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Distal Colonic K⁺ Secretion Occurs via BK Channels

Matthias Sausbier^*, Joana E. Matos, Ulrike Sausbier^*, Golo Beranek^*, Claudia Arntz^*, Winfried Neuhuber, Peter Ruth^* and Jens Leipziger

^* Pharmakologie und Toxikologie, Pharmazeutisches Institut, Universität Tübingen, Tübingen, Germany; Institute of Physiology and Biophysics, The Water and Salt Research Center, University of Aarhus, Aarhus C, Denmark; and Anatomisches Institut, Universität Erlangen-Nürnberg, Erlangen, Germany

Address correspondence to: Dr. Jens Leipziger, Ole Worms Alle 160, 8000 Aarhus C, Denmark. Phone: +45-89-42-2826; Fax: +45-86-12-9065; E-mail: leip{at}fi.au.dk

Received for publication October 24, 2005. Accepted for publication February 6, 2006.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
K⁺ secretion in the kidney and distal colon is a main determinant of K⁺ homeostasis. This study investigated the identity of the relevant luminal secretory K⁺ ion channel in distal colon. An Ussing chamber was used to measure ion transport in the recently generated BK channel–deficient (BK^–/–) mice. BK^–/– mice display a significant colonic epithelial phenotype with (1) lack of Ba²⁺-sensitive resting K⁺ secretion, (2) absence of K⁺ secretion stimulated by luminal P2Y₂ and P2Y₄ receptors, (3) absence of luminal Ca²⁺ ionophore (A23187)-stimulated K⁺ secretion, (4) reduced K⁺ and increased Na⁺ contents in feces, and (5) an increased colonic Na⁺ absorption. In contrast, resting and uridine triphosphate (UTP)-stimulated K⁺ secretion was not altered in mice that were deficient for the intermediate conductance Ca²⁺-activated K⁺ channel SK4. BK channels localize to the luminal membrane of crypt, and reverse transcription–PCR results confirm the expression of the BK channel -subunit in isolated distal colonic crypts. It is concluded that BK channels are the responsible K⁺ channels for resting and stimulated Ca²⁺-activated K⁺ secretion in mouse distal colon.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
In patients with ESRD, fecal K⁺ excretion is directly proportional to dietary K⁺ intake. This raises the possibility that colonic "K⁺ adaptation" makes a substantial contribution to K⁺ homeostasis in this disease (1) and underscores the importance of defining the basic elements of K⁺ handling in the colon. K⁺ transport in renal and colonic epithelium includes both active absorption and secretion, and their balance determines the net gain or loss of body K⁺ (2). In the colon, K⁺ absorption dominates in states of low oral K⁺ intake. Under normal conditions of high oral K⁺ intake, K⁺ secretion is predominant. K⁺ absorption requires active translocation of the K⁺ ion over the luminal membrane via the H⁺/K⁺ ATPase. K⁺ secretion follows the "pump-leak" mechanism. K⁺ is translocated actively over the basolateral membrane via the Na⁺/K⁺ pump or the Na⁺/2Cl^–/K⁺ co-transporter NKCC1 and leaks out of the cell through luminal K⁺ channels (2,3). In distal colonic epithelium, the relevant K⁺ channel in the luminal membrane remains to be established. For this purpose, we exploit the physiologic phenomenon that luminal nucleotide P2Y receptors in rat and mouse colon stimulate an electrogenic K⁺ secretory burst (4,5). This transient K⁺ secretion may be involved in a local intrinsic epithelial reflex that use-dependently becomes active during the stool passage. Mechanical perturbation of the epithelial layer might promote nonlytic release of nucleotides (6). The activation of Cl^– and K⁺ secretion and inhibition of Na⁺ absorption by stimulation of epithelial P2 receptors (7) should favor the hydration of the luminal space and may augment the generation of mucus (via the hydration of mucins) and thus allow for a better stool passage and protection of the epithelial surface barrier. The luminal P2Y receptors involved are established to trigger elevations of the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) (8). Therefore, the K⁺ channel in question is expected to be a Ca²⁺-activated K⁺ channel, i.e., of small, intermediate, or large conductance (9,10). In some epithelia, including rodent colon, evidence indicates that BK channels (large conductance) localize to the luminal membrane (11,12). We therefore considered the BK channel as the first-choice candidate. This also was suggested in a previous work that showed that luminal iberiotoxin, a specific blocker for BK (large conductance) channels, was able to inhibit strongly the luminal nucleotide-triggered K⁺ secretion (5). Here we use BK channel -subunit–deficient mice (BK^–/–) (13) to investigate the role of BK channels in K⁺ secretion in mouse distal colon. Our results strongly suggest that BK channels are the only Ca²⁺-activated K⁺ channels relevant for K⁺ secretion in mouse distal colon.

