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J. Biol. Chem., Vol. 282, Issue 34, 24563-24573, August 24, 2007

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Female Gender-specific Inhibition of KCNQ1 Channels and Chloride Secretion by 17-Estradiol in Rat Distal Colonic Crypts^*

From the Department of Molecular Medicine, Beaumont Hospital, Royal College of Surgeons in Ireland, Dublin 9, Ireland

Received for publication, December 21, 2006 , and in revised form, May 29, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The estrogen sex steroid 17-estradiol rapidly inhibits secretagogue-stimulated cAMP-dependent Cl^– secretion in the female rat distal colonic crypt by the inhibition of basolateral K⁺ channels. In Ussing chamber studies, both the anti-secretory response and inhibition of basolateral K⁺ current was shown to be attenuated by pretreatment with rottlerin, a PKC-specific inhibitor. In whole cell patch-clamp analysis, 17-estradiol inhibited a chromanol 293B-sensitive KCNQ1 channel current in isolated female rat distal colonic crypts. Estrogen had no effect on KCNQ1 channel currents in colonic crypts isolated from male rats. Female distal colonic crypts expressed a significantly higher amount of PKC in comparison to male tissue. PKC and PKA were activated at 5 min in response to 17-estradiol in female distal colonic crypts only. Both PKC- and PKA-associated with the KCNQ1 channel in response to 17-estradiol in female distal colonic crypts, and no associations were observed in crypts from males. PKA activation, association with KCNQ1, and phosphorylation of the channel were regulated by PKC as the responses were blocked by pretreatment with rottlerin. Taken together, our experiments have identified the molecular targets underlying the anti-secretory response to estrogen involving the inhibition of KCNQ1 channel activity via PKC- and PKA-dependent signaling pathways. This is a novel gender-specific mechanism of regulation of an ion channel by estrogen. The anti-secretory response described in this study provides molecular insights whereby estrogen causes fluid retention effects in the female during periods of high circulating plasma estrogen levels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Fluid and electrolyte secretion is an important function in the distal colon as it regulates whole body fluid homeostasis and also maintains mucosal hydration (1). Chloride secretion in the distal colon occurs as a two-step transport process. Cl^– is transported into the cell basolaterally with Na⁺ and K⁺ via the Na⁺/K⁺/2Cl^– isoform 1 (NKCC1) co-transporter. Cl^– is secreted across the apical membrane into the lumen via a chloride ion channel, the cystic fibrosis transmembrane conductance regulator (CFTR).³ Secretion of Cl^– ions is driven by the activity of Na⁺-K⁺-ATPase located in the basolateral membrane. Basolateral K⁺ ion channels carry out the K⁺ recycling required to establish the favorable membrane electrical potential for Cl^– secretion. The activity of ion transporters involved in Cl^– secretion may be modulated by secretagogues acting through cAMP activity or intracellular Ca²⁺ concentration. Disorders resulting in increased Cl^– secretion, for example in bacterial or viral infections, are a primary cause of secretory diarrhea (2).

The biologically active estrogen 17-estradiol (E2) plays an important role in the normal development and maturation of the female. In addition to this classical role, E2 has also been implicated in the regulation of whole body fluid and electrolyte balance (3, 4). The distal colon has recently been recognized as a target of E2. The colonic crypts express both isoforms of the nuclear estrogen receptor, ER and ER, similar to classical E2-responsive tissues such as uterus and breast tissue (5). We have previously demonstrated that E2 inhibits secretagogue stimulated Cl^– secretion in the rat distal colonic crypt (6). The anti-secretory response to E2 was rapid, occurring within 10 min and was dependent on protein kinase C (PKC). In human distal colonic tissue, blocking basolateral K⁺ channel activity decreases forskolin-stimulated Cl^– secretion (7). In rat distal colon the basolateral K⁺ conductance is formed by at least two different types of K⁺ channels, activated by Ca²⁺ or cAMP-dependent agonists (8). Electrophysiological studies in these cells have revealed the candidate K⁺ channel involved in Cl^– secretion to be KCNQ1, a low conductance (1–3 pS) K⁺ channel, which is activated during cAMP-stimulated Cl^– secretion (9). Other types of K⁺ channels have also been implicated in chloride secretion, including the Ca²⁺-dependent KCNN4 channel (10).

