK(Ca) channel inhibition may prevent or limit increased intestinal permeability during liver surgery.

Apical potassium (BK) channels and enhanced potassium secretion in human colon.

The human colon has the capacity to secrete potassium (K(+)) ions and enhanced K(+) secretion is a feature of a variety of diarrhoeal diseases. Recent work points to K(+) secretion in human colon being mediated by high conductance (BK) K(+) channels located in the apical membrane of colonic epithelial cells. The aim of this review is to highlight the importance of these channels in maintaining K(+) homoeostasis in health and disease.

Regulation of colonic apical potassium (BK) channels by cAMP and somatostatin.

High-conductance apical K+ (BK) channels are present in surface colonocytes of mammalian (including human) colon. Their location makes them well fitted to contribute to the excessive intestinal K(+) losses often associated with infective diarrhea. Since many channel proteins are regulated by phosphorylation, we evaluated the roles of protein kinase A (PKA) and phosphatases in the modulation of apical BK channel activity in surface colonocytes from rat distal colon using patch-clamp techniques, having first increased channel abundance by chronic dietary K+ enrichment. We found that PKA activation using 50 micromol/l forskolin and 5 mmol/l 3-isobutyl-1-methylxanthine stimulated BK channels in cell-attached patches and the catalytic subunit of PKA (200 U/ml) had a similar effect in excised inside-out patches. The antidiarrheal peptide somatostatin (SOM; 2 micromol/l) had a G protein-dependent inhibitory effect on BK channels in cell-attached patches, which was unaffected by pretreatment with 10 micromol/l okadaic acid (an inhibitor of protein phosphatase type 1 and type 2A) but completely prevented by pretreatment with 100 micromol/l Na+ orthovanadate and 10 micromol/l BpV (inhibitors of phosphoprotein tyrosine phosphatase). SOM also inhibited apical BK channels in surface colonocytes in human distal colon. We conclude that cAMP-dependent PKA activates apical BK channels and may enhance colonic K+ losses in some cases of secretory diarrhea. SOM inhibits apical BK channels through a phosphoprotein tyrosine phosphatase-dependent mechanism, which could form the basis of new antidiarrheal strategies.

Altered cryptal expression of luminal potassium (BK) channels in ulcerative colitis.

Decreased sodium (Na(+)), chloride (Cl(-)), and water absorption, and increased potassium (K(+)) secretion, contribute to the pathogenesis of diarrhoea in ulcerative colitis. The cellular abnormalities underlying decreased Na(+) and Cl(-) absorption are becoming clearer, but the mechanism of increased K(+) secretion is unknown. Human colon is normally a K(+) secretory epithelium, making it likely that K(+) channels are expressed in the luminal (apical) membrane. Based on the assumption that these K(+) channels resembled the high conductance luminal K(+) (BK) channels previously identified in rat colon, we used molecular and patch clamp recording techniques to evaluate BK channel expression in normal and inflamed human colon, and the distribution and characteristics of these channels in normal colon. In normal colon, BK channel alpha-subunit protein was immunolocalized to surface cells and upper crypt cells. By contrast, in ulcerative colitis, although BK channel alpha-subunit protein expression was unchanged in surface cells, it extended along the entire crypt irrespective of whether the disease was active or quiescent. BK channel alpha-subunit protein and mRNA expression (evaluated by western blotting and real-time PCR, respectively) were similar in the normal ascending and sigmoid colon. Of the four possible beta-subunits (beta(1-4)), the beta(1)- and beta(3)-subunits were dominant. Voltage-dependent, barium-inhibitable, luminal K(+) channels with a unitary conductance of 214 pS were identified at low abundance in the luminal membrane of surface cells around the openings of sigmoid colonic crypts. We conclude that increased faecal K(+) losses in ulcerative colitis, and possibly other diseases associated with altered colonic K(+) transport, may reflect wider expression of luminal BK channels along the crypt axis.

Enhanced large intestinal potassium permeability in end-stage renal disease.