	Materials and Methods

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Mice
The previously generated BK^–/– and wild-type (WT) littermate mice on hybrid 129Sv/C57BL6 background were used (F2 generation) (13). Generation of SK4 channel–deficient (SK4^–/–) mice was as follows: Using a genomic 129/ola cosmid library (RZPD), the targeting vector was constructed such that the pore exon was flanked by a single loxP site and a floxed neo/tk cassette. This construct was electroporated into R1 embryonic stem (ES) cells, and G418-resistant clones were screened. Two positive clones, analyzed by Southern blotting, were transiently transfected with a Cre-expressing plasmid to excise the neo/tk cassette and the pore exon yielding L1/+ clones. Correctly targeted L1/+ clones were injected into C57BL/6 blastocysts. The resulting chimeras were mated with 129/Sv mice to obtain germ-line transmission. Male heterozygous 129/Sv mice then were mated with 129/Sv or C57BL/6 mice to generate mice on 129/Sv inbred and 129/Sv/C57BL6 hybrid background, respectively. These heterozygous offspring were intercrossed to yield SK4^–/– mice on 129/Sv/C57BL6 hybrid background (always F2 generation) and 129/Sv inbred background, and both genetic backgrounds were used for the functional studies. Genotyping was performed by PCR with three primers (F1 5'-TAA GTG CTT GCT GAG TCT GGA-3'; F2 5'-CAG GAA GCA CAG GCA CTG C-3'; reverse 5'-AGG AGA GTG ACT GTA GGT GAG-3') amplifying either the WT (264 bp) or the knockout (507 bp) allele. All mice were bred and maintained at the animal facility of the Pharmaceutical Institute, Department Pharmacology & Toxicology, University of Tübingen. Either litter- or age-matched mice of either gender were randomly assigned to the experimental procedures with respect to the German legislation on protection of animals.

Ussing Chamber Experiments
Mice (age 4 to 10 wk) were killed by cervical dislocation. Two centimeters of the mouse distal colon were used. The muscular layers were removed gently and a piece was mounted in an Ussing chamber with an aperture of 0.126 cm². The two halves of the chamber were perfused continuously by a bubble lift system. The solutions on both sides were symmetrical and had the following composition (in mM): 120 NaCl, 25 NaHCO₃, 1.6 K₂HPO₄, 0.4 KH₂PO₄, 1.3 Ca-gluconate, 1 MgCl₂, 5 d-glucose, and 0.005 indomethacin. The reservoirs were gassed with carbogen (5% CO₂ and 95% O₂) and kept at 37°C by water jackets. The measurements were performed in "open circuit" mode with the transepithelial voltage (V_te) in reference to the serosal side. Transepithelial resistance (R_te) was calculated from the voltage deflections (V_te) that were induced by short current pulses (25 µA, 0.6 s) (14). These deflections were corrected by the chamber resistance measured in the absence of tissue. The equivalent short-circuit current (I_sc) was obtained by Ohm’s law from V_te/R_te. The calculated I_sc changes were derived from peak values. Because of the small aperture of 0.126 cm² in this "mouse" Ussing chamber setup, a likely underestimation of the true R_te value must be expected (significant edge leakiness of the tissue). This in turn will lead to a tendency to overestimate true equivalent I_sc values. Initially, tetrodotoxin (1 µM) was added to the serosal side to inhibit possible secretory activation by the enteric nervous system or other autonomous nerve cells. Subsequently, amiloride (100 µM) was added to the mucosal perfusate to abolish any rheogenic Na⁺ absorption. After an equilibration period (30 to 60 min), uridine triphosphate (UTP) or other agonists and antagonists were added to the mucosal side.

