Aldosterone induces active K⁺ secretion by enhancing mucosal expression of Kcnn4c and Kcnma1 channels in rat distal colon

Abstract

Although both Kcnn4c and Kcnma1 channels are present on colonic mucosal membranes, only Kcnma1 has been suggested to mediate K⁺ secretion in the colon. Therefore, studies were initiated to investigate the relative roles of Kcnn4c and Kcnma1 in mediating aldosterone (Na-free diet)-induced K⁺ secretion. Mucosal to serosal (m-s), serosal to mucosal (s-m), and net ⁸⁶Rb⁺ (K⁺ surrogate) fluxes as well as short circuit currents (I_sc; measure of net ion movement) were measured under voltage clamp condition in rat distal colon. Active K⁺ absorption, but not K⁺ secretion, is present in normal, while aldosterone induces active K⁺ secretion (1.04 ± 0.26 vs. −1.21 ± 0.15 μeq·h⁻¹·cm⁻²; P < 0.001) in rat distal colon. Mucosal VO₄ (a P-type ATPase inhibitor) inhibited the net K⁺ absorption in normal, while it significantly enhanced net K⁺ secretion in aldosterone animals. The aldosterone-induced K⁺ secretion was inhibited by the mucosal addition of 1) either Ba²⁺ (a nonspecific K⁺ channel blocker) or charybdotoxin (CTX; a common Kcnn4 and Kcnma1 channel blocker) by 89%; 2) tetraethyl ammonium (TEA) or iberiotoxin (IbTX; a Kcnma1 channel blocker) by 64%; and 3) TRAM-34 (a Kcnn4 channel blocker) by 29%. TRAM-34, but not TEA, in the presence of IbTX further significantly inhibited the aldosterone-induced K⁺ secretion. Thus the aldosterone-induced Ba²⁺/CTX-sensitive K⁺ secretion consists of IbTX/TEA-sensitive (Kcnma1) and IbTX/TEA-insensitive fractions. TRAM-34 inhibition of the IbTX-insensitive fraction is consistent with the aldosterone-induced K⁺ secretion being mediated partially via Kcnn4c. Western and quantitative PCR analyses indicated that aldosterone enhanced both Kcnn4c and Kcnma1α protein expression and mRNA abundance. In vitro exposure of isolated normal colonic mucosa to aldosterone also enhanced Kcnn4c and Kcnma1α mRNA levels, and this was prevented by exposure to actinomycin D (an RNA synthesis inhibitor). These observations indicate that aldosterone induces active K⁺ secretion by enhancing mucosal Kcnn4c and Kcnma1 expression at the transcriptional level.

the mammalian colon (large intestine) plays important roles in maintaining body K⁺ homeostasis, as it regulates both active K⁺ absorption and active K⁺ secretion under physiological as well as under pathophysiological conditions (4). Active K⁺ absorption is regulated by an ATP-dependent electroneutral H/K exchange (H-K-ATPase), while active K⁺ secretion is mediated via K⁺ channels localized on the mucosal membranes of the colon (4–5, 7, 10, 25). Aldosterone stimulates both active K⁺ absorption and active K⁺ secretion in rat distal colon (31–32). Increased H-K-ATPase α-subunit (HKα) mRNA abundance and HKα protein expression have been shown to be the mechanism for aldosterone-enhanced active K⁺ absorption in rat distal colon (28).

Active K⁺ secretion has been shown to be mediated by mucosal Ba²⁺ and tetraethyl ammonium (TEA)-sensitive K⁺ channels (32). In recent studies (16), iberiotoxin (IbTX) and clotrimazole (CLT)-sensitive K⁺ channels have been shown to mediate carbachol-enhanced K⁺ secretion and this is taken as evidence for the presence of large (known as BK, K_Ca1.1, and Kcnma1) and intermediate (known as IK, K_Ca3.1, and Kcnn4) conductance K⁺ channels on the mucosal membranes of rat proximal colon, respectively. Immunofluoresence studies (2, 11, 14, 26) have localized Kcnn4 and Kcnma1 proteins on the mucosal membranes of rat, guinea pig, and human colon. In recent studies (2) , we have cloned three distinct Kcnn4 splice variants (Kcnn4a, Kcnn4b, and Kcnn4c) and have shown that the Kcnn4b and Kcnn4c transcripts encode serosal and mucosal membrane Kcnn4 channels, respectively, in the rat distal colon. Further, we have shown that 5,6-dichloro-1-ethyl-1,3-dihydro-2H-benzimidazole-2-one (a Kcnn4 channel opener) induced K⁺ secretion in rat colon, indicating the presence of Kcnn4 channels on the mucosal membranes (22). Although both Kcnn4c and Kcnma1 channels are present on the mucosal membranes, only Kcnma1 has been suggested to be responsible for K⁺ secretion in human, mouse, and rat colon (6, 27, 29). Studies were therefore initiated to examine whether aldosterone-induced K⁺ secretion is mediated via Kcnn4c and/or Kcnma1 channels. Results presented in this study demonstrate that 1) active K⁺ absorption, but not active K⁺ secretion (in contrast to previous studies), is present in normal rat distal colon; 2) aldosterone induces active K⁺ secretion by enhancing mucosal expression of both Kcnn4c and Kcnma1 channel proteins; and 3) the aldosterone enhanced Kcnn4c and Kcnma1 channels are regulated at the transcriptional level.

MATERIALS AND METHODS

Animals.