The rat KCNQ1 channel was cloned from colonic tissue and expression was demonstrated in both the crypt and surface cells (9). KCNQ1 is a voltage-gated channel, which plays a crucial role in controlling salt and water homeostasis in a number of epithelia (11). KCNQ1 is located in the basolateral membrane of the rat distal colonic crypt (8) and is regulated by forming a complex with the -subunit KCNE3 (MiRP2) to form the basolateral K⁺ conductance that is required for transepithelial cAMP-stimulated chloride secretion (12). We propose KCNQ1 as a candidate membrane target for E2 in the inhibition of Cl^– secretion in the rat distal colonic crypt. A previous study in cardiac tissue demonstrated a concentration-dependent inhibition of the KCNQ1/KCNE1-mediated K⁺ current by E2 (13).

PKC is a serine/threonine kinase belonging to the novel PKC subgroup and plays a key role in cell cycle progression, transcriptional regulation and tissue remodeling (14). In the gastrointestinal tract PKC has been implicated in the maintenance of the epithelial barrier integrity (15) and is an important regulator of Cl^– secretion (16). We have previously demonstrated that physiological concentrations of E2 (0.01–100 nM) produced a rapid (15 min) stimulation of PKC and PKA activities in female rat distal colonic crypts (17).

The phosphorylation of ion channels is a major mechanism for their regulation. Indeed it has been demonstrated recently that KCNQ1 can be phosphorylated at Ser²⁷ in the N terminus of the channel by PKA (18).

In the present study, we examined the female gender-specific regulation of the KCNQ1 channel by E2 and the role of PKA and PKC as mediators of the rapid anti-secretory response to estrogen in rat distal colonic crypts.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Phospho-PKC (Ser⁶⁴³) antibody was obtained from Cell Signaling Technologies. Phosphoserine (Clone IC8) antibody was obtained from Calbiochem. Total PKC, total PKA regulatory subunit isoform I (PKARI), and total PKA catalytic subunit isoform I (PKACI) antibodies from BD Transduction. Anti-rabbit anti-goat horseradish peroxidase-linked secondary antibody from Santa Cruz Biotechnology. Anti-rabbit and anti-mouse horseradish peroxidase-linked secondary antibodies from Sigma-Aldrich. Total KCNQ1 antibody was obtained from Sigma-Aldrich and Santa Cruz Biotechnology. Protein-G Sepharose beads and the ECL plus detection system were from Amersham Biosciences and Bradford reagent from Bio-Rad. Chromanol 293B was obtained from Tocris and rottlerin from Calbiochem. All other reagents were obtained from Sigma-Aldrich.

Animals—Male (350 g) and female (300 g) Sprague-Dawley rats at three-months-old were used for all experiments. Animals were kept on a 12-h light, 12-h dark cycle, and were given ad libitum access to food and water. Following anesthesia rats were killed by cervical dislocation. Cervical smears were obtained from female rats, and the stage of the estrous cycle was determined histologically as previously described (19). All female rats were used at the estrus stage of the cycle where estradiol is present at a circulating level of 75 pg/ml and progesterone at 32.5 pg/ml (20).

The distal colon was removed to below the pelvic rim. The faecal contents were rinsed and distal colonic crypts were isolated as previously described (17). Isolations and treatments were carried out at room temperature to avoid colonic crypt disintegration (17, 21). Sheets of colonic mucosa were obtained by blunt dissection for transepithelial transport measurements. All procedures were approved by the RCSI Ethics Committee.