Crypt Preparation
Mice (aged 4 to 20 wk) were killed by cervical dislocation. The preparation of colonic crypts was similar to that described by Siemer and Gögelein (15) and Diener et al. (16). A 4-cm piece of mouse distal colon was everted and rinsed with ice-cold Ca²⁺-free Ringer’s-type solution with the following composition (in mM): 127 NaCl, 5 KCl, 5 Na-pyruvate, 5 d-glucose, 10 HEPES, 5 EDTA, and 1 MgCl₂. Both ends were tied to obtain a sac preparation. This sac was filled with the same Ca²⁺-free solution. This preparation was then incubated in the above-mentioned solution for 10 min at 37°C. Isolated crypts were obtained by shaking the sacs in the preparation solution.

Measurement of Fecal Ion Content
WT and BK^–/– mice were killed by inhalation of CO₂. The feces were carefully extracted from the distal colon with tweezers. The oven-dry mass of feces was registered after a 24-h drying period in an incubator, after which the ionic content of the feces was extracted overnight with 0.75 N HNO₃. The supernatant was used for measuring the fecal concentrations of Na⁺ and K⁺ by flame photometry (ELEX 6361; Eppendorf, Germany) (17).

Immunohistochemistry of BK Channels
On-slide 10-µm cryostat slices from nonfixed WT and BK^–/– mouse distal colon segments were used. After preincubation with 10% normal donkey serum in buffer (1% BSA, 0.5% Triton X-100, 0.05 M Tris-buffered saline) and rinsing with Tris-buffered saline, the slices were incubated with anti-BK_(674-1115) (1:1000 in buffer) and tagged with Alexa 555–conjugated donkey anti-rabbit IgG (1:1000 in buffer) (13). BK channel immunofluorescence was analyzed using a confocal-laser scanning microscope (Biorad MRC1000 [Hercules, CA] attached to Nikon Diaphot 300 and equipped with a krypton-argon laser).

Reverse Transcription–PCR Analysis of BK Channel -Subunit in Isolated Mouse Colonic Crypts
Reverse transcription–PCR analysis was used to detect the presence of specific mRNA for the mouse BK channel -subunit in isolated colonic crypts. To this end, total RNA was extracted from approximately 500 isolated colonic crypts and transcribed into cDNA using reverse transcriptase. Primer selection was based on published mouse sequences for the BK channel subunit, rendering 5'-GCCGAGGTCGGCTGGATGA-3' as forward primer and 5'-GAAGAAGACCATGAAGAGGCG-3' as reverse primer, which yield an amplicon of 660 bp. The primer pair spans several exons of the BK channel gene to avoid amplification of genomic DNA.

Solutions and Chemicals
Tetrodotoxin and iberiotoxin (IBTx) were purchased from Latoxan (Rosans, France). All other chemicals were obtained from Sigma-Aldrich Denmark A/S (Vallensbaek, Denmark) and Merck (Darmstadt, Germany).

Statistical Analyses
The data shown are mean values ± SEM (n), where n refers to the number of mucosal preparations. For experimental series, n reflects the number of animals used. Paired and unpaired t test was used to compare mean values within one experimental series. P < 0.05 was accepted to indicate statistical significance.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
Abolished UTP-Stimulated K⁺ Secretion in BK^–/– Mice
Figure 1A shows two original Ussing chamber measurements of V_te (top line) in WT and BK^–/– mouse distal colonic mucosa in the presence of luminal amiloride (100 µM). Luminal UTP stimulates a rapid and transient deflection of V_te to lumen-positive values in WT mice. As described previously, this phenomenon was inhibited greatly by luminal Ba²⁺ and luminal IBTx and thus was identified as K⁺ secretion in distal colon (4,5). In contrast, luminal UTP does not promote K⁺ secretion in BK^–/– mice. Figure 1A summarizes the luminal UTP-stimulated peak I_sc data (WT: I_sc 103.8 ± 22.4 µA cm^–2, n = 10; BK^–/–: I_sc –4.3 ± 1.7 µA cm^–2, n = 6). Because of the small size of the mucosal tissue, an overestimation of absolute I_sc values in this study may need to be considered (see Materials and Methods). These results strongly indicate that luminal BK channels conduct the luminal UTP-stimulated K⁺ secretion. Furthermore, our data show that luminal UTP in BK^–/– mouse colon stimulated a very small but significant negative short-circuit current. This is consistent with a very small activation of Cl^– secretion via an agonist that is expected to elevate cytosolic Ca²⁺ (18).

