Active K+ secretion through multiple KCa-type channels and regulation by IKCa channels in rat proximal colon

William J. Joiner,1,2,* Srisaila Basavappa,2,* Sadasivan Vidyasagar,3,* Keith Nehrke,4 Selvi Krishnan,3 Henry J. Binder,2,3 Emile L. Boulpaep,2 and Vazhaikkurichi M. Rajendran3

Departments of 1Pharmacology, 2Cellular and Molecular Physiology, and 3Internal Medicine, Yale University, New Haven, Connecticut 06520; 4Center for Oral Biology, Aab Institute for Biomedical Research, University of Rochester Medical Center, Rochester, New York 14642

Submitted 13 August 2002 ; accepted in final form 15 February 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Colonic K+ secretion stimulated by cholinergic agents requires activation of muscarinic receptors and the release of intracellular Ca2+. However, the precise mechanisms by which this rise in Ca2+ leads to K+ efflux across the apical membrane are poorly understood. In the present study, Northern blot analysis of rat proximal colon revealed the presence of transcripts encoding rSK2 [small conductance (SK)], rSK4 [intermediate conductance (IK)], and rSlo [large conductance (BK)] Ca2+-activated K+ channels. In dietary K+-depleted animals, only rSK4 mRNA was reduced in the colon. On the basis of this observation, a cDNA encoding the K+ channel rSK4 was cloned from a rat colonic cDNA library. Transfection of this cDNA into Chinese hamster ovary (CHO) cells led to the expression of Ca2+-activated K+ channels that were blocked by the IK channel inhibitor clotrimazole (CLT). Confocal immunofluorescence confirmed the presence of IK channels in proximal colonic crypts, and Western blotting demonstrated that IK protein sorted to both the apical and basolateral surfaces of colonic epithelia. In addition, transcellular active K+ secretion was studied on epithelial strips of rat proximal colon using unidirectional 86Rb+ fluxes. The addition of thapsigargin or carbachol to the serosal surface enhanced net 86Rb+ secretion. The mucosal addition of CLT completely inhibited carbachol-induced net 86Rb+ secretion. In contrast, only partial inhibition was observed with the BK and SK channel inhibitors, iberiotoxin and apamin, respectively. Finally, in parallel with the reduction in SK4 message observed in animals deprived of dietary K+, carbachol-induced 86Rb+ secretion was abolished in dietary K+-depleted animals. These results suggest that the rSK4 channel mediates K+ secretion induced by muscarinic agonists in the rat proximal colon and that transcription of the rSK4 channel is downregulated to prevent K+ loss during dietary K+ depletion.

intermediate conductance Ca2+-activated K+ channel; intermediate-conductance channel; carbachol; clotrimazole; 86RB+ flux; K+ depletion


THE MAMMALIAN LARGE INTESTINE participates in overall potassium (K+) homeostasis. After the kidney, the colon is the primary route of K+ excretion. At least three different K+ transport processes have been identified in the colon including active K+ absorption, active K+ secretion, and passive paracellular K+ secretion (11, 24). In the basal state, active K+ absorption is present in the distal colon, whereas active K+ secretion has been identified in the proximal colon. Active K+ absorption requires an uptake mechanism at the apical membrane, e.g., a H-K-ATPase, and an exit mechanism at the opposite membrane, e.g., a K+ channel or a K-Cl cotransporter (KCC). Active K+ secretion requires an uptake mechanism at the basolateral membrane, e.g., an Na-K-ATPase or an Na-K-2Cl cotransporter, and an exit step at the apical membrane, e.g., a K+ channel.

Aldosterone or dietary Na+ depletion stimulates active K+ secretion, possibly by activation or insertion of apical membrane K+ channels, whereas blocking these channels inhibits active K+ secretion (39). Dietary K+ depletion either inhibits active K+ secretion or stimulates active K+ absorption. For example, dietary K+ depletion is associated with an increase both in the abundance of an apical membrane {beta}-subunit of H-K-ATPase and KCC mRNA abundance and protein expression (32, 33). However, dietary K+ depletion does not alter colonic H-K-ATPase activity, message or protein levels of its {alpha}-subunit (32, 34). In the absence of an effect on absorption, it seems reasonable that K+ depletion would downregulate active K+ secretion. In this case, K+ conservation during dietary K+ depletion could be affected by downregulating K+ channels. To test this hypothesis and to study the effect of dietary K+ depletion on active K+ secretion independent of K+ absorptive processes, the present studies were performed in the proximal colon in which active K+ absorptive processes are absent.

In the colon, Ca2+-activated K+ (KCa) channels in particular have been postulated to play a role in K+ transport (35, 36). Large-conductance (BKCa) K+ channels have been identified in the apical (4) and basolateral (3, 22) membranes of colonic surface and crypt cells, respectively, whereas small (SKCa) and intermediate (IKCa) conductance K+ channels have been found in the basolateral membrane of crypt cells from the mammalian colon (2, 3, 7, 22, 27). Apical KCa channels may facilitate K+ secretion (4, 15, 23, 36). In contrast, basolateral KCa channels are thought to facilitate K+ absorption (35) through recycling of K+ that enters the cell via the Na-K-ATPase or the Na-K-2Cl cotransporter. The efflux of K+ through basolateral KCa channels may also hyperpolarize the basolateral and apical membranes, thereby enhancing the driving force for Ca2+-dependent apical Cl secretion (6, 7, 12, 13, 30, 36).

