Parallel intermediate conductance K+ and Cl− channel activity mediates electroneutral K+ exit across basolateral membranes in rat distal colon
Abstract
Transepithelial K+ absorption requires apical K+ uptake and basolateral K+ exit. In the colon, apical H+-K+-ATPase mediates cellular K+ uptake, and it has been suggested that electroneutral basolateral K+ exit reflects K+-Cl− cotransporter-1 (KCC1) operating in parallel with K+ and Cl− channels. The present study was designed to identify basolateral transporter(s) responsible for K+ exit in rat distal colon. Active K+ absorption was determined by measuring 86Rb+ (K+ surrogate) fluxes across colonic epithelia under voltage-clamp conditions. With zero Cl− in the mucosal solution, net K+ absorption was reduced by 38%, indicating that K+ absorption was partially Cl−-dependent. Serosal addition of DIOA (KCC1 inhibitor) or Ba2+ (nonspecific K+ channel blocker) inhibited net K+ absorption by 21% or 61%, respectively, suggesting that both KCC1 and K+ channels contribute to basolateral K+ exit. Clotrimazole and TRAM34 (IK channel blockers) added serosally inhibited net K+ absorption, pointing to the involvement of IK channels in basolateral K+ exit. GaTx2 (CLC2 blocker) added serosally also inhibited net K+ absorption, suggesting that CLC2-mediated Cl− exit accompanies IK channel-mediated K+ exit across the basolateral membrane. Net K+ absorption was not inhibited by serosal addition of either IbTX (BK channel blocker), apamin (SK channel blocker), chromanol 293B (KV7 channel blocker), or CFTRinh172 (CFTR blocker). Immunofluorescence studies confirmed basolateral membrane colocalization of CLC2-like proteins and Na+-K+-ATPase α-subunits. We conclude that active K+ absorption in rat distal colon involves electroneutral basolateral K+ exit, which may reflect IK and CLC2 channels operating in parallel.
NEW & NOTEWORTHY This study demonstrates that during active electroneutral K+ absorption in rat distal colon, K+ exit across the basolateral membrane mainly reflects intermediate conductance K+ channels operating in conjunction with chloride channel 2, with a smaller, but significant, contribution from K+-Cl− cotransporter-1 (KCC1) activity.
INTRODUCTION
Active K+ absorption occurs in mammalian distal colon (17, 22, 44) and involves apical K+ uptake and basolateral K+ exit mechanisms. Previous studies performed in vitro under voltage-clamp conditions showed that active K+ absorption is electroneutral, Na+-independent, and partially Cl−-dependent, which suggested that an apical K+-H+ exchange process mediates apical K+ uptake in rat distal colon (17). Studies with rat ileal membrane vesicles demonstrated proton gradient-driven K+ uptake, consistent with a K+-H+ exchange process (5). On the basis of the observation that apically applied ortho-VO4 (a P-type ATPase inhibitor) and ouabain (a Na+-K+-ATPase inhibitor) inhibited active K+ absorption, it was proposed that H+-K+-ATPase mediates apical K+ uptake (22, 45). Further characterization of H+-K+-ATPase, involving molecular studies to clone and characterize colonic H+-K+-ATPase α-(HKCα) and β-subunits (HKCβ), established that apical K+ uptake reflects H+-K+-ATPase activity (11–13, 29). However, the mechanism involved in the basolateral K+ exit step of electroneutral K+ absorption is not known.
Since electroneutral K+ absorption is partially Cl−-dependent, both the basolateral K+-Cl− cotransporter (KCC) and basolateral K+ and Cl− channels operating in parallel have been proposed as mechanisms underlying electroneutral basolateral K+ exit during active K+ absorption (17, 22, 42). Although changes in KCC mRNA abundance and protein expression have been described in rat distal colon during dietary K+ depletion, there is a lack of functional evidence linking KCC to the basolateral K+ exit step (42). Inhibition of K+ absorption in rabbit distal colon by serosally applied Ba2+ ions (a nonspecific K+ channel blocker) has been interpreted as evidence that K+ channels are involved in basolateral K+ exit (22). However, the molecular identities of the K+ channel(s) and possible parallel anion channel that might be involved in basolateral K+ exit are unknown. The present study provides evidence that both KCC, and intermediate conductance K+ (IK) and chloride channels (CLC2) operating in concert, mediate electroneutral basolateral K+ exit during active K+ absorption in rat distal colon.