View larger version (49K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. (A) Absence of luminal uridine triphosphate (UTP)-stimulated (100 µM) K+ secretion in distal colonic mucosa of BK channel–deficient (BK–/–) mice. Shown are the resting and UTP-stimulated transepithelial voltage (Vte) values. The top line shows Vte, and the bandwidth of voltage deflections reflects the transepithelial resistance (Rte; see Materials and Methods). Application of UTP to the luminal side in wild-type (WT) mice led to a transient deflection of Vte to lumen-positive values and a decrease of Rte. This effect reflects an activated K+ secretion. (Right) Statistical analysis of UTP-stimulated short-circuit current (Isc; n = 6/10). All data are means ± SEM; *P < 0.05. (B) Absence of luminal Ba2+ (5 mM) and iberiotoxin (IBTx; 240 nM)-sensitive K+ secretion in distal colonic mucosa of WT and BK–/– mice. (Right) Statistical analysis of Ba2+ and IBTx-sensitive Isc (n = 9/10 and n = 5). All data are means ± SEM; *P < 0.05. (C) Absence of luminal Ca2+ ionophore A23187-stimulated (1 µM) K+ secretion in distal colonic mucosa of BK–/– mice. (Right) Statistical analysis of Ba2+-sensitive A23187-stimulated Isc (n = 5/6). All data are means ± SEM; *P < 0.05.

Abolished Luminal Ca²⁺ Ionophore-Stimulated K⁺ Secretion in BK^–/– Mice
The data presented in Figure 1, A and B, indicate that BK channels mediate resting and luminal nucleotide-stimulated distal colonic K⁺ secretion. Because P2Y₂ and P2Y₄ receptors initiate an increase of cytosolic Ca²⁺ (8), it is suggested that the stimulated K⁺ secretion occurs via an increase in [Ca²⁺]_i. This was investigated by adding the Ca²⁺ ionophore A23187 (1 µM) to the luminal side of colonic mucosa of WT and BK^–/– mice. In WT mice, luminal A23187 induced a change of the V_te from –0.1 ± 0.2 to 0.8 ± 0.4 mV (n = 6), whereas this effect was absent in BK^–/– mice. The ionophore-induced effect was slow in onset and persistent (Figure 1C). This effect was inhibited by luminal Ba²⁺, and the ionophore-stimulated increase in Ba²⁺-sensitive I_sc amounted to 51.2 ± 3.9 µA cm^–2 (n = 6). Importantly, this ionophore-stimulated Ba²⁺-sensitive effect on I_sc was completely absent in BK^–/– colon (I_sc 1.2 ± 0.8 µA cm^–2; n = 5). These data strongly suggest that receptor-dependent and Ca²⁺ ionophore–stimulated increases in [Ca²⁺]_i promote BK channel–mediated distal colonic K⁺ secretion.