Colonic K+ secretion by transcellular pathways is regulated by several different means. Agonists that increase intracellular levels of cAMP have been reported to induce K+ secretion (10, 14, 21). In contrast, activation of muscarinic receptors elevates intracellular Ca2+ (8, 9), a messenger that induces K+ secretion (25), presumably by activating apical KCa channels. However, the identity of these apical K+ channels is unknown. In the present study, we used carbachol to stimulate proximal K+ secretion. Our results demonstrate that 1) BKCa, SKCa, and IKCa channel transcripts are present in rat proximal colon, but only IKCa mRNA is downregulated in dietary K+-depleted animals; 2) a cDNA encoding the IKCa channel rSK4 was cloned from rat colon, and its expression in Chinese hamster ovary (CHO) cells reveals IKCa channel activity that is blocked by IKCa channel inhibitor clotrimazole (CLT); 3) K+ secretion mediated by cholinergic agonists in the rat proximal colon is completely blocked by CLT and partially blocked by the BKCa and SKCa channel inhibitors iberiotoxin (IbTX) and apamin (APA), respectively; and 4) that dietary K+ depletion abolishes carbachol-induced K+ secretion. These results suggest that cholinergic K+ secretion in the rat colon is mediated by IKCa > BKCa≥ SKCa channels and that K+ depletion causes mRNA encoding the IK channel rSK4 to be downregulated, presumably to prevent loss of K+.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals. Nonfasting male Sprague-Dawley rats (200–250 g) were divided into two groups: 1) normal animals were fed Purina rat chow containing 200 µeq Na+ + 250 µeq K+/g food ad libitum; 2) K+-depleted animals were fed chow containing 200 µeq Na+ + 0.014 µeq K+/g food (ICN Pharmaceuticals, Costa Mesa, CA) ad libitum for 21 days, as previously described (12). All animals were given tap water ad libitum.

RNA isolation and Northern blot analysis. Total RNA was isolated by the CsCl cushioning method of Sambrook et al. (31), as described previously (34). Total rat RNA was purified from isolated proximal colonic epithelial cells. Proximal colonic segments from two rats were used for each RNA preparation. mRNA was purified from total RNA using a Qiagen kit (Chatsworth, CA). Five micrograms of mRNA were then electrophoresed on an agarose gel and transferred to a nylon membrane (New England Nuclear, Boston, MA) for use in Northern blots. Hybridizations were performed as described previously (33, 34). In brief, BstXI was used to excise full-length rSK4 from pCDNA1 (see below for cloning). Similarly, SacI was used to isolate an ~0.9-kb fragment from hSlo, and EvoRV and XhoI were used to isolate an ~1.0-kb fragment from rSK2. All three fragments were similarly labeled by random priming using [32P]dCTP and allowed to hybridize to membranes for 18 h at 42°C. The blots were washed with 2x saline-sodium citrate and 0.1% SDS at 45°C for 30 min. Subsequently, the blots were exposed to hyperfilm at –80°C and developed after 18 h. Individual blots were stripped and hybridized with channel cDNA probes for KCa channels and GAPDH. The abundance of hybridizing mRNA was quantified with a personal densitometer SI using ImageQuaNT software (Molecular Dynamics, Sunnyvale, CA) and normalized to the level of GAPDH mRNA on the same blot.

rSK4 cDNA isolation and characterization. A rat colonic cDNA library was screened for the rat SK4 cDNA using the human SK4 cDNA [hSK4 (18)] as probe as per the manufacturer's suggestion (Invitrogen, San Diego, CA). The hybridizing plasmid containing the largest insert (~1.9 kb) was sequenced completely in both directions using an automated fluorescence sequencer (W. M. Keck Biotechnology Resource Lab, Yale University). The resulting sequence (accession #AF156554) encodes an open reading frame of 424 amino acids and is 98% identical to the recently cloned rat rSK4 (42) and the rat SMIK (26). In particular, the amino acid sequence encoded by our cDNA differs from that of rSK4 and SMIK at positions 280, 395, and 399 and lacks a glutamine at position 416. Furthermore, SMIK and the amino acid sequence encoded by our cDNA differ from rSK4 in containing a leucine at position 213 in place of phenylalanine. These minor differences may reflect sequencing errors. Because our clone so closely resembles rSK4 and SMIK, we will refer to it hereafter as rSK4 out of deference to the nomenclature established for the first rat IKCa channel that was cloned.

The full-length cDNA was subsequently subcloned into the BamHI site of pCDNA1; this plasmid was then used for transfection studies. Briefly, CHO cells were grown to ~10–20% confluence in 25-cm2 vented flasks. Growth media for these cells consisted of Iscove's DMEM (GIBCO) supplemented with 10% FBS, hypoxanthine/thymidine (GIBCO), and antibiotic/antimycotic (GIBCO). CHO cells were transfected with 5 µl Lipofectamine (GIBCO) premixed with 0.5 µg rIK1/pCDNA1 + 0.5 µg pGreen Lantern (Invitrogen) in 1 ml Optimem (GIBCO) for 5 h, followed by a 12-h incubation in the same media supplemented with 10% FBS. Subsequently, the transfection media was replaced with normal growth media, and recordings were made from green fluorescent cells within the next 40 h.

Electrophysiology. All recordings were performed at 22°C using an Axopatch 1D amplifier (Axon Instruments, Foster City, CA). Electrodes were fabricated from WPI glass (Sarasota, FL) using a Narishige two-stage puller. Electrodes had a resistance of 3–5 M{Omega} for whole cell recordings and 10–50 M{Omega} for inside-out patch recordings when filled with recording solution.

For whole cell recordings, the bath solution consisted of (in mM): 140 NaCl, 1.0 CaCl2, 5 KCl, 29 glucose, and 25 HEPES (pH 7.4). Changes were made to the bath composition during ion-substitution experiments, where K+ was substituted for Na+. In pharmacological experiments, the bath solution was either supplemented with various drugs [charybdotoxin (CTX), clotrimazole (CLT), tetraethylammonium (TEA), IbTX, and APA] at concentrations not exceeding 1 mM or, in the case of TEA, 20 mM TEA was substituted for Na+. In whole cell experiments, pipettes were filled with intracellular solution containing (in mM): 30 KCl, 100 K gluconate, 5 EGTA, 4.27 CaCl2, and 10 HEPES (pH 7.2). The free Ca2+ concentration of the intracellular solution was determined to be 1.0 µM using the computer program Cabuffer (kindly provided by Dr. L. Schlichter). Pulse protocols for whole cell recordings consisted of 500-ms ramps between –120 and +80 mV from a holding potential of –70 mV. Interpulse intervals were 5–10 s.