Nonfasting normal male Sprague-Dawley rats (200–225 g) were given standard rat chow, while experimental aldosterone rats were produced by feeding Na-free rat chow (MP Biochemicals, Solon, OH) for 7 days. To establish that the Na-free diet produced hyperaldosteronism, serum aldosterone levels were measured using the aldosterone enzyme immunoassay (EIA) kit (Enzo Life Sciences). The Na-free diet enhanced the serum aldosterone levels by 15-fold (normal vs. Na-free diet: 286 ± 42 and 4,325 ± 393 pg/ml; P < 0.001; n = 5). These results are consistent with earlier observations (21). Animals fed with the Na-free diet are referred to as aldosterone animals. All animals were given food and water ad libitum. The 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; PerkinElmer, Billerica, MA) fluxes, short circuit currents (I_sc), and membrane conductance (G) were measured using the EasyMount Ussing chamber system (Physiological Instruments, San Diego, CA) in colonic mucosal layers mounted under voltage-clamp conditions, as previously described (18, 33). In brief, distal colons excised from euthanized rats were flushed with an ice cold saline. Mucosal layers were gently separated from serosal muscular layers of distal colon opened along the mesenteric border. Two distal segments (1 cm proximal to rectum) obtained from each animal were mounted on the snap wells (with an opening of 1.12 cm²). The snap wells placed in the sliders were inserted into the chambers that bath both sides of the mucosa with equal volumes of Tris·HCl Ringer solution (in mM: 115 NaCl, 25 NaHCO₃, 1.2 CaCl₂, 1.2 MgCl₂, 7.5 NMG-Cl, 10 glucose, and 5 Tris·HCl pH 7.4). In medium containing Ba²⁺, NMG-Cl was replaced with 5 mM BaCl₂. Since this study used Ba²⁺ to characterize K⁺ channels and because Ba²⁺ gets precipitated in phosphate-buffered Ringer, this study used Tris·HCl buffer in place of phosphate-buffered Ringer solution. The bathing solutions maintained at 37°C were gassed continuously with 5% CO₂-95% O₂. The I_sc and G were recorded every 20 s using an automated multichannel voltage/current-clamp instrument (Physiological Instruments). For flux studies, a trace of ⁸⁶RbCl was added to either mucosal (m) or serosal (s) bath solutions. After a 45-min equilibration period, serosal-to-mucosal (s-m) and mucosal-to-serosal (m-s) ⁸⁶Rb⁺ fluxes were measured under voltage-clamp conditions. Net fluxes were calculated from the difference between m-s and s-m fluxes in tissue pairs that were matched based on differences in basal G of < 10%. Positive and negative values represent active absorption and active secretion, respectively. Since the basal and the effect of inhibitors on ⁸⁶Rb⁺ fluxes were examined in the same tissue pair, three consecutive 15-min flux periods [basal (P-I), inhibitor equilibrium (P-II), and inhibitor effect (P-III)] were measured in each tissue pair.

The effect of inhibitors was examined on ⁸⁶Rb⁺ fluxes, I_sc, and G. In these studies, immediately following the basal flux period (P-I), inhibitors added to the mucosal bath were allowed to equilibrate during the second flux period (P-II). Fluxes measured at P-III were compared with P-I to determine the effect of inhibitors. The inhibitors used were as follows: 1 mM Na-orthovanadate (VO₄; a P-type ATPase inhibitor), 10 μM amiloride or 50 μM benzamil (epithelial Na⁺ channel blocker), 5 mM Ba²⁺ (a nonspecific K⁺ channel blocker), 1 mM TEA (a Kcnma1 channel blocker), 20 nM charybdotoxin (CTX; a Kcnn4 and Kcnma1 channel blocker), 50 μM TRAM-34 (a Kcnn4 channel blocker), 100 nM IbTX (a Kcnma1 channel blocker), and 100 μM of either cystic fibrosis transmembrane conductance regulator (CFTR)_inh-172 (a CFTR blocker), niflumic acid (a Ca²⁺-activated Cl⁻ channel blocker), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; a nonspecific anion channel blocker). When Ba²⁺ was used, the Na⁺ concentration was adjusted to maintain isotonicity. ⁸⁶Rb⁺ flux and I_sc values are presented as μeq·h⁻¹·cm⁻², while G values are presented as mSiemens (mS).

In vitro aldosterone induced I_sc.

Immediately following mounting the colonic mucosal layer in the Ussing chamber, aldosterone (15 nM) or aldosterone plus actinomycin D (2 μM) or vehicle (0.1% ethanol and 0.1% methanol) was added to the serosal bathing solution. I_sc was recorded under voltage clamp condition for up to 9 h. The aldosterone concentration (15 nM) was selected based on the plasma aldosterone level in Na⁺-free diet-fed animals. Total RNA extracted from mucosa exposed to bathing solution was used for real-time PCR (RT-PCR) analysis as described below.

Western blot.