Transepithelial Transport Studies—Colonic epithelia were mounted in Ussing chambers (Physiologic Instruments) on inserts exposing an area of 0.5 cm². Transepithelial potential difference was clamped to 0 mV using an EVC-4000 voltage-clamp apparatus (World Precision Instruments). The transepithelial short circuit current (I_SC) was recorded using Ag-AgCl electrodes in 3 M KCl agar bridges. Apical and basolateral baths were filled with Krebs bicarbonate buffer (in mM: 120 NaCl, 25 NaHCO₃, 3.3 KH₂PO₄, 0.8 K₂HPO₄, 1.2 MgCl₂, 1.2 CaCl₂, 10 glucose), pH 7.4 maintained at 37 °C by heated water jackets and oxygenated with a 95% O₂/5% CO₂ mixture. All preparations were allowed to equilibrate for 30–45 min before the experiments were performed. The I_sc was defined as positive for anion flow from the basolateral to apical chamber and for cation flow in the opposite direction.

To investigate the activity of basolateral K⁺ channels in Ussing chamber experiments, the apical membrane was permeabilized by addition of 10 µM of the K⁺ ionophore amphotericin B in the presence of a mucosal to serosal K⁺ gradient established by the following bath solutions: apical (in mM), 145 K-gluconate, 3 KH₂PO₄, 0.8 K₂HPO₄, 1.2 Mg(gluconate)₂, 4 Ca(gluconate)₂, 10 glucose, and 10 HEPES; basolateral, 145 Nagluconate, 3.3 NaH₂PO₄, 0.8 Na₂HPO₄, 1.2 Mg(gluconate)₂, 4 Ca(gluconate)₂, 10 glucose, and 10 HEPES. Ouabain (1 mM) was added to the serosal bath to inhibit Na⁺-K⁺-ATPase. The resulting I_sc was generated by the movement of K⁺ through channels in the basolateral membrane (I_K) as the current collapses to zero in equimolar K⁺ solutions bathing both sides of the epithelium. For measurement of I_K current-voltage relationships, currents were elicited in asymmetrical K⁺ gluconate solutions by imposition of 1-s test potentials between –100 and +100 mV in 20-mV increments.

Patch-clamp—A small aliquot (100 µl) of freshly isolated colonic crypts was transferred into 1 ml of superfusion chamber mounted on the stage of an inverted microscope (TE 2000-S, Nikon Ltd). Pipettes were prepared from capillary glass (GC150F-10, Harvard Apparatus Ltd, Edenbridge, UK). Patch pipettes were pulled and fire-polished using a programmable horizontal puller (DMZ-Universal, Zeitz-Instruments GmbH) and had an electrical resistance of 2–5 m when filled with K⁺ solutions. The patch-clamp apparatus consisted of a CV-203BU head stage (Axon Instruments Incorporated) connected to an Axopatch 200B series amplifier (Axon Instruments). Patch-clamp experiments were performed at 37 °C in the standard whole cell recording configuration (22) and recorded membrane currents were filtered at 1 kHz through an 8 pole, low-pass Bessel filter, and digitized at 5 kHz. The voltage clamp protocol consisted of a series of voltage steps from –100 mV to +100 mV in 20-mV increments from an initial holding potential of –50 mV. Colonic crypts were superfused at a rate of 1 ml/min in a standard bath solution containing (in mM): NaCl 140, KCl 5.4, MgCl₂ 1, CaCl₂ 1.25, HEPES 10, glucose 12.2, buffered at pH 7.4 with NaOH. The patch pipette solution contained (mM): K-gluconate 95, KCl 30, Na₂ATP 4.8, KH₂ PO₄ 1.2, EGTA 1, Ca-gluconate 0.73, MgCl₂ 1, ATP 3, D-glucose 5 (pH 7.2). Once whole cell access to the inside of the cell was obtained, cells were allowed to stabilize for up to 10 min before the experiment began. The protocols for patch-clamp and data analysis were established with routines using pClamp 9.2 software (Axon Instruments), and data were stored for subsequent analysis.