For inside-out patch recordings of transiently transfected cells, pipettes were filled with (in mM): 30 KCl, 100 K gluconate, 1.0 CaCl2, and 10 HEPES (pH 7.2). The cytoplasmic side of the membrane was perfused with intracellular solution in which the free Ca2+ concentration was varied between 0 and 1.0 µM using CaCl2 and readjusted to pH 7.2, as determined by the computer program Cabuffer. For insideout recordings, patches were held at –80 mV, and data were collected continuously for 30- to 60-s intervals.

All data were acquired online at a sampling rate of 10–20 kHz and filtered at either 2 (whole cell recordings) or 1 kHz (inside-out patch recordings) before analysis with pCLAMP6.0 software. Errors due to series resistance during whole cell recordings were minimized by employing 80–90% series resistance compensation. Results represent means ± SE from five to eight experiments.

86Rb+ flux studies. 86Rb+ was used as a K+ surrogate in in vitro flux studies across proximal colonic segments isolated from both normal and K+-depleted rats. 86Rb+ fluxes were performed at 0 mV under voltage-clamp conditions, as described previously (29). In brief, excised proximal colons were flushed with oxygenated cold Ringer solution, and the muscular layers were stripped. Two mucosal sheets were obtained from each animal, mounted in Lucite chambers (1.13 cm2), and bathed on both sides with equal volumes of Ringer solution. The bath solutions were maintained at 37°C and continuously gassed with 5% CO2-95% O2. The Ringer solution contained (in mM): 115 NaCl, 25 NaHCO3, 5 KCl, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose (pH 7.4). The transepithelial potential difference (Vte) of colonic tissues was determined, and, after voltage clamping, the short-circuit current (Isc) was measured. Transepithelial conductance (Gte) was calculated from the Vte and the Isc (DVC 1000, World Precision Instruments) and normalized to the exposed area of 1.13 cm2.

86Rb+ (1.0 µCi 86RbCl; New England Nuclear) was added to either the serosal or mucosal bath solution. After a 50-min equilibration, basal unidirectional fluxes [i.e., mucosal to serosal (m-s) and serosal to mucosal (s-m)] were measured for three 15-min control periods (basal). In other experiments, after basal measurements, 86Rb+ fluxes were measured in the presence of 200 µM carbachol in the serosal solution. In the latter experiments, after a 5-min equilibration in carbachol, unidirectional 86Rb+ fluxes were measured for two sequential 15-min periods (carbachol I and II). In experiments using KCa-channel blockers, the blocker was added to either the mucosal or serosal bath solution of both normal and K+-depleted animals at the end of carbachol I. Thus, after carbachol treatment, 100 nM IbTX, 50 nM APA, or 500 nM CLT were added to the mucosal bath solution, whereas in other experiments, 500 nM CLT was added to the serosal bath solution. In addition, the effect of 100 nM IbTX, 50 nM APA, or 500 nM CLT was tested in the absence of carbachol. In some experiments, 86Rb+ fluxes were measured for 15 min after adding 3 µM thapsigargin to both serosal and mucosal solutions. Net 86Rb+ transport (JnetRb+) was calculated from the difference between JmsRb+ and JsmRb+ using tissues matched on the basis of the initial Gte (with a difference <10%). Positive and negative values represent active absorption and active secretion, respectively. Results are expressed as means ± SE. A nonparametric Friedman test was performed, and a P value <0.05 was considered significant.

36Cl flux studies 36Cl (1.0 µCi; New England Nuclear) was added to either the serosal or mucosal bath solution. After incubation for 15 min, unidirectional fluxes were measured first under control conditions, then following the addition of 200 µM serosal carbachol, and finally in the presence of carbachol plus 500 nM CLT.

Generation of a polyclonal antibody to IK/SK4. At the time of the antibody production, the rat gene for IKCa had not been identified. Therefore, the anti-IK antibody was directed against a peptide derived from the mouse homolog of rat SK4 (rSK4). This peptide, RQVRLKHRKLTEQVNSMVD, is identical to the sequence encoded by the subsequently cloned rSK4, with the exception that at position 11, where T in mouse is substituted for R in rat. No obvious deficiencies in immunoreactivity against native rSK4 have been observed. The anti-IK antibody was custom produced by Research Genetics, a division of Invitrogen (Carlsbad, CA). New Zealand white rabbits were inoculated with a keyhole limpet hemocyanin peptide conjugate mixed with an equal volume of Freund's adjuvant. Animals were injected in three subcutaneous dorsal sites with a total of 0.1 mg per injection. The injection protocol was repeated at weeks 2, 6, and 8. After 10 wk, terminal bleeds were taken. The serum was collected from clotted blood by centrifugation, and an ELISA was used to assess anti-peptide serum titer. The serum from both rabbits was then combined and affinity purified over an immobilized peptide column.