MATERIALS AND METHODS
Animals.
Nonfasting normal male and female Sprague-Dawley rats (200–225 g) were given normal food and tap water ad libitum. All experimental protocols used in this study were approved by the West Virginia University Institutional Animal Care and Use Committee.
Ussing chamber studies.
86Rb+ (K+ surrogate; Perkin Elmer, Billerica, MA) fluxes and Cl− secretion (indicated by changes in short circuit current, Isc) were measured in stripped epithelial layers from male rat distal colon mounted under voltage-clamp condition, as previously described (4, 41). In brief, colon excised from deeply anesthetized rats was flushed with ice-cold Ringer solution (in mM: 115 NaCl, 25 NaHCO3, 2.4 K2HPO4, 0.4 KH2PO4, 1.2 CaCl2, 1.2 MgCl2, and 10 glucose; pH 7.4). Epithelial layers were gently separated from the serosal muscular layers after opening the colon along the mesenteric border. Two distal (1 cm proximal to rectum) segments obtained from each animal were mounted in slider inserts (aperture 1.1 cm2), and both sides were bathed with equal volumes (5 ml) of Ringer solution. Bathing solutions gassed continuously with 5% CO2-95% O2 were maintained at 37°C. Tissue conductance (G) and short-circuit current (Isc) were recorded every 20 s using an automated multichannel voltage/current-clamp instrument (Physiological Instruments, San Diego, CA). For flux studies, 1 µCi 86RbCl was added to either the mucosal or the serosal bath solution. After 45-min equilibration, mucosal-to-serosal (m-s) and serosal-to-mucosal (s-m) 86Rb+ fluxes were measured in different tissues under voltage-clamp conditions. Net (i.e., active) K+ fluxes were calculated from the difference between m-s and s-m fluxes across tissue pairs that were matched on the basis of differences in basal conductance of <10%. 86Rb+ fluxes were also measured, while the mucosal side was bathed with Cl−-free Ringer solution (115 mM Na+ gluconate, 1.2 mM CaSO4, and 1.2 mM MgSO4 replacing 115 mM NaCl, 1.2 mM CaCl2, and 1.2 mM MgCl2 present in regular Ringer solution).
In additional experiments, following basal measurements, 86Rb+ fluxes were also measured after adding one of the following to the serosal solution: 100 µM serosal 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazole-2-one (DC-EBIO), 100 μM [(dihydronindenyl)oxy] alkanoic acid (DIOA), 3 mM BaCl2, 3 μM clotrimazole (CLT), 3 µM TRAM-34, 100 nM iberiotoxin (IbTX), 10 nM apamin (APA), 30 µM chromanol 293B, 1 mM 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS), 1 mM 5-nitro-2-(3-phenylpropyl-amino) benzoic acid (NPPB), or 250 pM GaTx2. Since the addition of these K+ and Cl− channel blockers did not significantly affect PD or G, these results have not been presented. All of the flux studies were performed in the serosal presence of indomethacin (5 μM) and tetrodotoxin (1 μM) that inhibit prostaglandin synthesis and potential enteric neuronal activity that influence electrolyte movements, respectively.
Immunofluorescence studies.
Male and female rat distal colon opened longitudinally along the mesenteric border was fixed with 4% formaldehyde for 30 min at room temperature. After rinsing with PBS, tissue samples mounted in OCT embedding compound were frozen at −80°C. Sections (10 μm) were placed on charged slides and exposed to 5% goat serum for 2 h. After the sections were rinsed with PBS containing 0.1% Triton X-100 and 0.1% toluene (PBS-Tween) for 10 min, they were incubated overnight with either a primary antibody to Na+-K+-ATPase α-subunit (NaKα1; 1:1,000 dilution, kindly provided by Dr. Michael Kashgarian, Yale School of Medicine, New Haven, CT) (28, 46), or anti-CLC2 antibody (1:500 dilution; cat. no. ACL002; Alomone Laboratories, Jerusalem, Israel) diluted with 5% goat serum. After three 15-min washes with PBST, the sections were incubated with species-specific secondary antibody (1:1,000 dilution; NaKα1, goat anti-mouse IgG Alexa Fluor 488; FPN1, goat anti-rabbit IgG Texas Red; Thermo Fisher Scientific, Waltham, MA), together with Hoechst dye at room temperature for 2 h. The sections were rinsed with PBS and mounted with mounting media. Images were captured by laser confocal microscopy (Zeiss 710, Oberkochen, Germany) using excitation and emission wavelengths of 488 and 550 nm, respectively, and were processed using ImageJ.