Co-immunoprecipitation and Western Blotting—Isolated distal colonic crypts resuspended in Krebs solution were treated with drugs for the indicated time points. The samples were then immediately centrifuged for 30 s at 4 °C at 4,000 rpm, and the supernatant removed. Cells were lysed by hypotonic shock on ice for 45 min (lysis buffer: 20 mM Tris, pH 7.4, 0.5% Nonident P-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, leupeptin 1 µg/µl, 500 mM dithiothreitol, 5 mM phenylmethylsulfonyl fluoride, complete mini EDTA-free protease inhibitor mixture tablets (1 tablet/7 ml of lysis buffer; Roche Applied Science) and phosphatase inhibitors). Following incubation the samples were clarified at 12,000 rpm for 10 min. The cleared supernatant was collected and the protein content was quantified by the Bradford method (23). For activation assays 50 µg of sample were combined with 2x Laemmli buffer, boiled at 95 °C for 5 min, and spun at 12,000 rpm for 2 min. All KCNQ1 immunoprecipitations were carried out using lysis buffers as described previously (24). 500 µg of sample was removed for co-immunoprecipitation of KCNQ1 and associated kinases. KCNQ1 was immunoprecipitated as follows: 1 µg of total KCNQ1 antibody was added to 500 µg of sample and incubated on a rotor for one hour at 4 °C. 40 µl of washed protein G-Sepharose beads were combined with the immunocomplex. The samples were then incubated overnight on a rotor at 4 °C. Complexes were spun at 12,000 rpm for 10 min and washed three times with ice-cold sterile 1x phosphate-buffered saline. The supernatant was removed, and 35 µl of Laemmli buffer were added and samples were boiled at 95 °C for 5 min and spun at 14,000 rpm for 5 min. Western blot analysis was carried out as standard. Protein was transferred to polyvinylidene difluoride membranes, blocked in 1x TBS with Tween (0.3%) (1x TBST) and 5% nonfat dry milk for 1 h. Membranes were incubated with the appropriate primary antibody overnight at 4 °C and incubated for 1 h at room temperature with the appropriate secondary antibody. Membranes were washed in 1x TBST 0.3% three times for 15 min. Bands were detected using autoradiographic film and chemiluminescence.

PKA Activation Assay—PKA activation was detected using PepTag Assay (Promega) for non-radioactive detection of cAMP-dependent protein kinase according to the manufacturer's instructions with minor modifications. 2.5 µl of the F-Kemptide PepTag and 2.5 µl of the cAMP activator solution were added instead of 5 µl.

RNA Preparation and Reverse Transcriptase (RT) PCR—Total RNA was extracted from male and female rat distal colonic crypt preparations using Qiagen RNeasy kit (Qiagen). Single-strand cDNA was synthesized using the Improm II reverse transcriptase kit (Promega). cDNA was quantified and corrected for loading into RT-PCR reaction mixes. PKC was amplified using the following primers: forward; 5-caccatcttccagaaagaacg-3' and reverse; 5'-cttgccataggtcccgttgttg-3'. -Actin was amplified using the following primers: forward; 5'-cagtaatctccttctgcatcc-3' and reverse; 5'-actacctcatgaagatcctga-3'. GoTaq® polymerase mix from Promega was used in the amplification. Touch-down PCR was used to amplify PKC cDNA for 25 cycles over an annealing temperature range of 65–55 °C. -Actin was amplified for 25 cycles at an annealing temperature of 52 °C. The RT-PCR product was analyzed on a 1.5% 1x Tris acetate-EDTA (TAE) agarose gel and imaged using a UV light source.

Statistical and Densitometric Analysis—Data are presented as mean ± S.E. for a series of the indicated number of experiments. Statistical analysis of the data was obtained by analysis using paired Student's t test for analysis between two groups. One-way analysis of variance and Tukeys post-hoc test was used for multiple analysis of more than two groups. Patch-clamp data analysis was performed using Clampfit software of the p-clamp suite version 9.2 and Origin 7.5 (OriginLab Corp.). Densitometric analysis of Western blots, PKA and RT-PCR images were performed using GeneTools software (SYNGENE).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
17-Estradiol Inhibits Cl^– Secretion Evoked by Forskolin in Rat Colonic Epithelia—We first set out to examine the effect of 17-estradiol (E2) on intestinal secretion elicited by the cAMP agonist forskolin. Basolateral addition of 20 µM forskolin induced an almost instantaneous and sustained increase in I_SC (Fig. 1A). E2 (10 nM) reduced the forskolin-effect on I_SC by 60 ± 6% (n = 5, p < 0.01). The estrogen inhibition of forskolin-induced secretion was observed to be gender dependent. E2 inhibition of forskolin-induced chloride secretion was only observed in tissues from female rats with no significant response to E2 in male tissues (female 60 ± 6% I_SC decrease versus male 6 ± 3%, p < 0.01, n = 5) (Fig. 1A). All Ussing chamber values are maximal inhibition achieved at E2 treatment between 15 and 20 min.