The capacity of the colon for potassium (K+) secretion increases in end-stage renal disease (ESRD), to the extent that it makes a substantial contribution to K+ homeostasis. This colonic K+ adaptive response may reflect enhanced active K+ secretion, and be associated with an increase in apical membrane K+ permeability. In this study, this hypothesis was tested in patients with normal renal function or ESRD, by evaluating the effect of barium ions (a K+ channel inhibitor) on rectal K+ secretion using a rectal dialysis technique, and the expression of high conductance (BK) K+ channel protein in colonic mucosa by immunohistochemistry. Under basal conditions, rectal K+ secretion was almost threefold greater (p < 0.02) in ESRD patients (n = 8) than in patients with normal renal function (n = 10). Intraluminal barium (5 mmol/l) decreased K+ secretion in the ESRD patients by 45% (p < 0.05), but had no effect on K+ transport in patients with normal renal function. Immunostaining using a specific antibody to the BK channel alpha-subunit revealed greater (p < 0.001) levels of BK channel protein expression in surface colonocytes and crypt cells in ESRD patients (n = 9) than in patients with normal renal function (n = 9), in whom low levels of expression were mainly restricted to surface colonocytes. In conclusion, these results suggest that enhanced colonic K+ secretion in ESRD involves an increase in the apical K+ permeability of the large intestinal epithelium, which most likely reflects increased expression of apical BK channels.

Decreased expression of apical Na+ channels and basolateral Na+, K+-ATPase in ulcerative colitis.

Impaired absorption of sodium (Na+) and water is a major factor in the pathogenesis of diarrhoea in ulcerative colitis (UC). Electrogenic Na+ absorption, present mainly in human distal colon and rectum, is defective in UC, but the molecular basis for this is unclear. The effect of UC on the expression of apical Na+ channels (ENaC) and basolateral Na+, K+-ATPase, the critical determinants of electrogenic Na+ transport, was therefore investigated in this study. Sigmoid colonic and/or proximal rectal mucosal biopsies were obtained from patients with mild to moderate UC, and patients with functional abdominal pain (controls). ENaC subunit expression was studied by immunohistochemistry, western blot analysis, and in situ hybridization, and Na+, K+-ATPase isoform expression was studied by immunohistochemistry, western blotting, and northern blot analysis. UC was associated with substantial decreases in the expression of the ENaC beta- and gamma-subunit proteins and mRNAs, whereas the decrease in ENaC alpha-subunit protein detected by immunolocalization was less marked. The levels of expression of Na+, K+-ATPase alpha1- and beta1-isoform proteins were also lower in UC patients than in controls, although there were no differences in Na+, K+-ATPase alpha1- and beta1-isoform mRNA levels between the two groups. Taken together, these results show that UC results mainly in decreased expression of the apical ENaC beta- and gamma-subunits, as well as the basolateral Na+, K+-ATPase alpha1- and beta1-isoforms. In conclusion, these changes provide a basis for the low/negligible levels of electrogenic Na+ absorption seen in the distal colon and rectum of UC patients, which contribute to the pathogenesis of diarrhoea in this disease.

Molecular and functional studies of electrogenic Na(+) transport in the distal colon and rectum of young and elderly subjects.

BACKGROUND: Human distal nephron and distal colon both exhibit mineralocorticoid sensitive electrogenic Na(+) absorption and make significant contributions to Na(+) homeostasis. Na(+) resorption in the distal nephron diminishes with age but it is unclear whether a similar change occurs in the distal colon. AIMS: To evaluate the effect of age on expression of apical Na(+) channels and basolateral Na(+), K(+)-ATPase, and on the responsiveness of electrogenic Na(+) absorption to mineralocorticoid stimulation in human distal colon and rectum. MATERIALS AND METHODS: Mucosal biopsies were obtained from healthy sigmoid colon and proximal rectum in "young" (aged 20-40 years) and "old" (aged 70 years or over) patients during routine colonoscopy/flexible sigmoidoscopy. Na(+) channel subunits and Na(+), K(+)-ATPase isoforms were studied at the mRNA level by in situ hybridisation and northern blotting, and at the protein level by immunocytochemistry and western blotting. The mineralocorticoid responsiveness of electrogenic Na(+) absorption was evaluated in the two groups by measuring amiloride sensitive electrical potential difference (PD) in the proximal rectum before and 24 hours after oral administration of 1 mg of fludrocortisone. RESULTS: Na(+) channel subunit and Na(+), K(+)-ATPase isoform expression at the level of mRNA and protein was similar in "young" and "old" patients. Both basal and the fludrocortisone stimulated amiloride sensitive rectal PDs were similar in the two groups. CONCLUSIONS: In contrast with the distal nephron, mineralocorticoid sensitive electrogenic Na(+) absorption in the human distal colon does not diminish with age, and may be particularly important in maintaining Na(+) homeostasis in the elderly.