Persistent Resting Ba²⁺-Sensitive and Luminal UTP-Stimulated K⁺ Secretion in SK4^–/– Mice
A recent study suggested that in rat proximal colon, the intermediate conductance Ca²⁺-activated K⁺ channel (SK4) contributes to the Ca²⁺ agonist–stimulated K⁺ secretion (19). These results are in conflict with the data presented here as we found no indication of a residual K⁺ secretion in BK^–/– mice. We therefore generated a novel SK4^–/– mouse as described in the Materials and Methods section and in the strategy displayed in Figure 2. This allowed investigation of resting Ba²⁺-sensitive K⁺ secretion in distal colonic mucosa also in SK4^–/– mice. The magnitude of the Ba²⁺-sensitive K⁺ secretion was identical in the WT and SK4^–/– mouse colon (I_sc in WT: 30.5 ± 3.4 µA cm^–2 [n = 6]; I_sc in SK4^–/–: 30.9 ± 2.3 µA cm^–2 [n = 5]; Figure 2). Also, luminal UTP application produced no difference in the nucleotide-induced K⁺ secretion between WT and SK4^–/– mice (I_{sc UTP} in WT: 43.0 ± 21.2 µA cm^–2 [n = 15]; I_{sc UTP} in SK4^–/–: 47.8 ± 22.7 µA cm^–2 [n = 9]; Figure 2). The SK4 channel is well documented to be localized in the basolateral membrane of rodent colonic crypts (18,20). Activation of basolateral K⁺ conductances provides the electrical driving force for luminal Cl^– exit via cystic fibrosis transmembrane conductance regulator (CFTR) (20). Therefore, as expected, the basolateral carbachol (CCH) stimulated (CCH 100 µM) NaCl secretion after forskolin (2 µM) prestimulation was greatly reduced in the SK4^–/– as compared with the WT mice (SK4 WT: I_{sc CCH} 203.1 ± 18.3 µA cm^–2 [n = 6]; SK4^–/– I_{sc CCH} in 88.7 ± 10.0 µA cm^–2 [n = 5]). These data argue strongly against the SK4 channel mediating luminal K⁺ exit in mouse distal colon. In conjunction with our data from the BK^–/– mice, which show the absence of other Ba²⁺-inhibitable K⁺ conductances in the luminal membrane, we suggest that BK channels are the only Ca²⁺-activated luminal K⁺ channels in mouse distal colon.

View larger version (42K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. (A) Targeted deletion of the murine SK4 gene using the Cre/loxP system. Targeting strategy: (Top) Murine WT SK4 locus and targeting vector that contains the Neo/Tk cassette flanked by two loxP sites () downstream of the pore exon and a single loxP site upstream of the pore exon. (Middle) Targeted allele after selection for neomycin resistance. (Bottom) Transfection of recombinant embryonic stem (ES) clones with Cre recombinase yields (1) ES cells with a "floxed" locus for generation of conditional knockout mice and (2) ES cells with deletion of the pore exons for generation of general knockout mice. B, BamHI; E, EcoRV; H, HindIII; X, XhoI. (B) Southern blotting of HindIII-digested genomic ES cell DNA hybridized with the 3' probe ( in A) results in specific fragments that represent the knockout (4.8 kb) and the wild type (6.6 kb) allele as indicated. (C) PCR analysis of tail DNA from a WT, a heterozygous (SK4+/–), and a SK4–/– mouse using specific primers F1, F2, and R (open arrowheads in A). (D) Presence of resting Ba2+-sensitive (5 mM) K+ secretion in distal colonic mucosa of SK4–/– mice. (Right) Statistical analysis of Ba2+-sensitive Isc (n = 5/6) and luminal UTP-stimulated Isc in distal colonic mucosa of WT and SK4–/– mice (n = 9/14). The data were pooled from three 129/Sv and two 129/Sv/C57BL6 hybrid mice of each genotype. All data are means ± SEM; *P < 0.05.

Immunolocalization of BK Channel -Subunit in Mouse Distal Colonic Mucosa
In reverse transcription–PCR experiments that were performed in isolated colonic crypts, specific mRNA transcripts for the subunit of BK channels were detected (data not shown). Furthermore, a well-characterized specific antibody against the BK channel -subunit (13) was used to immunolocalize BK channels in mouse distal colon. Figure 3, C and D, shows an overview of mouse distal colonic mucosa from a WT and a BK^–/– mouse. The green color represents intrinsic fluorescence (autofluorescence) and provides a morphologic image of the colonic tissue in these cryosections. The red stain in WT tissue (Figure 3C) reflects BK channel localization. The complete absence of labeling in the BK^–/– tissue confirms the high specificity of this antibody. A very strong red labeling is visible in the two smooth muscle layers (tunica muscularis and lamina muscularis mucosae) of the gut wall. This is an expected finding as BK channels are known to be expressed strongly in smooth muscle tissue. Specifically, we investigated the localization of BK channels to the luminal membrane of colonic crypts. The two higher magnification cutout images shown in Figure 3, E and F, display distinct labeling of luminal membrane domains in colonic crypt epithelium. A dotted red BK labeling pattern of the luminal membrane is seen in both crypt images. The enlarged pictures in Figure 3, A and B, try to identify whether BK-specific labeling also can be detected in surface cells. In contrast to the crypt data, only a very faint staining may seem present in surface cells. Taken together, these results demonstrate that BK channels are localized in the luminal membrane of colonic crypts, whereas luminal membrane surface epithelial staining is not clearly visible.