Previous data from our group have demonstrated direct and rapid activation of PKC in response to E2 (25). We therefore investigated a potential role for this kinase in mediating the anti-secretory effects of E2. We used rottlerin as an inhibitor of PKC as it has been reported to inhibit this kinase more potently than other kinases. A previous publication on the inhibitor reported that it inhibits the PKC isoforms at different IC₅₀ concentrations; PKC:3–6 µM, PKC, PKC and PKC: 30–42 µM, PKC, PKC and PKC: 80–100 µM (26). Pretreatment of colonic mucosa with rottlerin (10 µM) completely abolished the anti-secretory action of E2 (E2: 59 ± 6%, E2 + rottlerin, 5 ± 3%, n = 5, p < 0.01) (Fig. 1B). These results indicate that PKC activity is required for the E2-mediated inhibition of forskolin-induced Cl^– secretion.

The Effect of 17-Estradiol on Basolateral Membrane K⁺ Currents in Rat Colonic Epithelia—Basolaterally directed K⁺ currents were generated in female rat colonic epithelia permeabilized apically by pretreatment with amphotericin B. Fig. 2A shows the current/voltage (I_K/V) relationship for apically permeabilized epithelia before and after the addition of the cAMP-dependent agonist forskolin (10 µM). Treatment with forskolin-activated outward currents (apical to basolateral) that displayed slight outward rectification at positive transepithelial voltages and shifted apparent reversal potentials to more negative values consistent with activation of basolateral membrane K⁺ current I_K. Treatment with chromanol 293B (10 µM), a specific KCNQ1 channel blocker, inhibited both the basal and forskolin-activated basolateral membrane K⁺ current suggesting the main component of this current is due to KCNQ1 channel activity (Fig. 2A). Treatment with E2 (10 nM) produced a marked inhibition of forskolin-stimulated I_K (at Vp +100, forskolin 185 ± 19 µA/cm²; forskolin + E2 44 ± 6 µA/cm², n = 3, p < 0.01) and subsequent addition of chromanol 293B had little effect on the remaining K⁺ current (Fig. 2B). Pretreatment of colonic epithelia with rottlerin (10 µM) for 15 min before addition of forskolin did not modify the increase in I_K induced by forskolin but prevented the inhibition by E2 (at Vp +100, forskolin + rottlerin 182 ± 17 µA/cm²; forskolin + rottlerin + E2 158 ± 21 µA/cm², n = 3, p < 0.01) (Fig. 2C). These results suggest that the anti-secretory effect of 17-estradiol is mediated by the inhibition of basolateral KCNQ1 channels via PKC.

View larger version (15K): [in this window] [in a new window]   FIGURE 1. Anti-secretory effect of 17-estradiol on forskolin-induced Cl– secretion is gender-specific and PKC-dependent. A, representative recording of the effect of E2 (10 nM) on forskolin (20 µM)-induced ISC in male (solid line) and female (dashed line) rat colonic epithelia. Colonic mucosas were treated with 20 µM forskolin basolaterally; 17-estradiol was added at the peak of the forskolin response. B, representative recording of the inhibitory effect of rottlerin (10 µM)(solid line) compared with a Me2SO (0.01%) vehicle control (dashed line) on the anti-secretory effect of E2 (10 nM) in female rat distal colon. Values are displayed as ± S.E. (n = 5 for A and B).