Western analysis. Mouse brain, kidney, and colon homogenates were used to identify native IK/SK4 protein. Tissues were prepared as previously described (41). After dissection, the tissues were homogenized twice by 10-s strokes at power level 5 with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) using 1 g tissue/5 ml homogenization solution (10 mM HEPES adjusted to pH 7.4 with Tris, 10% sucrose, 1 mM EDTA, 1 mM PMSF) plus 1 tablet of Complete protease inhibitor (Roche Applied Science, Indianapolis, IN) per 50 ml solution. Homogenates were centrifuged at 2,500 g for 15 min at 4°C, and the supernatants were saved. The pellets were resuspended in 5 ml homogenization buffer per gram of starting tissue, then homogenized and recentrifuged as above. The supernatants were combined, and crude membrane proteins were precipitated by centrifugation at 22,000 g for 20 min at 4°C. The supernatants from this step were discarded, and the pellets were resuspended in PBS containing 1 mM EDTA, 1 mM PMSF, and Complete protease inhibitor (1 tablet/50 ml), then passed once through a 25-gauge needle and once through a 30-gauge needle. Aliquots were quickly frozen in liquid N2 and stored at –85°C until use. Rat colonic proximal apical membranes were isolated by the modified method of Stieger et al. (38). Basolateral membranes were prepared from scraped mucosa from rat proximal colon by the sucrose density gradient centrifugation described by Biber et al. (1).

Approximately 20 µg of crude membrane proteins were separated by two-phase Tricine polyacrylamide gel electrophoresis (10%T/6%C resolving layer, 4%T/3%C stacking layer) and transferred onto nitrocellulose membrane (Hybond ECL, Amersham Pharmacia Biotech, Piscataway, NJ) in buffer containing 10 mM 3'(cyclohexylamino)-1-propanesulfonic acid (adjusted to pH 11) and 10% methanol. The blot was blocked overnight at room temperature in blocking buffer (PBS containing 0.1% Tween-20, 4% BSA, and 1% normal goat serum). After the blot was blocked, it was incubated for 1 h at room temperature in blocking buffer containing a 1:5,000 dilution of a stock 3.2 mg/ml solution of rabbit anti-mouse intermediate Ca2+ activated K channel (anti-mIKCa) polyclonal affinity-purified antibody, washed three times with PBS containing 0.1% Tween-20 (PBS-T), incubated for 1 h at room temperature in blocking buffer containing a 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) and again washed three times with PBS-T. Immune complexes were detected on film using enhanced chemiluminescence (Amersham Pharmacia Biotech; Fig. 1). To assess specificity, the anti-mIKCa2+ (mouse intermediate Ca2+ activated K channel) antibody was preincubated for 1 h with a 50-fold molar excess of competitor peptide corresponding to the epitope recognition sequence before use in blotting (Fig. 1).



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Fig. 1. Characterization of the anti-mIKCa antibody. A: mouse brain (lanes 1 and 3), kidney (lanes 2 and 4), and colon (lane 5) membrane preparations were assayed via Western analysis for intermediate-conductance K+ channels (IKCa) protein expression. A strong 45-kDa band in lanes 2 and 5 (arrow) corresponds to the apparent molecular mass expected for IKCa; the lack of a band of that size in lane 1 is consistent with a much lower expression level of IKCa in the brain. Several minor larger molecular weight bands are also present in both brain and kidney. However, we did not observe formation of a homotetrameric complex in this gel system. Preincubation of the anti-mIKCa antibody with purified peptide containing the recognition epitope results in a dramatic loss of reactivity, particularly of the 45-kDa band (lanes 3 and 4). Note that lanes 3 and 4 display a 60 times the amount of exposure relative to lanes 1 and 2; similar exposure times demonstrate a nearly complete loss of apparent reactivity following peptide preincubation (data not shown). B: in the rat proximal colon, IK(SK)4 protein was detected in both apical (AM) and basolateral membrane (BM) preparations. However, expression of IK was greater in the AM. Na+/H+ exchanger (NHE)1 and NHE3, which are expressed in the BM and AM, respectively, were used as markers to confirm the relative purity of these membranes.

 

Confocal Immunofluorescence. Single crypts were isolated as previously described (16). Crypts were adhered with Cell-Tak (Collaborative Biomedical), washed with PBS, and fixed for 10 min at room temperature with 2.5% formaldehyde. After rinsing with PBS, crypts were permeablized with 0.25% Triton X-100 and 0.1% BSA in PBS for 5 min at room temperature. Nonspecific binding was blocked with three washes of PBS containing 1% BSA. Crypts were incubated with a 1:2,000 dilution of anti-mIKCa2+ primary antibody overnight at 4°C. Cells were then washed with PBS containing 0.1% BSA and exposed to goat anti-rabbit immunoglobulin G (Fab')2 fragments conjugated to R-phycoerythrin for 30 min at room temperature. Immunofluorescence was detected by confocal microscopy using excitation and emission wavelengths of 488 and >550 nm, respectively.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Expression and regulation of KCa channels in the rat proximal colon. The proximal colon participates in Ca2+-dependent K+ secretion (15, 25). Thus Northern blot analysis was used to identify which KCa-channel isoforms are expressed in the rat proximal colon. BKCa, SKCa, and IKCa mRNAs were detected as ~4-, 2.1-, and 2.2-kb transcripts, respectively (Fig. 2), whereas SK1 and SK3 mRNAs were not detected (data not shown). Subsequently, the effect of dietary K+ depletion on KCa-channel abundance in the proximal colon was investigated on rats maintained on reduced levels of dietary K+. Of the KCa-channel isoforms detectable in rats maintained on normal diets, only rSK4 mRNA abundance was significantly decreased in the proximal colons of dietary K+-depleted animals (Fig. 2). The degree to which rSK4 mRNA abundance was reduced (84%; n = 3) suggests that decreased expression of rSK4 in the proximal colon may help conserve K+ in K+-depleted animals.



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Fig. 2. Regulation of KCa mRNA abundance in the rat colon by dietary K+ depletion. mRNA purified from the proximal colon of rats grown on diets with normal (N) or depleted (D) amounts of K+ were analyzed by Northern blot hybridization using rSK1, rSK2, rSK3, rSK4, large conductance (BK), and GAPDH cDNAs as probes. The expected size of each transcript is denoted at the left (A). rSK1 and rSK3 were not detected and thus are not shown here. KCa mRNA abundance in normal or K+-depleted rats was quantitated by densitometry from 3 different mRNA preparations per dietary condition (B). For each condition, KCa abundance in the proximal colon is normalized to GAPDH mRNA abundance and then to KCa abundance in rats raised on normal diets.