Western blot analyses were performed on epithelial cell homogenates and mucosal membranes of rat distal colon using anti-Kcnn4-abc antibody (2), Kcnma1α (MaxiKα; Santa Cruz Biotechnology, Hercules, CA), and actin (Santa Cruz Biotechnology) antibodies, as described previously (2). Epithelial cells and apical membranes of distal colonic segments were isolated using the divalent chelation technique and differential centrifugation method of Stieger et al. (30), as described previously (23). The apical membrane purity was assessed by 10- to 12-fold enrichment of colonic H-K-ATPase activity compared with that of whole homogenate (normal: 11.6 ± 1.9 vs. 123.2 ± 3.4 nmol P_i liberated/mg protein·min; aldosterone: 24.4 ± 2.1 vs. 272.8 ± 6.6 nmol P_i liberated·mg protein⁻¹·min⁻¹). H-K-ATPase activity was measured by the method of Forbush (9), as described previously (7). Protein was assayed by the method of Lowry et al. (20). Epithelial cells and apical membranes suspended (1:20) in ice-cold lysis buffer (50 mM Tris pH 8.0, 0.5% SDS, 1 mM PMSF, 4 μg/ml pepstatin A, and one tablet of Complete protease inhibitor/50 ml solution; Roche Applied Science, Indianapolis, IN) were homogenized with a tight fit Teflon homogenizer. The homogenate was centrifuged for 15 min at 2,000 g, and 16-μl aliquots of supernatant were mixed with equal volumes of Laemmli buffer and heated at 95°C for 5 min. The heated aliquots were immediately placed in liquid nitrogen and stored at −80°C. Frozen samples were heated at 40°C for 1–2 min, and 10-μl samples (20 μg protein) resolved on 14% polyacrylamide gels were transferred onto nitrocellulose membranes. Blots were incubated with primary antibody [anti-Kcnn4-abc (1:3,000); Kcnma1α (1:400); and β-actin (1:2,500)] and then with horseradish peroxidase-conjugated goat anti-rabbit IgG, and immune complexes were detected using enhanced chemiluminescence (GE Healthcare, Buckinghamshire, UK). The stripped blots were processed with anti-actin antibody and horseradish peroxidase-conjugated donkey anti-mouse IgG. Arbitrary units of Kcnn4b, Kcnn4c, and Kcnma1 proteins normalized to actin were quantitated using Personal Densitometer SI (Molecular Dynamics).

Real-time-PCR.

Total-RNA purified using TRIzol (Invitrogen, Carlsbad, CA) from colonocytes of normal and aldosterone rat distal colon were used for RT-PCR analysis. RT-PCR was performed by a two-step method using total RNA. In brief, first-strand cDNA was synthesized from total-RNA using SuperScript III and random hexamers (Invitrogen). The first-strand cDNA template (5 ng) and TaqMan Universal PCR master mix along with Kcnn4b or Kcnn4c specific primers were used for RT-PCR according to the manufacturer's suggestions (Applied Biosystems, Foster City, CA). Custom designed primers used were as follows: Kcnn4b (sense 5′-GGCCACATAGCTGCCTGTTA and antisense 5′-TCCTTGAGCTCAGTCCTTCG-3′) primers (500 nM each) and 100-nM TaqMan probes 5′ FAM-TCAGGACCCACAGAAGAATCAGGCT-TAMRA 3′; and Kcnn4c (sense 5′-CTGGGTTGCAAGGAGGTC-3′ and antisense 5′-CATACCAGCAGCTCCAGCA-3′) primers (500 nM each) and 100-nM TaqMan probes 5′ FAM-CTGTTCATGACTGACAACGGGCTCC-TAMRA 3′. Rat BKα-subunit was amplified using Applied Biosystem's TaqMan Gene Expression Assay ID Rn01537142_ml, while rat β-actin amplified using TaqMan Gene Expression Assay ID Rn00667869_ml served as an endogenous control. Threshold cycle (Ct) values of the BKα-subunit, Kcnn4b, and Kcnn4c transcripts were normalized to the endogenous control. Differential expression of BKα and Kcnn4b/c was calculated according to the 2^−ΔΔCt method, as described previously (19).

Statistics.

Results presented represent means ± SE of six tissue pairs from 6 rats. Statistical analyses were performed using unpaired or paired Student's t-test or Bonferroni's one-way ANOVA post hoc test using Originpro 8.0 (OriginLab Corp, Northampton, MA). P < 0.05 was considered to be statistically significant.

Inhibitors and stock solutions.

Inhibitors and stock solutions were as follows: aldosterone (10 mM) in ethanol (Sigma-Aldrich, St. Louis, MO); actinomyicin D 10 mM in methanol (Thermo-Fisher); benzamil (20 mM) in methanol (Sigma-Aldrich); TRAM-34 (50 mM) in DMSO (Tocris Bioscience, Ellisville, MO); iberiotoxin (100 μM) in Ringer solution (Sigma-Aldrich); charybdotoxin (20 μM) in Ringer solution (Tocris); and CFTR_inh-172 (Tocris), niflumic (Calbiochem), and NPPB (100 mM) in DMSO (Tocris). All other molecular grade chemicals used were purchased from Sigma-Aldrich.

RESULTS

Since basal and experimental fluxes were examined in the same tissue, initial ⁸⁶Rb⁺ (K⁺ surrogate) fluxes and associated electrical parameters were measured for three consecutive 15-min periods (P-I, P-II, and P-III) in both normal and aldosterone rat distal colon. As presented in Table 1, m-s, s-m, and net K⁺ fluxes, and electrical parameters were not significantly altered between the three periods in normal or aldosterone rat colon. Under basal conditions, net K⁺ absorption and net K⁺ secretion are present in normal and aldosterone rat distal colon, respectively (Table 1). Net K⁺ secretion present in aldosterone animals occurred as a result of both significant reduction in m-s and increase in s-m fluxes in all the three flux periods (Table 1). Aldosterone also significantly enhanced both I_sc and G in rat distal colon (Table 1).

Table 1.