The NaKα1 monoclonal antibody has been used extensively and has been shown to recognize newly synthesized Na+-K+-ATPase α-subunit that binds the N-azidobenzoyl derivative of ouabain used as substrate during immunoprecipitation in Madin-Darby canine kidney (MDCK) cells (9). CLC2 antibody was validated by demonstrating the absence basolateral protein in CLC2 knockout mouse (34). Similar cell and membrane-specific localization of CLC2 and NaKα proteins were detected in both male and female rat distal colon. However, to be consistent with the flux data, only data with male rat distal colon are presented.
Statistics.
Results are presented as means ± SE. Statistical analyses were performed via GraphPad Prism 8.0 software using one-way ANOVA with Tukey’s post hoc test. P < 0.05 was considered statistically significant.
RESULTS
Effect of mucosal C− on electroneutral K+ absorption.
Initial studies were designed to demonstrate the Cl− dependency of active K+ absorption in rat distal colon. In these experiments, K+ fluxes (86Rb+; K+ surrogate) were measured either in the presence or absence of mucosal Cl− ions. Similar to previous studies (15), active K+ absorption (i.e., net K+ absorption) in the absence of Cl− was only 61% of that in the presence of Cl− (0.38 ± 0.03 vs. 0.62 ± 0.07 μEq·h−1·cm−2, respectively; P < 0.05) (Table 1). Decreased K+ absorption in the absence of mucosal Cl− reflected a decrease in the m-s flux without a change in the s-m flux. Mucosal addition of 1 mM ortho-VO4 (a P-type ATPase inhibitor) completely inhibited active K+ absorption by inhibiting the m-s flux, but not the s-m flux, irrespective of the presence of Cl− in the mucosal solution. Despite these decreases in m-s K+ fluxes, there were no significant changes in Isc and G (Table 1). These results confirm those of previous studies, and demonstrate that active K+ absorption in rat distal colon is partially Cl− dependent and that apical H+-K+-ATPase mediates the apical K+ uptake step of K+ absorption both in the presence and in the absence of mucosal Cl−. Since the aim of this study was to determine whether serosal KCC and/or K+ channels mediate electroneutral basolateral K+ exit, all further studies to characterize this aspect of active K+ absorption were done with regular (Cl− containing) Ringer solution bathing the mucosal and serosal sides of the tissues.
| Buffer | K+ fluxes, μEq·h−1·cm−2 | G, mS/cm2 | Isc, μA/cm2 | ||
|---|---|---|---|---|---|
| m-s | s-m | net | |||
| Regular Ringer | 0.93 ± 0.08 | 0.31 ± 0.04 | 0.62 ± 0.07 | 9.1 ± 0.7 | 47.6 ± 2.4 |
| Regular Ringer + VO4 | 0.28 ± 0.02* | 0.32 ± 0.04 | −0.04 ± 0.01† | 8.3 ± 0.6 | 42.4 ± 1.5 |
| Cl−-free Ringer | 0.72 ± 0.07‡ | 0.34 ± 0.05 | 0.38 ± 0.03$ | 9.3 ± 2.4 | 50.1 ± 4.6 |
| Cl−-free Ringer + VO4 | 0.33 ± 0.03** | 0.28 ± 0.05 | 0.05 ± 0.02** | 8.7 ± 0.8 | 49.6 ± 2.8 |
Role of serosal K+ transporters on electroneutral K+ absorption.