View larger version (16K): [in this window] [in a new window]   FIGURE 2. Steady-state short circuit current/voltage relationship (IK/V) of apically permeabilized female rat colonic epithelia in the presence of a basolaterally directed K+ gradient. A, basal IK was strongly activated by forskolin (20 µM) and almost completely blocked by chromanol 293B (293B, 10 µM). B, forskolin-stimulated IK was largely inhibited by 10 nM 17-estradiol, after which chromanol 293B showed little effect. C, inhibitory effect of 17-estradiol on forskolin-stimulated IK was prevented by 15 min of pretreatment with rottlerin (10 µM). Values are displayed as mean ± S.E. (n = 5 for A and n = 3 for B and C).

E2 Effect on KCNQ1 Whole Cell Currents in Female Rat Colonic Crypts—Following membrane breakthrough to the whole cell patch-clamp configuration, the patched cells were allowed to stabilize and dialyze for 5 min. Currents were recorded over this time period before the addition of E2 to ensure stability of the current recording and to check for channel rundown. The experiments involved exposing the isolated colonic crypts to E2 (100 nM) and measuring the whole cell currents at 30-s intervals.

Fig. 3A shows the effects of E2 (100 nM) treatment over time on the whole cell current-voltage relationships. E2 application caused a decrease in whole cell current and its outward rectification over 9 min with a concomitant fall in whole cell membrane conductance at +100 mV from control 278 ± 22 pS to: 160 ± 13 pS at 1 min; 158 ± 11 pS at 3 min; 107 ± 9 pS at 5 min; 82 ± 16 pS at 7 min and 81 ± 25 pS at 9 min (n = 4, p < 0.01). The effect of E2 treatment on whole cell current over time is shown in Fig. 3B. The mean maximal whole cell currents recorded over 5 min prior to E2 addition was 526 ± 50 pA (at Vp =+100 mV). Addition of E2 (100 nM) inhibited the mean maximal current after 5 min to 248 ± 38 pA and to 73 ± 10 pA after 15 min (n = 4, p < 0.05).

Taken together, these results support the conclusion that KCNQ1 channel activity generates the main basolateral membrane K⁺ current, in female colonic crypts, consistent with the Ussing chamber results, and the channel is a target for E2 causing an anti-secretory response.

Gender Comparisons of PKA, PKC, and KCNQ1 Basal Expression Levels in Rat Distal Colonic Crypts—Total untreated cellular lysates of isolated rat distal colonic crypts were prepared, subjected to Western blot analysis, and probed using specific antibodies to endogenous levels of PKARI, PKACI, PKC, and KCNQ1. In all cases expression differences were normalized for loading by probing for total -actin levels. Differences are expressed as fold values of female expression levels compared with male.

PKA isoform I is a cytoplasmic PKA, unlike isoform II which is membrane bound (28). Previous studies in rat colonic crypts demonstrated no significant activation of PKA in the membrane fractions of crypts and a significant activation of PKA in cytosolic fractions (25). Isoform I, the soluble cytosolic isoform, was therefore investigated in this study. Basal expression amounts of PKARI and PKACI were similar between male and female rat distal colonic crypts (PKACI: 1.1 ± 0.2, p > 0.05, n = 3; PKARI: 1 ± 0.2, n = 3, p > 0.05) (Fig. 4, A and B).

View larger version (18K): [in this window] [in a new window]   FIGURE 3. The effect of 17-estradiol on whole cell KCNQ1 current amplitudes in female rat colonic crypts. A, colonic crypt cells were allowed to stabilize and dialyze in whole cell patch-clamp configuration for at least 5 min before exposure to E2 (100 nM). Currents were recorded at 37 °C. Current/voltage relationships were recorded in control (untreated) and E2 exposure at 1, 3, 5, 7, and 9 min as indicated in the graph. B, time dependence of change in mean maximal current (measured at Vp +100 mV). Arrow indicates time of addition of E2. The results shown are mean ± S.E. (n = 4 for A and B) obtained from the last voltage step (+100 mV).

We have found gender differences to exist in the basal expression for novel PKC. In comparison to male, distal colonic crypts from female rats displayed a 3.2 ± 0.42-fold higher expression of the PKC protein (n = 6, p < 0.01) (Fig. 4C).