Basal ⁸⁶Rb fluxes and associated electrical parameters in normal and aldosterone rat distal colon

	86Rb Fluxes, μeq·h−1·cm−2
	m-s	s-m	Net	Isc, μeq·h−1·cm−2	G, mS
Normal
P-I	1.47 ± 0.26	0.43 ± 0.03	1.04 ± 0.26	2.0 ± 0.3	8.8 ± 0.5
P-II	1.30 ± 0.33 (NS)	0.40 ± 0.02 (NS)	0.90 ± 0.22 (NS)	2.1 ± 0.3 (NS)	8.9 ± 0.5 (NS)
P-III	1.21 ± 0.21 (NS)	0.40 ± 0.02 (NS)	0.81 ± 0.22 (NS)	2.6 ± 0.8 (NS)	8.1 ± 0.6 (NS)
Aldosterone
P-I	0.72 ± 0.14*	1.93 ± 0.09†	−1.21 ± 0.15†	8.4 ± 0.9†	23.5 ± 3.5†
P-II	0.69 ± 0.12* (NS)	1.83 ± 0.14† (NS)	−1.14 ± 0.19† (NS)	7.7 ± 0.9† (NS)	21.3 ± 1.5† (NS)
P-III	0.60 ± 0.13* (NS)	1.93 ± 0.14† (NS)	−1.33 ± 0.17† (NS)	7.0 ± 0.8† (NS)	20.3 ± 1.8† (NS)

Values are means ± SE. Mucosal to serosal (m-s), serosal to mucosal (s-m), and net ⁸⁶Rb fluxes were measured for 3 consecutive 15-min flux periods (P-I, P-II, and P-III), as described in materials and methods. Net ⁸⁶Rb absorption and secretion are represented by positive and negative values, respectively. I_sc, short circuit current; G, conductance (mSiemen).

^*P < 0.05, compared with respective normal period;

^†P < 0.001, compared with respective normal period; NS, not significant compared with respective P-I.

The aldosterone-induced K⁺ secretion was characterized in the absence of a K⁺ absorptive process mediated via H-K-ATPase by blocking with mucosal VO₄ (31). As shown in Fig. 1, mucosal VO₄ significantly reduced the m-s K⁺ fluxes (1.17 ± 0.10 vs. 0.25 ± 0.03 μeq·h⁻¹·cm⁻²; P < 0.001) and completely inhibited the net K⁺ absorption (0.85 ± 0.13 vs. −0.02 ± 0.04 μeq·h⁻¹·cm⁻²; P < 0.001) in normal colon. In contrast, mucosal VO₄ enhanced the net K⁺ secretion by both significantly reducing m-s (0.86 ± 0.08 vs. 0.46 ± 0.03 μeq·h⁻¹·cm⁻²; P < 0.02) and enhancing s-m (−0.98 ± 0.16 vs. −1.62 ± 0.21 μeq·h⁻¹·cm⁻²; P < 0.05) fluxes in aldosterone rat colon (Fig. 1B). The VO₄-insensitive m-s K⁺ flux was also significantly higher in aldosterone rat distal colon (0.25 ± 0.03 vs. 0.46 ± 0.03 μeq·h⁻¹·cm⁻²; P < 0.05; Fig. 1, A and B). Mucosal VO₄ did not alter either I_sc (Fig. 1, C and D) or tissue conductance (G: Fig. 1) in normal and aldosterone rat distal colon. These observations indicate that active K⁺ absorption, but not active K⁺ secretion, is present in normal rat distal colon, while aldosterone induces active K⁺ secretion in rat distal colon.

Fig. 1.

Effect of mucosal VO₄ on ⁸⁶Rb fluxes and short circuit currents (I_sc) in normal and aldosterone rat distal colon. Mucosal to serosal (m-s), serosal to mucosal (s-m), and net ⁸⁶Rb fluxes (A and B) and I_sc (C and D) were measured in the presence (closed bars) and absence (open bars) of mucosal VO₄ under short circuit conditions in normal (A and C) and aldosterone (B and D) rat distal colon. Basal and post-VO₄ tissue conductance (G) were 8.5 ± 0.4 and 9.6 ± 0.5 mS (P < 0.02) for normal and 18.6 ± 1.1 and 20.0 ± 1.8 mS (NS) for aldosterone rat distal colon, respectively. *P < 0.001, compared with respective basal values; £P < 0.05, compared to respective basal values; ‡P < 0.001, compared with normal m-s value in presence of VO₄.

Since mucosal VO₄ significantly enhanced the net K⁺ secretion, experiments were designed to identify whether the long-term presence of VO₄ would affect the tissue integrity and/or transport properties in aldosterone rat distal colon. In this study, 40 min following the addition of ⁸⁶Rb⁺, 1 mM VO₄ was added to the mucosal bath and allowed to equilibrate for 15 min, and then three consecutive 15-min flux periods were measured. The results presented in Table 2 indicate that the mucosal presence of VO₄ did not alter either K⁺ fluxes or the electrical parameters. Thus further characterization of aldosterone-induced K⁺ secretion was performed in the presence of mucosal VO₄.

Table 2.

Basal ⁸⁶Rb fluxes and associated electrical parameters in presence of mucosal VO₄ in aldosterone rat distal colon

	86Rb Fluxes, μeq·h−1·cm−2
	m-s	s-m	Net	Isc, μeq·h−1·cm−2	G, mS
P-I	0.26 ± 0.04	2.57 ± 0.10	−2.31 ± 0.09	7.5 ± 0.6	17.8 ± 1.3
P-II	0.27 ± 0.06 (NS)	2.58 ± 0.12 (NS)	−2.31 ± 0.07 (NS)	7.7 ± 0.5 (NS)	17.7 ± 1.6 (NS)
P-III	0.26 ± 0.03 (NS)	2.39 ± 0.26 (NS)	−2.13 ± 0.25 (NS)	7.7 ± 0.6 (NS)	20.0 ± 1.8 (NS)

Values are means ± SE. Mucosal to serosal (m-s), serosal to mucosal (s-m), and net ⁸⁶Rb fluxes were measured for 3 consecutive 15-min flux periods (P-I, P-II, and P-III) in the presence of 1 mM mucosal VO₄, as described in materials and methods. Net ⁸⁶Rb absorption and secretion are represented by positive and negative values, respectively. NS, not significant compared with respective P-I.