View larger version (42K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Immunolocalization of the BK channel protein in mouse distal colon. (A) Overview of colonic mucosa from a WT (C) and a BK–/– (D) mouse. The upper side reflects the gut lumen. Several crypts are shown in their longitudinal cut of the cryosection. The green color indicates the tissue autofluorescence to display its morphology. Specificity of the antibody is demonstrated by the absolute absence of red staining in the BK–/– tissue. The enlarged cutouts (A and B) represent magnifications of the surface cell region showing only very faint labeling of surface cells for the BK channel. (E and F) Two enlarged views of a single colonic crypt. In contrast, distinct labeling is apparent in the luminal membrane domain of colonic crypts (white arrowheads). Note the strong labeling in the colonic smooth muscle layers (Tunica muscularis [TMu] and Lamina muscularis mucosae [LMM]). Also positively stained for BK channels and localized between the crypts are presumptive contractile elements in the Lamina propria (LP) splitting off from the LMM (C and E).

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Reduced K+ and increased Na+ excretion in feces from BK–/– mice. The amount of feces produced per day did not differ between both genotypes (WT 46.5 ± 2.3 mg/g body wt; BK–/– 49.3 ± 1.7 mg/g body wt). All data are means ± SEM; *P < 0.05.

View larger version (37K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Increased amiloride-sensitive Isc in distal colonic mucosa of BK–/– mice. (A) Two representative Ussing chamber recordings of the resting and the luminal amiloride-mediated Vte values in WT and BK–/– mice. Note the different Vte under resting conditions compared with Figure 1, in which amiloride was present at the mucosal side already from the beginning of the experiments. (B) Summary of amiloride-sensitive Isc in WT and BK–/– mouse distal colonic mucosa (n = 9/11). All data are means ± SEM; *P < 0.05.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

A study that was performed in rat proximal colon suggested that multiple Ca²⁺-activated K⁺ channels are involved in the K⁺ exit step from colonic mucosa (19). This study used carbachol-stimulated ⁸⁶Rb⁺ fluxes and the inhibitors IBTx (BK), clotrimazole (IK = SK4), and apamin (SK) and found that each drug was able to inhibit >50% of the stimulated flux. On the basis of these data, the authors suggested that all three Ca²⁺-activated K⁺ channels may be present in the luminal membrane of rat proximal colon (19). We therefore addressed the question of whether other Ca²⁺-activated K⁺ channels could be relevant for K⁺ secretion in mouse distal colon. In our newly generated SK4^–/– mice, we found that the resting Ba²⁺-sensitive and UTP-stimulated I_sc was identical to that of WT littermates. These results argue against an important role of SK4 channels for luminal K⁺ secretion in response to UTP-induced Ca²⁺ rises, a finding also supported by recent results from another group (23). The evidence obtained here from BK^–/– and SK4^–/– mice supports that the BK channel is the only Ca²⁺-activated K⁺ secretory channel in mouse distal colon.