To further verify the gender difference in expression of PKC we also amplified the messenger transcript levels of the kinase by RT-PCR. Female distal colonic crypts had a significantly higher level of the PKC transcript present compared with male distal colonic crypts (2.1 ± 0.1-fold higher, n = 3, p < 0.01) (Fig. 4D).

We conclude from these expression studies that a significant gender difference exists for PKC, with a higher expression level in female rat distal colonic crypts. No gender differences were detected for PKACI, PKARI, or the KCNQ1 channel protein expression.

View larger version (23K): [in this window] [in a new window]   FIGURE 4. Male and female rat distal colonic crypts express similar levels of both PKA regulatory and catalytic Isoform I subunits and the KCNQ1 channel. Female rat distal colonic crypts express higher levels of PKC. A and B, representative blot of PKACI and PKARI protein levels in cellular extracts from male and female rat distal colonic crypts. C, representative blot of PKC protein levels in cellular extracts from male and female distal colonic crypts. D, representative image of PKC transcript levels from total RNA extracts from male and female distal colonic crypts. E, representative blot of KCNQ1 protein levels in cellular extracts from male and female distal colonic crypts. Values are given as mean pixel intensities of bands for male and female. Values are displayed as mean ± S.E. (n = 3 for A, B, D, and E, and n = 6 for C). ** denotes significance (p < 0.01) between male and female values.

View larger version (33K): [in this window] [in a new window]   FIGURE 5. The anti-secretory effect of 17-estradiol (10 nM) is related to the expression levels of PKC in the colonic tissue. A, inhibitory effect of E2 on forskolin (20 µM) induced Cl– secretion in male and female (proestrus and estrus) colonic mucosa (bar graph) compared with PKC protein levels (line graph and representitive blot). B, PKC transcription is regulated by E2. Colonic crypts from male and female rats were treated with E2 for 30 min. Total mRNA was extracted and converted into cDNA. PKC cDNA was amplified by PCR using specific primers. The figure shows a representative agarose gel of the amplified products. Values on the graph are given as a mean fold increase compared with control samples. Values are displayed as mean ± S.E. (n = 5 for (A) and n = 4 for B). ** and ## denotes significance (p < 0.01) between male and female values.

Gender Differences in PKC and PKA Activation in Response to 17-Estradiol in Rat Distal Colonic Crypts—Isolated male and female rat distal colonic crypts were exposed to E2 (10 nM) or equivalent vehicle (0.01%) for the duration of 2, 5, and 15 min. Total lysates were prepared, subjected to Western blot analysis and probed using phosphospecific antibodies to the Ser⁶⁴³ autophosphorylation site on the PKC protein. In all cases differences were normalized for loading by probing total -actin. For PKA analysis total lysates were analyzed using the PepTag assay for the non-radioactive detection of cAMP-dependent protein. Differences are expressed as fold values of treated over control.

View larger version (25K): [in this window] [in a new window]   FIGURE 6. PKC is rapidly activated in response to 17-estradiol (10 nM) in female rat distal colonic crypts compared with male. A, representative blot of PKC phosphorylation levels at Ser643 in cellular extracts from female rat distal colonic crypts. B, representative blot of PKC phosphorylation levels at Ser643 in cellular extracts from male rat distal colonic crypts. The graphs represent densometric analysis at specific time points of E2 treatment. Values are given as fold changes in PKC phosphorylation (activity) for female and male samples. Values are displayed as mean ± S.E. (n = 3 for A and B). ** denotes significance (p < 0.01) between male and female values.

PKC and PKACI Associate with the KCNQ1 Channel in Female Rat Distal Colonic Crypts in Response to 17-estradiol—Co-immunoprecipitation experiments were preformed to establish whether PKC and PKACI associate with KCNQ1 in response to E2.

Following E2 (10 nM) treatment PKC associated with the KCNQ1 channel protein at 5 and 15 min, with maximal association at 5 min (6.2 ± 1.2-fold higher, n = 3, p < 0.001) (Fig. 8A). The timing of the association correlates with the time response of PKC activation (Fig. 6A). No increase in association between PKC and KCNQ1 was observed in the male tissue after E2 treatment (1.1 ± 0.3, n = 3, p > 0.05) (Fig. 8B).