To identify whether KCC and/or K+ channels mediate basolateral K+ exit, active K+ absorption was measured when either 100 μM DIOA (a KCC inhibitor) or 3 mM BaCl2 (a nonspecific K+ channel blocker) were present in the serosal bathing solution. Serosal addition of DIOA (basal vs. DIOA: 0.57 ± 0.03 vs. 0.45 ± 0.02 μEq·h−1·cm−2; P < 0.05) and BaCl2 (basal vs BaCl2: 0.57 ± 0.03 vs. 0.22 ± 0.04 μEq·h−1·cm−2; P < 0.001) inhibited active K+ absorption by 21% and 61%, respectively (Fig. 1, A and B). Simultaneous serosal addition of both DIOA and BaCl2 almost completely inhibited active K+ absorption (0.06 ± 0.04 μEq·h−1·cm−2). These observations indicate that both KCC and K+ channels mediate basolateral K+ exit during active K+ absorption. They also suggest that K+ channel-mediated basolateral K+ exit is three-fold higher than that mediated by KCC.
Fig. 1.Effect of serosal K-Cl cotransporter-1 inhibitor and K+ channel blocker on short-circuit current and active K+ absorption in rat distal colon. After an equilibration period of 45 min, short-circuit current (Isc) and active K+ absorption across colonic epithelial sheets, bathed on both sides with regular Ringer solution, were determined under voltage-clamp conditions, as described in materials and methods. A: active K+ absorption was calculated as the difference between serosal (m-s) and serosal to mucosal (s-m) fluxes measured in the absence (Control), or presence of DIOA (KCC inhibitor) alone, or a combination of DIOA and BaCl2 (nonspecific K serosal to mucosal (s-m) fluxes measured in the absence (Control), or presence of BaCl2 alone, + channel blocker). B: active K+ absorption was calculated from mucosal to serosal (m-s) and or a combination of BaCl2 and DIOA. Results are presented as means ± SE of net fluxes of three different experiments with three different rat distal colons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. *P < 0.05 compared with control; **P < 0.05 compared with control. C: representative experiment showing continuous Isc measurements first in the absence of DIOA, then in the presence of DIOA alone, then in the presence of a combination of DIOA and BaCl2. D: representative experiment showing continuous Isc measurements first in the absence of BaCl2, then in the presence of BaCl2 alone, then in the presence of a combination of BaCl2 and DIOA. E and F: mean Isc values derived from experiments such as those shown in C and D, respectively. Results representing means ± SE of six different tissues from three different rat distal colon.
Serosal K+ channels provide the driving force for Cl−/water secretion in intestine, including colon (15, 19, 36). Therefore, in parallel to K+ fluxes, Isc was also measured to determine whether inhibition of serosal K+ channels would affect electrogenic colonic Cl− secretion. As shown in Fig. 4, C–F, serosal addition of BaCl2 did not affect Isc. Similar to BaCl2, serosal addition of either DIOA or the simultaneous presence of BaCl2 and DIOA also did not affect Isc. These observations suggest that serosal K+ channels may not be critical in providing the driving force for basal colonic Cl−/water secretion.
Identification of specific K+ channel(s) responsible for electroneutral K+ absorption.
Since K+ channel-mediated serosal K+ exit appeared to be substantially higher than that mediated by KCC (Fig. 1, A and B), further studies were done to identify the specific type(s) of K+ channel, and the anion channel, that maintains electroneutrality by mediating Cl− exit. Therefore, we determined the effects on active K+ absorption of a variety of specific K+ channel inhibitors added to the serosal bathing solution. As shown in Fig. 2, serosal addition of 1 μM CLT or 1 μM TRAM34 [both intermediate conductance K+ (IK) channel inhibitors] virtually abolished active K+ absorption, whereas 100 nM IbTX [a large-conductance K+ (BK) channel inhibitor], 10 nM APA [a small-conductance K+ (SK) channel inhibitor], or chromanol 10 μM 293B [a cAMP-activated K+ (KCNQ1/KV7) channel inhibitor] had no effect. These observations indicate that IK channels mediate the basolateral K+ exit during active K+ absorption.