Aldosterone has been shown to induce electrogenic Na⁺ absorption through epithelial Na⁺ channels (ENaC) in rat distal colon (12, 24). Thus the effect of benzamil (50 μM; an ENaC blocker) was examined on aldosterone-induced K⁺ secretion both in the absence (Fig. 2A) and in the presence (Fig. 2B) of mucosal VO₄. Mucosal benzamil significantly enhanced the net K⁺ secretion both in the absence (−0.96 ± 0 .23 vs. −1.35 ± 0.24 μeq·h⁻¹·cm⁻²; P < 0.05) and in the presence (−1.62 ± 0.11 vs. −2.13 ± 0.19 μeq·h⁻¹·cm⁻²; P < 0.05) of mucosal VO₄ in aldosterone rat colon (Fig. 2, A and B). This observation indicates that the aldosterone-induced electrogenic Na⁺ absorption and K⁺ secretion are mediated via independent pathways in rat colon.

Fig. 2.

Effect of mucosal benzamil on ⁸⁶Rb fluxes and I_sc in the presence and absence of mucosal VO₄ in aldosterone rat distal colon. Mucosal to serosal, serosal to mucosal, and net ⁸⁶Rb fluxes (A and B) and I_sc (C and D) were measured in the presence (B and D) and absence (A and C) of mucosal VO₄ in aldosterone rat distal colon. ⁸⁶Rb fluxes and I_sc were measured either in the presence (closed bars) or in the absence (open bars) of mucosal benzamil (50 μM). Basal and postbenzamil tissue conductances (G) were 21.0 ± 2.3 and 16.9 ± 2.6 mS in the absence (P < 0.01) and 22.4 ± 2.3 and 17.1 ± 1.6 mS in the presence (P < 0.02) of VO₄, respectively. I_sc and G recorded for every 20 s were averaged to represent each flux period. *P < 0.001, compared with respective basal values; £P < 0.05, compared with respective basal values; ‡P < 0.001, compared with normal m-s value in presence of VO₄.

Mucosal benzamil also inhibited the serosal positive I_sc and revealed mucosal positive I_sc both in the presence (8.8 ± 1.1 vs. −2.0 ± 0.3 μeq·h⁻¹·cm⁻²; P < 0.001) and in the absence (10.6 ± 1.1 vs. −3.1 ± 0.3 μeq·h⁻¹·cm⁻²; P < 0.001) of VO₄, which is consistent with K⁺ secretion in aldosterone rat colon (Fig. 2, C and D). Mucosal benzamil also significantly reduced the trans-epithelial conductance both in the presence (21.0 ± 2.3 and 16.9 ± 2.6 mS; P < 0.01) and in the absence (22.4 ± 2.3 and 17.1 ± 1.6 mS; P < 0.02) of VO₄. These observations indicate that the overwhelming rate of ENaC activity masks the I_sc attributed to electrogenic K⁺ secretion and that the inhibition of ENaC activity reveals the I_sc attributed to K⁺ secretion. Further characterization of aldosterone-induced K⁺ secretion was therefore performed in the presence of mucosal VO₄ and benzamil.

The effect of mucosal K⁺ channel blockers was examined to identify whether aldosterone-induced K⁺ secretion was mediated via Kcnn4c and/or Kcnma1 channels. As shown in Fig. 3A, Ba²⁺ and CTX inhibited the aldosterone-induced K⁺ secretion by 89%. The aldosterone-induced K⁺ secretion was also inhibited partially by TEA (64%), IbTX (66%), and TRAM-34 (29%; Fig. 3A). TRAM-34 (IbTX vs. IbTX/TRAM-34: −0.63 ± 0.07 vs. −0.42 ± 0.06 μeq·h⁻¹·cm⁻²; P < 0.05) but not TEA (IbTX vs. IbTX/TEA: −0.63 ± 0.07 vs. −0.76 ± 0.08 μeq·h⁻¹·cm⁻²) significantly inhibited the IbTX-insensitive fraction (Fig. 3A). Similar to the inhibition of K⁺ secretion, the I_sc attributed to aldosterone-induced K⁺ secretion was also significantly inhibited by the K⁺ channel blockers (Fig. 3B). These observations indicate that the Ba²⁺/CTX-sensitive aldosterone-induced K⁺ secretion consists of both IbTX/TEA-sensitive and IbTX/TEA-insensitive fractions that might represent K⁺ secretions mediated via Kcnma1 and Kcnn4c channels, respectively.

Fig. 3.

Effect of mucosal K⁺ channel blockers on ⁸⁶Rb secretion in aldosterone rat distal colon. Net ⁸⁶Rb secretion (A) and I_sc (B) were measured in the presence of mucosal VO₄ (1 mM) and benzamil (50 μM; open bars). Net ⁸⁶Rb secretion and I_sc were also measured in the presence of various K⁺ blockers (closed bars). K⁺ channel blockers used were Ba²⁺ (5 mM), TRAM-34 (50 μM), iberiotoxin (IbTX; 100 nM), tetraethyl ammonium (TEA; 10 μM), and charybdotoxin (CTX; 20 nM). Basal and post-K⁺ channel blocker tissue conductances (G) were 17.1 ± 1.1 (basal), 15.3 ± 1.6 (Ba²⁺; P < 0.05), 15.6 ± 1.8 (TEA; P < 0.05), 16.7 ± 1.6 (TRAM-34; NS), 15.6 ± 0.9 (IbTX; P < 0.05), 15.2 ± 1.3 (IbTX/TRAM-34; P < 0.05), 15.4 ± 1.1 (IbTX/TEA; P < 0.05), and 14.6 ± 1.1 (CTX; P < 0.05) mS. *P < 0.001, compared with respective control; £P < 0.001, compared with respective control.