Non-genomic regulation of intermediate conductance potassium channels by aldosterone in human colonic crypt cells.

BACKGROUND: Aldosterone has a rapid, non-genomic, inhibitory effect on macroscopic basolateral K(+) conductance in the human colon, reducing its capacity for Cl(-) secretion. The molecular identity of the K(+) channels constituting this aldosterone inhibitable K(+) conductance is unclear. AIM: To characterise the K(+) channel inhibited by aldosterone present in the basolateral membrane of human colonic crypt cells. METHODS: Crypts were isolated from biopsies of healthy sigmoid colon obtained during colonoscopy. The effect of aldosterone on basolateral K(+) channels, and the possible involvement of Na(+):H(+) exchange, were studied by patch clamp techniques. Total RNA from isolated crypts was subjected to reverse transcriptase-polymerase chain reaction (RT-PCR) using primers specific to intermediate conductance K(+) channels (KCNN4) previously identified in other human tissues. RESULTS: In cell attached patches, 1 nmol/l aldosterone significantly decreased the activity of intermediate conductance (27 pS) K(+) channels by 31%, 53%, and 54% after 1, 5 and 10, minutes, respectively. Increasing aldosterone concentration to 10 nmol/l produced a further 56% decrease in channel activity after five minutes. Aldosterone 1-10 nmol/l had no effect on channel activity in the presence of 20 micro mol/l ethylisopropylamiloride, an inhibitor of Na(+):H(+) exchange. RT-PCR identified KCNN4 mRNA, which is likely to encode the 27 pS K(+) channel inhibited by aldosterone. CONCLUSION: Intermediate conductance K(+) channels (KCNN4) present in the basolateral membranes of human colonic crypt cells are a target for the non-genomic inhibitory effect of aldosterone, which involves stimulation of Na(+):H(+) exchange, thereby reducing the capacity of the colon for Cl(-) secretion.

Segmental variability of ENaC subunit expression in rat colon during dietary sodium depletion.

In rat distal colon, aldosterone has little effect on Na(+) channel (ENaC) alpha-subunit levels but increases the expression of the beta- and gamma-subunits and stimulates electrogenic Na(+) transport. By contrast, the molecular basis of aldosterone's inability to stimulate electrogenic Na(+) transport in the proximal colon is unclear. We therefore compared the effects of hyperaldosteronism secondary to 10 days dietary Na(+) depletion on ENaC subunit expression in rat proximal and distal colon. Northern analyses revealed appreciable and similar levels of alpha-subunit mRNA throughout the colon in control and Na(+)-depleted animals. By contrast, Na(+) depletion substantially enhanced beta-subunit mRNA expression in the distal colon, but had no effect on the low expression levels of beta-subunit mRNA in the proximal colon. Expression of the gamma-subunit, evaluated by PCR, was also restricted to the distal colon of Na(+)-depleted animals. Western analyses demonstrated similar levels of alpha-subunit protein in the proximal and distal colon of both groups of animals, whereas beta-subunit and gamma-subunit proteins were detected solely or predominantly in the distal colon of the Na(+)-depleted animals. Immunocytochemistry confirmed that significant levels of all three subunit proteins only occurred in the apical membrane of surface cells in the distal colon of Na(+)-depleted animals. Our findings are consistent with previous studies demonstrating that aldosterone stimulates electrogenic Na(+) transport in rat distal colon by increasing the expression of beta- and gamma-subunit mRNA and protein, and thus the amount of functional heteromeric ENaC protein in the apical domain. They also show that aldosterone is incapable of stimulating electrogenic Na(+) transport in rat proximal colon (despite the presence of alpha-subunit mRNA and protein) because of its inability to enhance beta- and gamma-subunit expression in this segment.

Diarrhea in ulcerative colitis. The role of altered colonic sodium transport.

In normal human colon, water and sodium (Na+) absorption are directly related. Defective Na+ absorption may therefore be an important factor in the pathogenesis of diarrhea in ulcerative colitis (UC). Electrophysiological studies have revealed profound decreases in channel-mediated apical Na+ entry and Na(+)-K(+)-ATPase-mediated basolateral Na+ extrusion in surface epithelial cells in inflamed human distal colon. Recent molecular biological studies indicate that mucosal inflammation in UC leads to significant decreases in Na+ channel beta- and gamma-subunit expression in the apical membrane of surface colonocytes, with a marked reduction in the levels of beta- and gamma-subunit-specific mRNAs. In addition, basolateral expression of the Na(+)-K(+)-ATPase alpha 1-isoform is reduced along the surface cell-crypt cell axis in UC, although there is no change in the level of the corresponding mRNA. Diarrhea in ulcerative colitis is therefore related, at least in part, to a major defect in electrogenic Na+ absorption, which reflects changes in the levels of expression of critical subunits of both the apical Na+ channel and basolateral Na(+)-K(+)-ATPase.