Fig. 2.Effect of serosal K+ channel blockers on active K+ absorption in rat distal colon. Active K+ absorption was determined under control conditions (see legend to Fig. 1), and also when 100 μM IbTX, 3 μM CLT, 3 μM TRAM-34 (M-34), 100 μM APA, or 10 μM chromanol 293B (293B) were present in the serosal solution. Results represent means ± SE of net fluxes derived from three different experiments using three different rat distal colons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. *P < 0.001 compared with control.
Role of serosal IK channel activation on electroneutral K+ absorption.
Further studies were done with serosally applied DC-EBIO (an IK channel opener) to determine whether activation of basolateral IK channels potentiated K+ absorption. As shown in Fig. 3A, serosal DC-EBIO produced a small, but statistically insignificant increase in K+ absorption (basal vs. DC-EBIO: 0.44 ± 0.03 vs. 0.51 ± 0.07 μEq·h−1·cm−2; not significant). However, although active K+ absorption was not altered significantly, serosal DC-EBIO markedly stimulated anion secretion, Isc increasing from a basal value of 45.6 ± 6.2 μA·h−1·cm−2 to 212.6 ± 18.6 μA/h.cm2; P < 0.001) (Fig. 3B). Since DC-EBIO-stimulated anion secretion is associated with increased serosal Na-K-2Cl cotransporter (NKCC) activity, it is possible that activated IK channels may preferentially have maintained the concentration of intracellular K+, which maintained a cell membrane potential that sustained the driving force for continuous Cl− secretion. Thus, the role of DC-EBIO-activated IK channel activity in active K+ absorption was examined using bumetanide (an NKCC inhibitor). Serosal addition of bumetanide in the presence of DC-EBIO not only increased K+ absorption (Fig. 3A), but also inhibited DC-EBIO-induced anion secretion (Fig. 3B). These observations indicate that under basal conditions, IK channels mediate basolateral K+ exit during active K+ absorption and maintain membrane potential.
Fig. 3.Effect of intermediate conductance K+ (IK) channel opener DC-EBIO on active K+ absorption and Cl− secretion in rat distal colon. A: active K+ absorption was determined under control conditions (see Fig. 1) and also when 100 μM DC-EBIO alone or 100 μM DC-EBIO + 10 µM bumetanide were present in the serosal solution. B: during the experiments described in A, Cl− secretion was evaluated by changes in Isc. Results represent means ± SE of net K+ fluxes, and Isc was derived from three different experiments using three different rat distal colons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. $P < 0.05, compared with control; *P < 0.001, compared with control; **P < 0.001, compared with DC-EBIO alone.
Identification of the role of serosal Cl− channels on electroneutral K+ absorption.
Since K+ absorption is electroneutral and partially Cl−-dependent, it follows that to maintain electroneutrality, IK channel-mediated basolateral K+ exit should be accompanied by electrogenic Cl− exit through anion channel(s). Therefore, we studied the effect of anion channel blockers on the active K+ absorption. As shown in Fig. 4, serosal addition of the nonspecific anion channel blocker NPPB, or the specific CLC2 channel blocker GaTx2, both significantly inhibited active K+ absorption. By contrast, K+ absorption was not inhibited either by 1 μM MONNA (a blocker of a transmembrane protein known as anoctamin-1 or TMEM16A, which appears to function as a non-CLC2 Ca2+-activated channel), or 10 μM CFTRinh172 (a CFTR Cl− channel blocker), when added to the serosal bath. Net K+ absorption was also not affected by the serosal addition of the anion exchange inhibitor DIDS (1 mM). These observations indicate that CLC2 channel-mediated Cl− exit occurs in parallel with IK channel-mediated K+ exit across the basolateral membrane.
Fig. 4.Effect of anion channel blockers on active K+ absorption in rat distal colon. Active K+ absorption was determined under control conditions (see Fig. 1), and also when either 1 mM DIDS, 1 mM NPPB, 10 μM CFTRinh-175, 1 μM MONNA, or GaTx2 were present in the serosal solution. Results represent means ± SE of net K+ fluxes derived from three different experiments using three different rat distal colons. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. *P < 0.001 compared with control.