 

Electrophysiological characterization of rSK4. Because rSK4 message was selectively regulated by dietary K+ depletion, rSK4 was cloned from rat colonic cDNA library, and its electrophysiological properties were studied in a transiently transfected CHO cell line. As shown in Fig. 3A, in transfected cells, several nanoamperes of current were observed in whole cell mode in the presence of 1.0 µM free intracellular Ca2+. In contrast, whole cell currents were not observed in untransfected cells in 1.0 µM free intracellular Ca2+ (Fig. 3A). In the absence of intracellular Ca2+, no significant current was observed in transfected or untransfected cells (Fig. 3B). These results indicate that the currents measured in CHO cells were caused by rSK4 and that intracellular Ca2+ activates the channels encoded by this cDNA.



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Fig. 3. rSK4 generates Ca2+-dependent, clotrimazole (CLT)-sensitive K+ currents when expressed in chinese hamster ovary (CHO) cells. A: typical whole cell recording of a CHO cell transfected with rSK4 shows large inward and outward currents that are absent in untransfected cells. The recording pipette contained 1.0 µM free Ca2+, and currents were elicited during 500-ms ramps between –120 and +80 mV from a holding potential of –70 mV. B: whole cell recording of a typical cell transfected with rSK4 shows no significant inward or outward currents in the absence of free Ca2+ in the recording pipette. C: unclamped (zero current) membrane potentials of CHO cells transiently transfected with rSK4 were measured with K+ substituting for Na+ in the bath solution. The points were fit with a line with a slope of 55 mV per 10-fold change in K+ concentration (n = 5; error bars are not evident due to their small size relative to the size of the symbols). D: representative whole cell recording of a CHO cell transfected with rSK4 shows nearly linear currents with 130 mM K+ in the pipette and 5 mM K+ in the bath, whereas currents from the same cell rectify inwardly when extracellular K+ is raised to 100 mM. E: typical whole cell recording of a CHO cell transfected with rSK4 before and after bath application of 1.0 µM CLT. F: sensitivity of rSK4 to various blockers of KCa channels. Membrane current measurements were made at 0 mV before and after bath application of 20 nM charybdotoxin (CTX), 1.0 µM CLT, tetraethylammonium (TEA), 100 nM iberiotoxin (IbTX), or 20 mM or 10 nM apamin (APA) and are expressed as the fractional current remaining after application of the blocker.

 

To establish the K+ selectivity of rSK4, the extracellular concentration of K+ was varied from 3 to 100 mM by substituting K+ for Na+ in the bath solution. As predicted by the Nernst equation, the unclamped membrane potential shifted by 55.0 ± 0.7 mV per 10-fold change in extracellular K+ concentration (Fig. 3C). With 3 mM K+ and 140 mM Na+ in the bath, the permeability ratio of K+ to Na+ was 72, as calculated by a derivation of the Goldman-Hodgkin-Katz equation. These results indicate that rSK4 is highly selective for K+ over Na+.

The current-voltage (I-V) relationship of rSK4 currents changed significantly on changing the extracellular K+ concentration. In 5 mM K+, the I-V relationship was nearly linear over the physiological voltage range (Fig. 3D). In contrast, in 100 mM extracellular K+, the I-V relationship rectified significantly in the inward direction. In this example, the slope conductance in 100 mM extracellular K+ measured between –120 and –80 mV was 100 nS and between 0 and +40 mV it was 50 nS. These values yield a conductance ratio of 2.0 for inward/outward currents, indicating that rSK4 channels rectify strongly in high extracellular K+, a property observed for some native colonic KCa channels (5, 26).

To establish that the pharmacological characteristics of rSK4 are similar to those of other mammalian IKCa channels, the effects of various KCa-channel inhibitors were examined on rSK4 whole cell currents in the presence of 1.0 µM free intracellular Ca2+ (Figs. 3, E and F). After perfusion with 1.0 µM CLT, a specific blocker of IKCa channels, whole cell currents were inhibited by 76 ± 4% (n = 5) at 0 mV (Figs. 3, E and F). Similarly, currents from cells perfused with 20 nM CTX, a blocker of some maxi-KCa and IKCa channels, were inhibited by 88 ± 2% (n = 5) at 0 mV (Fig. 3F). In contrast, 20 mM TEA or 100 nM IbTX, blockers of maxi-KCa channels, inhibited whole cell currents by only 36 ± 2 (n = 5) and 22 ± 2% (n = 5), respectively (Fig. 3F). Similarly, 10 nM APA, a toxin that blocks some SKCa-channel isoforms but not members of the IKCa subfamily, did not significantly inhibit rSK4 currents (Fig. 3F). The efficacy of the specificIKCa-channel blocker CLT contrasted with the weak or negligible effects of the inhibitors of other K+ channels, supporting the notion that the colonic rSK4 is the rat isoform of an IKCa channel found in colonic epithelial cells (7, 30).

Single-channel characteristics. To establish the single-channel conductance of rSK4, inside-out patch-clamp recordings were performed with 130 mM K+ in both the pipette and bath solutions. As illustrated in Fig. 4A, an increase in channel activity was observed when free Ca2+ was raised from 0 to 300 to 600 nM, and this effect was reversible. An all-points histogram of the recording yielded two distinct peaks (Fig. 4B). Gaussian fits to these and other patch recording data yielded a chord conductance of 36 ± 2 pS (n = 4) between 0 and –80 mV. These results confirm that rSK4 encodes an IKCa channel similar to that found in colonic epithelia (5). Due to the increase in conductance and rectification observed for rSK4 when extracellular K+ is elevated (Fig. 3D), it is likely that some of the smaller conductances recorded for KCa channels in colonocytes under conditions of low K+ or at depolarized potentials also reflect rSK4 channels (2, 3, 22, 27).