Somatostatin peptides inhibit basolateral potassium channels in human colonic crypts.

Somatostatin is a powerful inhibitor of intestinal Cl(-) secretion. We used patch-clamp recording techniques to investigate the effects of somatostatin on low-conductance (23-pS) K(+) channels in the basolateral membrane of human colonic crypts, which are an important component of the Cl(-) secretory process. Somatostatin (2 microM) elicited a >80% decrease in "spontaneous" K(+) channel activity in cell-attached patches in nonstimulated crypts (50% inhibition = approximately 8 min), which was voltage-independent and was prevented by pretreating crypts for 18 h with pertussis toxin (200 ng/ml), implicating a G protein-dependent mechanism. In crypts stimulated with 100-200 microM dibutyryl cAMP, 2 microM somatostatin and its synthetic analog octreotide (2 microM) both produced similar degrees of K(+) channel inhibition to that seen in nonstimulated crypts, which was also present under low-Cl(-) (5 mM) conditions. In addition, 2 microM somatostatin abolished the increase in K(+) channel activity stimulated by 2 microM thapsigargin but had no effect on the thapsigargin-stimulated rise in intracellular Ca(2+). These results indicate that somatostatin peptides inhibit 23-pS basolateral K(+) channels in human colonic crypt cells via a G protein-dependent mechanism, which may result in loss of the channel's inherent Ca(2+) sensitivity.

Direct inhibitory effect of nicardipine on basolateral K+ channels in human colonic crypts.

The most abundant basolateral K+ channels in human colonic crypt cells have a low conductance (23 pS), respond to increases in intracellular Ca2+ and cAMP, and have been implicated in intestinal electrogenic Cl- secretion. The effect of nicardipine on the activity of these K+ channels was examined by patch-clamp recording in the cell-attached and excised inside-out configurations from the basolateral membrane of single crypts isolated from biopsied samples of human distal colon. During cell-attached recordings, addition of 2 micromol/l nicardipine to crypts pretreated with 200 micromol/l dibutyryl cAMP decreased single-channel open probability by 87%, but in parallel studies nicardipine had no effect on the intracellular Ca2+ concentration. Using inside-out patches from crypts pretreated with dibutyryl cAMP (bathed in 1.2 mmol/l Ca2+), the addition of increasing concentrations of nicardipine (200 nmol/l, 2 micromol/l and 20 micromol/l) decreased single-channel open probability in a concentration-dependent manner (IC50 0.47 micromol/l). In additional experiments using stripped rat distal colonic mucosa mounted in conventional Ussing chambers, serosal addition of nicardipine at increasing concentrations (ranging from 200 nmol/l to 20 micromol/l) produced a concentration-dependent inhibition of dibutyryl-cAMP-stimulated electrogenic Cl- secretion (IC50 2 micromol/l). Taken together, these results indicate that nicardipine has a direct inhibitory action on 23-pS basolateral K+ channels in human intestinal crypt cells, which is likely to decrease cAMP-stimulated electrogenic Cl- secretion. These basolateral K+ channels may provide a focal point for the development of new strategies in the treatment of secretory diarrhoeal diseases.

Potassium secretion in rat distal colon during dietary potassium loading: role of pH regulated apical potassium channels.