Cell and membrane-specific localization of CLC2 channels in rat distal colon.
Immunofluorescence studies were performed to establish that CLC2-like protein was present in the basolateral membranes of rat distal colonic epithelium. Immunolocalization of Na+-K+-ATPase α-subunit (NaKα) was also performed to provide a marker of the basolateral membrane (29). As shown in Fig. 5A, basolateral localization of CLC2-like protein was seen predominantly in surface cells, but far more patchily in crypt cells. By contrast, and similar to our earlier observations, NaKα-specific protein was localized to basolateral membranes of both surface and crypt cells (Fig. 5, B and E) (29). The superimposed immunofluorescence image indicated that both CLC2 and NaKα-specific proteins colocalized to the lateral and basement membranes of rat distal colon (Fig. 5, D and F). Our finding that CLC2 localized primarily to the basolateral membranes of surface cells is consistent with previous results demonstrating CLC2-like protein expression and a CLC2-like Cl− conductance in surface cells of rat distal colon (25, 30). Our results, therefore, suggest that CLC2-mediated Cl− exit may offset the IK channel-mediated K+ exit generated negative membrane potential, thereby maintaining the driving force for electroneutral K+ exit at the basolateral membrane.
Fig. 5.Localization of CLC2- and Na+-K+-ATPase α-subunit (NaKα)-like proteins in rat distal colon. A and A’: CLC2-like proteins (green) localized predominantly in surface cells (arrows), while minimally expressed in crypt regions (asterisks). B and B’: NaKα-like proteins (red) localized to both surface (arrows) and crypt (asterisks) cells. C and C’: nuclei (blue) stained with Hoechst 33342. D: composite of A, B, and C. D’: composite of A’, B’, and C’. Higher magnification of the boxed region from D and D’ presented in E and E’, respectively. E: CLC2 and NaKα colocalized (orange) in both lateral (arrows) and basement (arrowheads) membranes of surface cells. E’: in crypt cells, NaKα (arrow) and CLC2 (arrowhead) localized in the basolateral membranes of crypts. Identical results obtained in different specimens obtained from three different experiments with three different male and female (not shown) rat distal colons.
DISCUSSION
Mammalian colon has an important role in maintaining body K+ homeostasis because it is capable of active K+ absorption and active K+ secretion, both of which readily adapt to changes in dietary K+ intake (36). Active K+ secretion involves transporters that mediate basolateral K+ uptake and apical K+ exit, while active K+ absorption involves transporters mediating apical K+ uptake and basolateral K+ exit. Basolateral NKCC and apical K+ (BK) channels are known to act in concert to regulate active colonic K+ secretion (36, 43, 49). Electrophysiological and molecular studies have established that H+-K+-ATPase mediates electroneutral apical K+ uptake (6), while both KCC1 and Ba2+-sensitive K+ channel(s) mediate basolateral K+ exit (22, 42). Although dietary K+ depletion alters KCC1 mRNA abundance and protein expression (42), there has been a lack of functional evidence for KCC1-mediated basolateral K+ exit. In addition, while inhibition of active K+ absorption by the serosal addition of Ba2+ ions points to the involvement of K+ channels in basolateral K+ exit, the nature of the basolateral anion channel(s) operating in concert with K+ channels to maintain cell membrane potential is not known (22). In the present study, we show that during electroneutral K+ absorption in rat distal colon, basolateral IK and CLC2 channels working together are largely responsible for basolateral K+ exit, with an additional contribution from DIOA-sensitive KCC1.
Functional and molecular studies have localized IK channels to the basolateral membranes of colonic epithelia (1, 2, 8, 26). In general, basolateral K+ exit mediated by these IK channels maintains the membrane potential that provides the driving force for agonist (cAMP/Ca2+)-induced Cl− secretion (16, 19, 23, 32, 36). Maintenance of the membrane potential by these IK channels also appears to be essential for Na+ channel (ENaC)-mediated Na+ absorption (1), and downregulation of IK channels in ulcerative colitis (UC) may contribute to the decreases in Na+ and water absorption, leading to diarrhea in patients with this disease (1). These basolateral IK channels also regulate epithelial volume and cell migration and proliferation (7, 47). The present study demonstrates for the first time that these IK channels also mediate basolateral K+ exit, which has a critical role in active K+ absorption in rat distal colon. This conclusion is supported by the following observations: 1) serosal addition of the nonspecific K+ channel blocker (Ba2+ ions) and IK channel-specific blockers (CLT and TRAM34) inhibit active K+ absorption (Figs. 1 and 2); and 2) previous studies showing that the maintenance of intracellular pH requires the coordinated regulation of apical H+-K+-ATPase and Ba2+-sensitive basolateral K+ channels in rat colonic crypts (24).