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Fig. 4. rSK4 generates IK Ca2+-activated K+ channels. A: an insideout patch excised from a CHO cell transfected with rSK4 shows increasing channel activity as free Ca2+ on the intracellular surface of the patch is elevated. Activity is reversible with washout of Ca2+. This patch was held at –80 mV (inside negative) with 130 mM K+ in both the pipette and bath solution. B: an all-points histogram of the recording in A. Between 0 and –80 mV, rSK4 has a chord conductance of 36 pS in this patch.

 

86Rb+ flux studies. After pharmacological properties of rSK4 were established, various KCa-channel blockers were used to study 86Rb+ fluxes across isolated intact proximal colonic mucosa. 86Rb+ fluxes were first performed under basal conditions. Over three consecutive 15-min time periods, unstimulated basal net 86Rb fluxes did not change significantly (n = 4; Table 1a). Fluxes were also measured following exposure of tissues to carbachol, an agonist known to elevate intracellular Ca2+ (8, 9). Carbachol increased net 86Rb+ secretion from –0.07 ± 0.01 to –0.28 ± 0.01 µeq·h1·cm2 (n = 4; Table 1b and Fig. 5), primarily as a result of an increase of JsmRb+ (Table 1b). Carbachol-induced secretion was sustained during a subsequent 15-min period.


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Table 1. Effect of KCa channel blockers on carbachol-induced K+ (86Rb+) secretion in rat proximal colon

 


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Fig. 5. Effect of CLT on carbachol-induced 86Rb+ secretion. Basal net fluxes across rat proximal colonic epithelia were determined in Ringer solution (basal). Subsequently, carbachol was added to the serosal side (Carb) and net fluxes were determined. After exposure to carbachol, IbTX, APA, or CLT was added to the mucosal side and net fluxes were again determined. In the absence of carbachol pretreatment, IbTX, APA, or CLT did not reduce net fluxes significantly (inset). All values shown are normalized to the net fluxes determined under basal conditions. Number of measurements is shown in parentheses. *P < 0.05 compared with carbachol.

 

IbTX (100 nM) partially but significantly reduced carbachol-induced net 86Rb+ secretion when applied to the apical membranes of proximal colonic mucosa (n = 4; Table 1c and Fig. 5). A similar partial but significant inhibition of net 86Rb+ secretion was observed with 50 nM APA (n = 7; Table 1d and Fig. 5). Simultaneous exposure to IbTX and APA resulted in an additional but statistically insignificant decrease in the carbachol-induced net 86Rb+ compared with IbTX or APA alone (Table 1e). In contrast, 500 nM CLT applied to the mucosal or serosal solution significantly reduced JnetRb+ to basal values (Table 1, f and g, and Fig. 5). Neither the partial inhibition of the carbachol-stimulated JnetRb+ by mucosal IbTX and APA nor the complete inhibition by CLT can be attributed to a reduction in the unstimulated flux, because these drugs did not significantly affect the basal JnetRb+ (Fig. 5, inset). Together, these results suggest that apical BKCa, SKCa, and IKCa channels and basolateral IKCa channels contribute to Ca2+-activated 86Rb+ secretion across the normal rat proximal colon but that IKCa channels may play a more prominent role in this phenomenon.

Regulation of 86Rb+ secretion by dietary K+ depletion. Northern blot analyses demonstrated that under conditions of dietary K+ depletion, the abundance of rSK4 transcript in the proximal colon decreased (Fig. 2). To determine whether this downregulation of rSK4 transcript might be accompanied by a functional change in K+ flux, 86Rb+ flux studies were performed on proximal colonic segments of rats deprived of dietary K+. Basal 86Rb+ secretion was still present in K+-depleted rats. However, in K+-depleted animals, carbachol did not elevate JsmRb+ or JnetRb+ significantly above basal values and CLT did not inhibit secretion (Table 2a). Diminished active secretion appeared to be due to downregulation of IKCa channels rather than downregulation of muscarinic receptors or their transduction. This was illustrated by the addition of thapsigargin, an inhibitor of the endoplasmic reticulum Ca-ATPase and thus an activator of intracellular Ca2+ release. Thapsigargin failed to elevate JnetRb+ significantly above basal values in K+-depleted animals (Table 2b). In contrast, in animals raised on normal levels of dietary K+, JnetRb+ rose significantly above basal values in response to thapsigargin (Table 2c). Thus the insensitivity of K+ secretion to elevated intracellular Ca2+ and to CLT in K+-depleted animals may be caused by the downregulation of IKCa channel expression, which would thereby facilitate retention of K+ during periods of diminished K+ intake.


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Table 2. Effect of diet on Ca2+-activated K+ (86Rb+) secretion in rat proximal colon

 

36Cl flux studies. As with 86Rb+ studies, proximal colonic epithelia were studied under basal conditions and following exposure to serosal carbachol. Exposure to carbachol induced a large, sustained, net 36Cl secretion above basal values. The subsequent addition of CLT to either the apical or basolateral membranes of proximal colonic mucosa abolished carbachol-induced net 36Cl secretion (n = 7). The abolition of carbachol-induced secretion by CLT was so profound that proximal colons switched from secreting to absorbing Cl (Table 3, a and b). The inhibition of both 36Cl and 86Rb+ fluxes by CLT suggests that both Cl and K+ transport mechanisms are coupled to IKCa channels.