BACKGROUND: Chronic dietary K+ loading increases the abundance of large conductance (210 pS) apical K+ channels in surface cells of rat distal colon, resulting in enhanced K+ secretion in this epithelium. However, the factors involved in the regulation of these K+ channels are at present unclear. AIMS: To evaluate the effect of dietary K+ loading on intracellular pH and its relation to large conductance apical K+ channel activity in surface cells of rat distal colon. METHODS/RESULTS: As assessed by fluorescent imaging, intracellular pH was higher in K+ loaded animals (7.48 (0.09)) than in controls (7.07 (0.04); p<0.01) when surface cells were bathed in NaCl solution, and a similar difference in intracellular pH was observed when cells were bathed in Na2SO4 solution (7.67 (0.09) and 6.92 (0.05) respectively; p<0.001). Ethylisopropylamiloride (EIPA; an inhibitor of Na+-H+ exchange; 1 microM) decreased intracellular pH when surface cells from K+ loaded animals were bathed in either solution, although the decrease was greater when the solution contained NaCl (DeltapH 0.50 (0.03)) rather than Na2SO4 (DeltapH 0. 18 (0.02); p<0.05). In contrast, EIPA had no effect in cells from control animals. As assessed by patch clamp recording techniques, the activity of large conductance K+ channels in excised inside-out membrane patches from distal colonic surface cells of K+ loaded animals increased twofold when the bath pH was raised from 7.40 to 7. 60. As assessed by cell attached patches in distal colonic surface cells from K+ loaded animals, the addition of 1 M EIPA decreased K+ channel activity by 50%, consistent with reversal of Na+-H+ exchange mediated intracellular alkalinisation. CONCLUSION: Intracellular alkalinisation stimulates pH sensitive large conductance apical K+ channels in rat distal colonic surface cells as part of the K+ secretory response to chronic dietary K+ loading. Intracellular alkalinisation seems to reflect an increase in EIPA sensitive Na+-H+ exchange, which may be a manifestation of the secondary hyperaldosteronism associated with this model of colonic K+ adaptation.

Characterization of apical potassium channels induced in rat distal colon during potassium adaptation.

1. Chronic dietary K+ loading stimulates an active K+ secretory process in rat distal colon, which involves an increase in the macroscopic apical K+ conductance of surface epithelial cells. In the present study, the abundance and characteristics of K+ channels constituting this enhanced apical K+ conductance were evaluated using patch clamp recording techniques. 2. In isolated non-polarized surface cells, K+ channels were seen in 9 of 90 (10%) cell-attached patches in cells from control animals, and in 247 of 437 (57%) cell-attached patches in cells from K(+)-loaded animals, with a significant (P < 0.001) shift in distribution density. Similarly, recordings from cell-attached patches of the apical membrane of surface cells surrounding the openings of distal colonic crypts revealed identical K+ channels in 1 of 11 (9%) patches in control animals, and in 9 of 13 (69%) patches in K(+)-loaded animals. 3. In isolated surface cells and surface cells in situ, K+ channels had mean slope conductances of 209 +/- 6 and 233 +/- 14 pS, respectively, when inside-out patches were bathed symmetrically in K2SO4 solution. The channels were sensitive to 'cytosolic' Ca2+ concentration, were voltage sensitive at 'cytosolic' Ca2+ concentrations encountered in colonic epithelial cells, and were inhibited by 1 mM quinidine, 20 mM TEA or 5 mM Ba2+ ions. 4. The data show that dietary K+ loading increases the abundance of Ca(2+)- and voltage-sensitive large-conductance K+ channels in the apical membrane of surface cells in rat distal colon. These channels constitute the enhanced macroscopic apical K+ conductance previously identified in these cells, and are likely to play a critical role in the active K+ secretory process that typifies this model of colonic K+ adaptation.

Characteristics of two basolateral potassium channel populations in human colonic crypts.

The basolateral membrane of human colonic crypt cells contains Ca2+ and cAMP activated, Ba2+ blockable, low conductance (23 pS) K+ channels, which probably play an important part in intestinal Cl- secretion. This study has defined more clearly the basolateral K+ conductive properties of human colonic crypts using patch clamp recording techniques. High conductance (138 pS) K+ channels were seen in 25% of patches (one or two channels per patch), and significantly inhibited by the addition of 5 mM Ba2+, 1 mM quinidine or 20 mM tetraethylammonium chloride (TEA) to the cytosolic side of excised inside-out patches, whereas 1 mM diphenylamine-2-carboxylic acid (DPC) had no effect. In contrast, clusters of the 23 pS K+ channel (two to six channels per patch) were present in > 75% of patches, and channel activity was inhibited by quinidine and DPC, but not by TEA. Activity of the 138 pS K+ channel in inside-out patches was abolished almost completely by removal of bath Ca2+, but in contrast with its effect on the 23 pS K+ channel, addition of 0.1 mM carbachol had no effect on the 138 pS K+ channel in cell attached patches. It is concluded that human colonic crypt cells possess two discrete basolateral K+ channel populations, which can be distinguished by their responses to K+ channel blockers, and their different sensitivities to changes in intracellular Ca2+ concentration.