The effect of varying TRAM-34 concentrations was examined to establish whether IbTX-insensitive K⁺ secretion occurs via Kcnn4c in aldosterone rat distal colon. As shown in Fig. 4A, increasing TRAM-34 concentrations (0–100 μM) progressively inhibited the IbTX-insensitive K⁺ secretion. Dixon plot analyses of these data yielded a half-maximal inhibitory concentration (IC₅₀) for TRAM-34 of ∼5.6 ± 0.4 μM (Fig. 4B). This IC₅₀ value for TRAM-34 of IbTX-insensitive K⁺ secretion is similar to the IC₅₀ for TRAM-34 of K⁺ efflux in oocytes expressing Kcnn4c (22). Thus these observations establish that the mucosal Kcnn4c channels mediate the aldosterone-induced IbTX-insensitive K⁺ secretion in colon.

Fig. 4.

Effect of TRAM-34 concentrations on IbTX-insensitive net ⁸⁶Rb secretion in aldosterone rat distal colon. A: net ⁸⁶Rb secretion was measured in the presence of mucosal VO₄ (1 mM), benzamil (50 μM), IbTX (100 nM), and varying concentrations of mucosal TRAM-34 (0–100 μM). B: Dixon plot of TRAM-34 sensitive components of net ⁸⁶Rb secretion. Calculated IC₅₀ for TRAM-34 was 5.6 μM.

The effects of anion channel blockers were also examined to determine if the aldosterone-induced K⁺ secretion is associated with anion secretion, as active K⁺ exits that maintain cell hyperpolarization provide the driving force for active anion secretion (4). Mucosal CFTR_(inh)-172 (a CFTR blocker), niflumic acid (a Ca²⁺-activated Cl⁻ channel blocker), and NPPB (a nonspecific anion channel blocker) did not inhibit either I_sc or K⁺ secretion (Table 3). These observations indicate that the aldosterone-induced K⁺ secretion is not driving anion secretion in rat distal colon.

Table 3.

Effect of mucosal anion channel blockers on net ⁸⁶Rb secretion and I_sc in aldosterone rat distal colon

	Net 86Rb Secretion, μeq·h−1·cm−2	Isc, μeq·h−1·cm−2
Basal	−2.1 ± 0.3	−2.4 ± 0.4
+CFTRinh−172	−2.3 ± 0.3 (NS)	−2.1 ± 0.3 (NS)
Basal	−1.7 ± 0.2	−1.6 ± 0.2
+Niflumic acid	−1.9 ± 0.2 (NS)	−1.6 ± 0.3 (NS)
Basal	−1.9 ± 0.2	−2.1 ± 0.3
+NPPB	−1.8 ± 0.3 (NS)	−1.9 ± 0.3 (NS)

Values are means ± SE. Net ⁸⁶Rb fluxes were measured as described in materials and methods. Basal fluxes were measured in the presence of mucosal benzamil (50 μM) and VO₄ (1 mM). Basal fluxes were also measured in the presence of cystic fibrosis transmembrane conductance regulator (CFTR)_inh−172 (100 μM), niflumic acid (100 μM), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; 100 μM). NS, not significant compared with respective basal.

Western blot analyses with Kcnma1α (a MaxiKα antibody) and Kcnn4-abc antibodies were performed to determine whether aldosterone-induced K⁺ secretion was associated with the activation of existing channels and/or enhanced expression of Kcnn4c and Kcnma1 in distal colon. The Kcnma1α antibody detected a 40-kDa protein in the apical membranes of both normal and aldosterone rats (Fig. 5A), while densitometry analyses indicated that Kcnma1 protein was enhanced by 3.2-fold in the apical membranes of aldosterone rat distal colon (Fig. 5D). The Kcnn4-abc antibody, which detected 37 (Kcnn4c)- and 40 (Kcnn4b)-kDa proteins in epithelial cell homogenates, detected a Kcnn4c protein in apical membranes of both normal and aldosterone rat distal colon (Fig. 5B). Densitometry analyses indicated that aldosterone enhanced Kcnn4c protein expression by 2.4-fold (Fig. 5E). The Kcnn4-abc antibody also weakly detected Kcnn4b protein in apical membranes (Fig. 5B). However, Western blot analyses of biotinylated apical membranes isolated from normal rat distal colon indicated that only Kcnn4c (37 kDa), but not Kcnn4b (40 kDa), is present on the apical membranes of rat distal colon (3). Thus, the Kcnn4b protein detected in the apical membrane represents contamination from basolateral membranes. Although it is contamination, the expression level of Kcnn4b (40-kDa protein) seems substantially higher in aldosterone rat distal colon (Fig. 5B). Thus Western blot analyses were performed on colonic epithelial homogenates. As shown in Fig. 5, C and F, aldosterone enhanced both Kcnn4c (37 kDa) and Kcnn4b (40 kDa) protein expression by 2.3- and 3.1-fold, respectively (Fig. 5F). These observations indicate that aldosterone enhances Kcnn4b, Kcnn4c, and Kcnma1 channel protein expression in distal colon.

Fig. 5.