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Table 3. Effect of clotrimazole on Cl- (36Cl-) secretion in rat proximal colon

 

Membrane localization of IKCa channels. To study the polarization of IKCa channel distribution in the proximal colon, both confocal immunohistochemistry and Western bolts were performed. Intact rat colonic crypts were isolated as previously described (16) and imaged by confocal microscopy. Figure 6A depicts a typical crypt preparation, as shown by phase-contrast microscopy. With the use of immunofluorescence, IKCa channels were observed predominantly in the basolateral membranes of crypt cells (Fig. 6B). In addition, IKCa channels were also observed on surface cells but with more intense staining compared with crypt cells. In the absence of IKCa antibody and in the presence of the secondary antibody, no significant fluorescence was detected. The absence of IKCa channel expression on the apical membrane of crypt epithelial cells may result from limited access of the antibody to the lumen of colonic crypts. This issue was resolved with Western blots using apical and basolateral membranes from rat proximal colonic crypt cells. As illustrated in Fig. 1B, IKCa was expressed in both the apical and basolateral membrane preparations as a protein of ~45 kDa, a molecular mass similar to that predicted by the open reading frame of SK4. However, a greater level of expression of IKCa was detected in the apical membrane. An additional band at ~70 kDa of unknown origin was also observed in the apical membrane preparation. The purity of the membrane preparations was confirmed using antibodies that specifically recognize the apical membrane Na+/H+ exchanger (NHE)3 transporter or the basolateral membrane NHE1 transporter.



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Fig. 6. IKCa expression in proximal colonic crypts by confocal immunofluorescence. Colonic crypts were isolated from the rat proximal colon as described in MATERIALS AND METHODS. A: phase contrast image of a typical colonic crypt. After exposure to the anti-mIKCa antibody, fluorescence was mainly localized to the basolateral membrane (B). In the absence of anti-mIKCa antibody, minimal fluorescence was observed.

 


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In the proximal colon, cholinergic agonists increase intracellular Ca+ concentration ([Ca2+]i) and stimulate K+ secretion (15). The rise in [Ca2+]i acts on the Na-K-2Cl cotransporter to enhance uptake of K+ across the basolateral membrane (15). Elevated [Ca2+]i also opens as yet unidentified apical KCa channels in the proximal colon, providing intracellular K+ with a means of exit to the lumen (Fig. 7) (15).



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Fig. 7. Model of K+ secretion in the rat proximal colonic epithelium. Transcellular K+ secretion requires a basolateral uptake mechanism and an apical exit step. An increase in cytosolic Ca2+ by cholinergic agonists, such as carbachol, leads to opening of apical IbTX-sensitive BKCa, CLT-sensitive IKCa, and APA-sensitive SKCa channels, which allow K+ to be secreted into the lumen. In addition, the elevation in intracellular Ca2+ also opens basolateral CLT-sensitive IKCa channels, allowing K+ recycling. Intracellular K+ is replenished by the combined basolateral activities of the Na-K-2Cl cotransporter and the Na-K-ATPase. The activation of apical and basolateral KCa channels also leads to hyperpolarization of the cell membrane, which favors Cl secretion.

 

Both paracellular and transcellular pathways contribute to ion transport in polarized epithelia. In the case of the former, ions do not cross a membrane barrier, and they therefore passively distribute between the lumenal and serosal compartments according to their individual electrochemical gradients. In the case of the latter, the segmental localization, polarized distribution, and differential regulation of the various molecular components of the absorptive and secretory machinery are critical for determining how the proximal colon performs net K+ secretion and the distal colon performs net K+ absorption. Both paracellular and transcellular transport mechanisms contribute to K+ secretion in the proximal colon. Although the cotransporters and pumps have been extensively studied, the molecular identities of the channels participating in colonic K+ absorption and secretion have been less well defined. However, it is apparent that KCa channels are important conduits for transcellular K+ transport and that different isoforms may have distinct roles on different membranes. For example, in patch-clamp studies, BKCa channels have been identified in both apical and basolateral membranes of rat enterocytes (3, 4, 19, 20, 22), whereas SKCa/IKCa channels have been identified only in basolateral membranes (2, 3, 22, 27). Although the specific roles of these KCa channels are not known, some insight into which channels participate in K+ absorption and secretion has been gleaned from flux studies. In the distal colon, for example, carbachol stimulates an m-s 86Rb+ flux that is inhibited by quinine at the basolateral membrane, whereas carbachol stimulates a mucosal 86Rb+ efflux that is inhibited by TEA at the apical membrane (15). In contrast, in the proximal colon, mucosal and serosal effluxes are blocked by the inhibitor of SKCa/IKCa channels, Ba2+ (15, 17), but not by the BKCa-channel inhibitor TEA (15). This suggests that SKCa or IKCa channels may play significant roles in active K+ secretion in the proximal colon.

In the present study, the potential roles of KCa channels in mediating K+ secretion in the proximal colon were investigated. There are five known KCa channels in mammals: one BKCa isoform [Slo (28, 40)], three SKCa isoforms [SK1–3 (20)], and one IKCa [SK4 (18)]. With the use of Northern analysis, the present study demonstrates that under normal dietary conditions, only the BKCa, SK2Ca, and IKCa isoforms are expressed in rat proximal colon.

The roles of BKCa, SK2Ca, and IKCa in regulating proximal colonic K+ secretion were confirmed in flux studies under zero voltage-clamp conditions using pharmacological activators and inhibitors of the three classes of KCa channels. Because of the absence of any overall transepithelial gradient for K+, the observed net secretion only represents active transcellular transport. In the absence of elevated intracellular Ca2+, none of the KCa-channel antagonists have any effect on 86Rb+ fluxes, indicating that KCa channels do not participate in basal secretion. In contrast, carbachol-induced 86Rb+ secretion is partially inhibited by the mucosal application of IbTX or APA, indicating that apical BKCa and SK2Ca channels probably contribute to K+ secretion. The greater degree of inhibition of carbachol-induced secretion by CLT compared with the inhibition measured during simultaneous exposure to IbTX and APA suggests that IKCa channels may play a prominent role in K+ secretion in the proximal colon. The complete abolition of carbachol-induced 86Rb+ secretion by mucosal CLT also suggests that in addition to its basolateral distribution, which has been confirmed by patch-clamp studies (2, 22, 27), IKCa channels may also be expressed at the apical membrane, from which few recordings have been made. The present data suggest that all three types of KCa channels are involved in K+ secretion (Fig. 7).