Since active K+ absorption in rat distal colon is electroneutral and partially dependent on the presence of mucosal (i.e., apical) Cl−, and basolateral IK channel-mediated K+ exit is electrogenic, it follows that basolateral K+ exit must be accompanied by electrogenic basolateral anion exit via Cl− and/or channel(s) to ensure electroneutrality of the K+ absorptive process. Several anion channels exist in the basolateral membrane of mammalian distal colon (10, 14, 33, 48). Electrophysiological studies have demonstrated volume-sensitive Cl− channels (25–30 pS) and DIDS-sensitive Cl− channels (20–90 pS) in the basolateral membranes of rat and mouse colonic crypts, respectively (14, 33). Functional and molecular studies have also identified CLC2 and BEST2 (bestrophin-2) in the basolateral membranes of guinea pig and mouse colonic epithelia, respectively (10, 48). BEST2 localized to goblet cells mediated transfer across basolateral membranes in mouse colon (48), while it has been suggested that basolateral CLC2 channels in absorptive surface cells, but not in secretory crypt cells, are involved in transepithelial Cl− absorption in guinea pig and mouse distal colon (10, 35). In keeping with these observations, we have shown that in rat distal colon, CLC2 protein is expressed in the basolateral membranes of surface cells, but not crypt cells (Fig. 5).
The fact that CLC2 is localized to the basolateral membranes of absorptive surface cells, but not secretory crypt cells, it has been suggested that CLC2 may be involved in transepithelial NaCl absorption (10). However, the present study suggests that CLC2-mediated basolateral Cl− exit provides the driving force for IK channel-mediated basolateral K+ exit during active K+ absorption in rat distal colon. This conclusion is supported by the following observations: 1) active K+ absorption was partially dependent on mucosal Cl− (Table 1), 2) the specific CLC2 channel blocker GaTx2 significantly inhibited active K+ absorption (Fig. 4), and 3) CLC2 protein localized to the basolateral membranes of absorptive surface cells, but not secretory crypt cells (Fig. 5). It is conceivable that CLC2-mediated Cl− exit, which is activated by hyperpolarization (10), offsets the negative membrane potential created by IK channel-mediated K+ exit across basolateral membranes, and vice versa, thereby ensuring electroneutrality of the basolateral K+ exit step of distal colonic K+ absorption.
It seems likely that basolateral IK and CLC2 channels operating in conjunction with apical H+-K+-ATPase account for colonic K+ absorption (Fig. 6). This notion is supported by previous results, indicating a relationship between basolateral K+ channel activity and apical H+-K+-ATPase in the regulation of intracellular pH (24). Although CLC2 is present only in surface cells (Fig. 5) (35), IK channels are present in the basolateral membranes of both surface and crypt cells (2, 20). H+-K+-ATPase is also present in the apical membranes of both surface and crypt cells (6, 13, 37). However, H+-K+-ATPase in surface cells is ouabain-insensitive, whereas that in crypt cells is ouabain-sensitive (37). Similarly, although basolateral CLC2 channels are present only in surface cells (Fig. 5) (35), other Cl− channels exist in the basolateral membranes of crypts cells in rat colon (14). Although the nature of the Cl− dependency of apical H+-K+-ATPase-mediated K+ uptake is unknown, it seems likely that the combination of basolateral IK and CLC2 channels, and other as yet unidentified Cl− channels, together with ouabain-insensitive and ouabain-sensitive apical H+-K+-ATPase, provide mechanisms for electroneutral K+ absorption in colonic surface and crypt cells, respectively. In summary, added to the fact that H+-K+-ATPase mediates apical K+ uptake (6), our results strongly suggest that both basolateral KCC1 and IK channels contribute to electroneutral basolateral K+ exit during active K+ absorption in rat distal colon (Fig. 6).