Western blot analyses of apical membranes (APM) and colonocyte homogenates (Homo) of normal (Norm) and aldosterone (Aldo) rat distal colon. Blots were stained with either anti-BKα (a Kcnma1α antibody; 1:400; A) or anti-Kcnn4-abc (1:3,000; B and C) primary antibodies followed by horseradish peroxidase-conjugated secondary antibody. Same blots were stripped and stained with anti-actin antibody and secondary antibody. Relative expression of Kcnma1α (D)- and Kcnn4c (37 kDa; E)-specific proteins in APM, band of Kcnn4b and Kcnn4c in colonocyte homogenates (F) of normal and aldosterone rat distal colon. Arbitrary units presented were normalized to actin. Three different APM preparations from normal and Na⁺-depleted animals were used in this study. Each APM preparation used epithelial cells isolated from 8 rat distal colons.

To determine if changes in mRNA levels occurred in aldosterone rats, the levels of Kcnma1α, Kcnn4b, and Kcnn4c mRNAs were measured in normal and aldosterone rat distal colon. It was found that Kcnma1α mRNA was increased 3.2-fold, Kcnn4b 2.6-fold, and Kcnn4c 1.9-fold in aldosterone rats compared with normal (Fig. 6). This suggests that aldosterone might upregulate these channels either by increasing transcription or mRNA half-life. To identify whether aldosterone-enhanced mRNA expression occurred at the transcriptional or posttranscriptional level, the effect of in vitro treatment with aldosterone and subsequent addition of actinomycin D effects were examined on I_sc and K⁺ channel specific mRNA abundances in normal rat distal colon. As shown in Fig. 7, I_sc was induced at ∼2 h 30 min after aldosterone addition and reached a steady state at ∼5 h that was stable at least for 4 h. Ten micromolar mucosal amiloride completely inhibited the aldosterone-induced I_sc. The enhanced amiloride-sensitive I_sc is attributed to ENaC mediated Na⁺ absorption. These results are consistent with earlier observations with human, guinea pig, and rat colon (1, 8, 13). The presence of actinomycin D completely blocked the aldosterone-induced I_sc (Fig. 7). The normal rat colonic mucosa control exhibited steady state I_sc throughout the experimental period (data not shown). These observations suggest that aldosterone-induced Na⁺ absorption (i.e., ENaC) and K⁺ secretion (i.e., mucosal K⁺ channels) are regulated at the transcriptional level.

Fig. 6.

Real-time (RT-PCR) analyses of Kcnma1-, Kcnn4b-, and Kcnn4c-specific mRNA in normal and aldosterone rat distal colon. RT-PCR analyses of total-RNA isolated from epithelial cells of normal and aldosterone rat distal colons were performed as described in materials and methods. Kcnma1α-subunit (A)-, Kcnn4b (B)-, and Kcnn4c (C)-specific mRNA abundances in epithelial cells from normal (open bars) and aldosterone (closed bars) rat distal colon. Aldosterone enhanced Kcnma1α-subunit, Kcnn4b and Kcnn4c specific mRNA abundances by 3.1-, 2.6-, and 1.9-fold in rat distal colon, respectively. Results presented represent means ± SE of analyses performed from total-RNA isolated from epithelial cells from 3 different rat distal colons.

Fig. 7.

Effect of actinomycin D (Act D) on in vitro aldosterone induced I_sc, in normal rat distal colon. Immediately following mounting the tissue, either aldosterone (Aldo) or aldosterone plus actinomycin D (Aldo/Act D) was added to the serosal bath (arrow). I_sc was measured under voltage clamp condition for up to 9 h 30 min. At the end of 9 h, 10 μM amiloride was added to mucosal bath (Amil). Experiment was also performed in the presence of vehicle (0.1% ethanol and 0.1% methanol) in place of aldosterone and Act D (data not shown). Data represent means ± SE of 6 tissues obtained from 3 different rats.

Although ENaC is transcriptionally regulated (1, 8), aldosterone regulation of mucosal K⁺ channels has not been established. Thus Kcnn4c- and Kcnma1α-specific mRNA abundances were determined in RNA isolated from normal rat colonic mucosa exposed to vehicle, aldosterone, and aldosterone plus actinomycin D in vitro. Aldosterone enhanced the Kcnn4c and Kcnma1α mRNA abundance by 1.3- and 2.4-fold, respectively. The presence of actinomycin D completely blocked the aldosterone-enhanced mRNA abundance of both Kcnn4c and Kcnma1α transcripts (Fig. 8). This observation establishes that aldosterone regulates both Kcnn4c and Kcnma1 channels at the transcriptional level.

Fig. 8.

Effect of actinomycin D on in vitro aldosterone enhanced Kcnn4c and Kcnma1 specific mRNA abundance. Total RNA was isolated from mucosal tissues used for I_sc measurement in the presence of in vitro vehicle (Control), aldosterone (Aldo), and aldosterone plus actinomycin D (Aldo/Act D) in Fig. 7. Tissue from 2 chambers was pooled to prepare total RNA. Results presented represent mean ± SE obtained from 3 different RNA preparations.

DISCUSSION

The Ba²⁺- and TEA-sensitive K⁺ channels localized on colonic mucosal membranes mediate active K⁺ secretion, which is enhanced by aldosterone (5, 31–32). However, the signaling pathway for this effect is not known. While both Kcnn4c and Kcnma1 channels are present on the colonic mucosal membranes, electrophysiological studies have characterized only Kcnma1 channels (26–27). This study was therefore directed to assess the relative contributions of Kcnn4c and Kcnma1 in the aldosterone-induced K⁺ secretion in rat distal colon. This study demonstrates an absence of net K⁺ fluxes in normal rat distal colon in the presence of mucosal VO_4, indicating the absence of K⁺ secretion. The absence of K⁺ secretion in normal distal colon differs from interpretations of previous work, where the inhibition of s-m K⁺ fluxes by TEA and Ba²⁺ was taken as evidence for active K⁺ secretion (32).