It is possible that CLT is sufficiently permeant that, when added to the mucosal surface, it enters the cell. Indeed, heterologous expression studies with SK4 indicate that CLT probably crosses the plasma membrane to inhibit IKCa channels (44). As a consequence, CLT applied to the mucosal surface may also have access to and subsequently block IKCa channels at the basolateral membrane.

CLT applied to the serosal surface also abolishes carbachol-induced K+ secretion as an indirect consequence of blocking basolateral IKCa channels. Indeed, closing basolateral IKCa channels should initiate the following sequence of events in proximal colonic epithelial cells. Depolarization of the basolateral membrane would abolish the driving force for apical Cl efflux, an effect that has been well described by others (7, see also Ref. 36 for review). The cessation of intracellular Cl loss would lead to a decrease in basolateral Na-K-2Cl cotransporter activity, which would diminish not only Cl uptake but also K+ uptake across the basolateral membrane. Consequently, the intracellular K+ concentration would fall, and apical efflux through BKCa and SKCa channels would decrease. This model links Cl and K+ at their point of uptake at the basolateral membrane and at the level of secretion across the apical membrane: inhibiting the transport of either ion at either membrane would shut down transport of both ions at both membranes (Fig. 7). Consistent with this model, the present study shows that mucosal or serosal application of CLT inhibits both K+ and Cl secretion in the proximal colon.

The model predicts that blocking of IKCa channels present at either membrane would inhibit net 86Rb+ secretion. The present immunofluorescence data suggest that IKCa channels are expressed predominantly at the basolateral membrane. In contrast, Western blots demonstrate that IKCa channels are present in both apical and basolateral membranes, with a higher expression in the apical membrane. This discrepancy between immunohistochemistry and Western blot data suggests that the anti-IKCa antibody had limited accessibility to the crypt lumen in immunofluorescence experiments. Regardless, the present data indicate that IKCa channels are present on both the apical and basolateral membranes in rat proximal colonic epithelia.

In addition to possible modes of action on apical or basolateral IKCa channels, there have been reports in other systems of CLT acting on BKCa channels and on P-450 enzymes. However, other evidence bolsters the notion that the effects of CLT described in this study are caused by CLT acting exclusively on IKCa. First, the concentration of CLT used in this study is less than 10% of the measured Kd of CLT for 12 K+ channels, including BKCa and SKCa channels (43). Second, inhibition of P-450 enzymes with the IKCa-insensitive CLT analog econazole does not disrupt inducible transepithelial transport properties in the rat colon (37). Thus, in the present study, the inhibition of proximal colonic 86Rb+ secretion by CLT cannot be ascribed to CLT acting on P-450 enzymes.

Because the present study suggests that several KCa channels are involved in active proximal colonic K+ secretion, the regulation of KCa-channel expression by changes in dietary K+ was examined. Under normal dietary conditions, activation of KCa channels by thapsigargin or carbachol stimulates a large increase in Jnet in the proximal colon. In contrast, in K+-depleted rats, neither thapsigargin nor carbachol significantly elevates Jnet. Because basal 86Rb+ secretion is similar in K+-depleted rats and animals raised on normal K+ diets (see Table 2), dietary K+ depletion must specifically abolish the Ca2+-activated component of proximal colonic K+ secretion without affecting basal secretion.

In the present study, depletion of dietary K+ in rats results in the downregulation of mRNA encoding the IKCa isoform rSK4 but not of the mRNAs of the other two colonic KCa channels BKCa and rSK2. Thus downregulation of rSK4 mRNA probably underlies the inability of the proximal colon to secrete K+ in response to carbachol in the K+-depleted state. Selective transcriptional downregulation of rSK4 is expected to help conserve K+ in the K+-depleted condition if this channel normally either underlies or regulates a major conductance pathway for K+ secretion in the proximal colon. Because CLT, IbTX, and APA each are capable of reducing net secretion, IKCa, BKCa, and SKCa channels probably each contribute to secretion in the normal rat proximal colon. However, the inability of IbTx and APA together to reduce Jnet to baseline values plus the strong correlation between dietary K+, the abundance of rSK4 transcript, and carbachol-induced CLT-sensitive Jnet suggests that IKCa channels are the major regulators of Ca2+-activated K+ secretion by the proximal colon in response to changes in dietary K+.


    ACKNOWLEDGMENTS
 
We thank Dr. Gordon MacGregor for valuable input and S. Mentone for assistance with confocal immunofluorescence. W. J. Joiner also thanks Dr. Leonard Kaczmarek for training and support.

This research was supported by a Research Fellowship from the National Kidney Foundation and its Connecticut affiliate (to S. Basavappa.) and National Institutes of Health Grants DE-14119 (to K. Nehrke), DK-17433 (E. L. Boulpaep), and DK-18777 (to V. M. Rajendran and H. J. Binder).


    FOOTNOTES
 

Address for reprint requests and other correspondence: V. M. Rajendran, Dept. of Internal Medicine, Yale Univ., 333 Cedar St., P. O. Box 208019, New Haven, CT 06520 (E-mail: Vazhaikkurichi.Rajendran{at}yale.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. Section 1734 solely to indicate this fact.

* W. J. Joiner, S. Basavappa, and S. Vidyasagar contributed equally to this work. Back


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