Localization of BK Channels
Our data show that BK channels localize to the luminal membrane of colonic crypts. This is a prerequisite for a BK channel–mediated distal colonic K⁺ secretion, and, therefore, BK localization is in close agreement with its role for K⁺ secretion. The immunolabeling results in Figure 3E leave no doubt that BK channels are associated with the luminal membrane domain in crypt epithelial cells. This finding agrees closely with previous studies in rats that indicated that colonic K⁺ secretion occurs primarily in colonic crypts and not in surface cells. It was shown that K⁺ secretion occurs independently from electrogenic Na⁺ absorption, i.e., is not amiloride sensitive (24), and that the aldosterone-stimulated K⁺ secretion precedes the increase of electrogenic Na⁺ absorption (25). Our study provides no convincing evidence for surface cell expression of BK channels. Close inspection, however, cannot exclude a low-level BK expression also in surface cells (Figure 3, A and B). This contrasts with two studies that suggested that BK channels are expressed in colonic surface cells. However, noteworthy is that both studies were performed in "high aldosterone" states, i.e., in rabbits (26) and in rats that were on a high-K⁺ diet (12). It therefore is speculated that BK channel protein and luminal expression in surface cells are upregulated in states of high aldosterone.

In crypt epithelium, BK channels localize to the same area as the secretory luminal Cl^– channel CFTR (27,28). During secretory diarrhea, an elevation of cytosolic cAMP leads to the activation of CFTR in the enterocyte. Because BK channels are localized in the same membrane, CFTR activation will also promote K⁺ loss by augmenting the driving force for K⁺ exit. In addition, a CFTR-dependent depolarization of the luminal membrane is likely to favor the opening of the voltage-dependent BK channel. It therefore is suggested that the well-documented cAMP-stimulated colonic K⁺ secretion (3,29) may occur via BK channels in crypt epithelial cell indirectly via the activation of CFTR. Further experiments are needed to clarify this specific issue.

Altered Fecal K⁺ and Na⁺ Content
The electrolyte data from mouse feces showed a significantly reduced daily colonic K⁺ excretion in BK^–/– mice, and this is expected for a mouse with a defective colonic K⁺ secretion mechanism. In addition, the daily colonic Na⁺ excretion in BK^–/– mice was significantly increased. The reason for this intestinal Na⁺ wasting is currently not fully understood, but several issues need mentioning. The measurement of electrogenic Na⁺ absorption showed an upregulation of the amiloride-sensitive I_sc; therefore, BK^–/– mice display an increased Na⁺ absorption. This must indicate that factors that increase the delivery of luminal Na⁺ must overrule the increased Na⁺ absorption in BK^–/– mice. It is interesting that the resting I_sc in the presence of luminal amiloride is significantly larger in BK^–/– mice. In the absence of K⁺ secretion and Na⁺ absorption, an increased I_sc value is a strong indicator for an elevated NaCl secretion (30). It therefore is suggested that this increased NaCl secretion is the cause of colonic Na⁺ wasting in BK^–/– mice, but other undiscovered factors may be involved, such as a higher delivery of Na⁺ from more proximal parts of the intestine.

	Conclusion

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 
In distal colonic epithelia, the lack of luminal BK channels leads to a defective colonic K⁺ secretion during resting and agonist-stimulated Ca²⁺-dependent secretion. The key finding of this study is that luminal BK channels are the only functionally relevant K⁺ channels for Ca²⁺-activated colonic K⁺ secretion. By revealing the identity of the luminal K⁺ conductance in the distal colonic epithelium, our data significantly extend the mechanistic understanding of colonic K⁺ secretion. An important finding of this study is that BK channels significantly contribute to the determination of the V_te and therefore to the resting membrane potential. This is in agreement with findings in cerebellar Purkinje cells (13) and vascular smooth muscle cells (22), where altered membrane voltage in cells from BK^–/– mice contributes to altered cellular functions. It might be speculated that BK channel–mediated colonic K⁺ loss contributes to the clinically important and life-threatening K⁺ wasting during severe diarrhea (3,21).

	Acknowledgments

 
This study was supported by the Danish Medical Research Council and Deutsche Forschungsgemeinschaft.

We thank Dan-Yang Huang and Dr. Volker Vallon for assisting us in flame photometry analysis; Isolde Breuning for excellent technical assistance; and Deutsche Forschungsgemeinschaft, Danish Medical Research Council, the Novo Nordisk Foundation, and the Portuguese Foundation for Science and Technology for financial support.

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

M.S. and J.E.M. contributed equally to this work.

	References

Top Abstract Introduction Materials and Methods Results Discussion Conclusion References

 

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