This study demonstrates that aldosterone induces Ba²⁺-sensitive K⁺ secretion and that the Ba²⁺-sensitive K⁺ secretion consists of both IbTX/TEA-sensitive and IbTX/TEA-insensitive K⁺ secretions that are mediated through Kcnma1 and Kcnn4c channels, respectively. Similar inhibition by IbTX and TEA individually or in combination supports the conclusion that both IbTX and TEA inhibit the same fraction (i.e., IbTX/TEA-sensitive fraction) of aldosterone-induced K⁺ secretion mediated via Kcnma1 channels. Complete inhibition by the simultaneous use of IbTX and TRAM-34 and partial inhibition by their individual use support the conclusion that the IbTX/TEA-insensitive fraction of aldosterone-induced K⁺ secretion is mediated via Kcnn4c channels. Since both Kcnn4 and Kcnma1 channels are inhibited by Ba²⁺ (17, 26), the present observations establish that both Kcnn4c and Kcnma1 channels mediate aldosterone-induced K⁺ secretion. This conclusion differs from that of an earlier study (32), which has shown aldosterone to induce Ba²⁺-sensitive and TEA-sensitive (i.e., Ba²⁺-insensitive) K⁺ secretion in rat distal colon. The nonbuffered solution used in this earlier study might be the reason for the presence of a major fraction of Ba²⁺-insensitive K⁺ secretion in rat distal colon (32).

As mentioned, Kcnn4c in part mediates the aldosterone-induced IbTX/TEA-insensitive K⁺ secretion in rat distal colon. This conclusion is supported by the demonstration that TRAM-34 inhibits the IbTX/TEA-insensitive K⁺ secretion with an apparent IC₅₀ of 5.6 μM. While this is higher than that for human lymphocyte Kcnn4 channels (34), it is similar to that of Kcnn4c-mediated K⁺ efflux characterized in Xenopus oocytes (2). Therefore, the present study supports the conclusion that IbTX/TEA-insensitive K⁺ secretion, which exhibits relative resistance to TRAM-34, is mediated via mucosal Kcnn4c channels. As indicated, although both Kcnn4c and Kcnma1 channels are present, patch-clamp studies (6, 26–27) have characterized only Kcnma1, but not Kcnn4c channels, on the mucosal membranes of rat and human colonic epithelial cells. It is possible that the dominant presence of Kcnma1 might have masked the Kcnn4c activity in these mucosal membrane patches (6), as evidenced by our recent observation (3) in which inhibition of Kcnma1 activity uncovers Kcnn4c function in apical membrane patches from IEC-18 cells.

Although both Kcnn4c and Kcnma1 proteins are present on the apical membranes of both normal and aldosterone rat distal colon, active K⁺ secretion is present only in the aldosterone but not in the normal rat distal colon. This suggests that in addition to enhanced Kcnn4c and Kcnma1 expression, aldosterone also induces additional factor(s) that activate these channels in rat distal colon. Alkaline intracellular pH (pH_i) generated by enhanced mucosal NHE3 has been suggested to activate Kcnma1 channels in high-K⁺ diet-fed rat colon (26). Since Na⁺ depletion, in contrast to a high-K⁺ diet, enhances the aldosterone level by severalfold and inhibits Na-H exchanger isoform 3 NHE3 (15, 24), it is highly unlikely that alkaline pH_i would be generated to influence the activated K⁺ secretion in aldosterone rat distal colon. It is more plausible that the enhanced mucosal H-K-ATPase activity alkalinized the pH_i to activate Kcnma1-mediated K⁺ secretion in aldosterone rat distal colon. However, the significant increase in K⁺ secretion by the inhibition of H-K-ATPase by mucosal addition of VO₄ argues against this possibility that an alkaline pHi would have to exist to activate the Kcnn4c and/or Kcnma1 channels in aldosterone rat distal colon. Further, this conclusion is supported by the observation that the resting pH_i was not significantly different in surface cells of aldosterone and normal rat distal colon (Singh SK, unpublished observations). Although aldosterone-increased intracellular free Ca²⁺ has been suggested as a mechanism for enhanced K⁺ secretion in high-K diet fed rats, it is highly unlikely because the increased Ca²⁺ concentration is only transient. Therefore, we speculate that increased sensitivity to Ca²⁺ and/or phosphorylation of Kcnn4c and Kcnma1 channels might be responsible for the aldosterone-induced K⁺ secretion in rat distal colon.

In summary, this study demonstrates that aldosterone induces IbTX/TEA-insensitive and IbTX/TEA-sensitive K⁺ secretion that are mediated via mucosal Kcnn4c and Kcnma1 channels in rat distal colon, respectively. The aldosterone-induced K⁺ secretion is associated with increased mucosal expression of both Kcnn4c and Kcnma1 channels. In addition to enhanced expression, aldosterone also activates both Kcnn4c and Kcnma1 channels by unknown mechanism(s) that might involve either enhanced Ca²⁺ sensitivity and/or phosphorylation, which requires additional extensive study.

GRANTS

This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-018777.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

Author contributions: S.K.S. and V.M.R. interpreted results of experiments; S.K.S. and V.M.R. edited and revised manuscript; S.K.S., B.O., J.R.T., and V.M.R. approved final version of manuscript; B.O., J.R.T., and V.M.R. performed experiments; B.O. drafted manuscript; J.R.T. prepared figures; V.M.R. conception and design of research; V.M.R. analyzed data.