Fig. 6.Cellular model of mucosal and serosal transporters involved in transepithelial electroneutral K+ absorption in mammalian colon. H+-K+-ATPase mediates apical K+ uptake (5). Both K+-Cl− cotransport 1 (KCC1) and concerted action of intermediate conductance K+ (IK) channel and chloride channel 2 (CLC2) contribute to electroneutral basolateral K+ exit. “AE?” indicates that the molecular identity of the apical anion exchanger that regulates Cl−-dependent electroneutral K+ absorption is not known.
H+-K+-ATPase-mediated apical K+ uptake is the initial step in active K+ absorption in rat distal colon (6), and the present study shows that the absorptive process also involves electroneutral K+ exit mediated by basolateral KCC1 and IK channels operating in parallel. If we speculate that a similar mechanism for electroneutral active K+ absorption exists in human colon, what might be its relevance to altered colonic K+ transport in disease? For example, studies in a rat model of chronic renal insufficiency (3) and in patients with end-stage renal disease (ESRD) (38, 39) showed that the capacity of the colon for K+ secretion was enhanced to compensate for the loss of renal K+ excretion. The increase in net K+ secretion seen in ESRD patients compared with control patients was inhibited by apical Ba2+ ions, in keeping with enhanced expression of apical BK (KCNMA1) channels seen in the ESRD patients (31). In this clinical scenario, we might intuitively expect no change or downregulation of the putative K+ absorptive process. As a further example, some patients with severe extensive UC develop hypokalemia related, at least in part, to increased fecal K+ losses, which may reflect enhanced expression of apical BK channels reported along the entire colonic surface cell-crypt cell axis in UC patients, whereas BK channels were largely restricted to surface cells in control patients (40). Increased colonic K+ secretion, reflecting enhanced apical BK expression, has also been demonstrated in dextran sulfate-induced colitis in rat distal colon, which shares the same histopathologic changes as its human counterpart (27). In this particular example, we might anticipate downregulation of apical H+,K+-ATPase and, by inference, electroneutral K+ absorption, since decreased expression of apical amiloride-sensitive Na+ (ENaC) channel β- and γ-subunits (21), basolateral Na+-K+-ATPase α1- and β1-isoform proteins (21), and basolateral IK channels (1), occur in patients with active UC. Regarding possible upregulation of electroneutral K+ absorption, chronic dietary K+ depletion enhances active K+ absorption in rat distal colon (18), but a similar degree of dietary K+ depletion would be impractical in humans. Nevertheless, patients overtreated with ‘K+-losing’ diuretics (e.g., furosemide and bumetanide) may develop severe hypokalemia and become profoundly K+ depleted, but it remains to be seen whether active K+ absorption in the colon is upregulated as a compensatory K+-conserving mechanism. Increased active K+ absorption does occur in rat distal colon during secondary hyperaldosteronism induced by chronic dietary Na+ depletion, but this is seen only after inhibiting the dominant electrogenic K+ secretory process (45). However, it is unclear whether active K+ absorption is upregulated in the colon of patients with Conn’s syndrome (primary hyperaldosteronism), or in patients with postural hypotension related to autonomic neuropathy being treated with fludrocortisone, a synthetic mineralocorticoid agonist.
GRANTS
This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases Grants DK104791 and DK112085.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
S.D.C. and V.M.R. conceived and designed research; S.R., K.N., A.J.N., S.D.C., and V.M.R. performed experiments; K.N., S.D.C., and V.M.R. prepared figures; A.J.N., S.D.C., G.I.S., and V.M.R. interpreted results of experiments; K.M.H., G.I.S., and V.M.R. analyzed data; G.I.S. and V.M.R. drafted manuscript; A.J.N., S.D.C., K.M.H., G.I.S., and V.M.R. edited and revised manuscript; S.R., K.N., A.J.N., S.D.C., K.M.H., G.I.S., and V.M.R. approved final version of manuscript.
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