Following E2 (10 nM) treatment, PKACI rapidly and transiently associated with KCNQ1 at 5 min (3.1 ± 0.4-fold higher, n = 3, p < 0.001) (Fig. 9A). The timing of the PKACI/KCNQ1 association was similar to the time course for PKA activation (Fig. 7A). No association of PKACI with the KCNQ1 channel was detected in the male tissue in response to E2 (0.9 ± 0.1, n = 3, p > 0.05) (Fig. 9B). These results indicate that PKC and PKACI form a multiprotein complex with the KCNQ1 channel in response to E2 in female colonic crypts.

View larger version (31K): [in this window] [in a new window]   FIGURE 7. PKA is rapidly activated in response to 17-estradiol (10 nM) in female rat distal colonic crypts compared with male. A, representative image of PKA phosphorylation of an F-Kemptide PepTag in cellular extracts from female rat distal colonic crypts. B, representative image of PKA phosphorylation of an F-Kemptide PepTag in cellular extracts from male rat distal colonic crypts. The graphs represent densitometric analysis at specific time points of E2 treatment. Values are given as fold changes in PKA phosphorylation of the F-Kemptide PepTag for female and male samples. Values are displayed as mean ± S.E. (n = 5 for A and n = 3 for B). ** denotes significance (p < 0.01) between male and female values.

The latter result indicates that PKC could also regulate PKACI association with the KCNQ1 channel. PKACI associated with KCNQ1 at 5 min and this was abolished by pre-treatment with rottlerin (10 µM) (E2 alone; 2.3 ± 0.3-fold higher, n = 3, p < 0.01; E2 + rottlerin; 0.5 ± 0.2, n = 3, p > 0.05) (Fig. 10B). These results indicate that in addition to regulating PKA activation in response to E2, PKC also regulates PKACI association with the KCNQ1 channel.

View larger version (22K): [in this window] [in a new window]   FIGURE 8. PKC rapidly associates with the KCNQ1 ion channel in response to 17-estradiol (10 nM) in female rat distal colonic crypts and not in the male. Total KCNQ1 pools were immunoprecipitated from total cellular lysates using an antibody specific to KCNQ1. Associated PKC was analyzed by Western blot analysis. A, representative blot of PKC association with the KCNQ1 ion channel in the female rat distal colonic crypts. B, representative blot of PKC association with the KCNQ1 ion channel in the male rat distal colonic crypts. The graphs represent densitometric analysis at specific time points of E2 treatment. Values are given as fold changes in PKC association with KCNQ1 for female and male crypts. Values are displayed as mean ± S.E. (n = 3 for A and B). ** denotes significance (p < 0.01) between male and female values.

View larger version (22K): [in this window] [in a new window]   FIGURE 9. PKACI rapidly associates with the KCNQ1 ion channel in response to 17-estradiol (10 nM) in female rat distal colonic crypts only. Total KCNQ1 pools were immunoprecipitated from total cellular lysates using an antibody specific to KCNQ1. Associated PKACI was analyzed by Western blot analysis. A, representative blot of PKACI association with the KCNQ1 ion channel in the female rat distal colonic crypts. B, representative blot of PKACI association with the KCNQ1 ion channel in the male rat distal colonic crypts. The graphs represent densitometric analysis at specific time points of E2 treatment. Values are given as fold changes in PKACI association with KCNQ1 for female and male samples. Values are displayed as mean ± S.E. (n = 3 for A and B). ** denotes significance (p < 0.01) between male and female values.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

View larger version (22K): [in this window] [in a new window]   FIGURE 10. PKA activation and PKACI association with the KCNQ1 channel in response to 17-estradiol (10 nM) is PKC-dependent in female rat distal colonic crypts. A, representative image of PKA phosphorylation of an F-Kemptide PepTag in cellular extracts from female rat distal colonic crypts. Rottlerin (10 µM), a specific PKC inhibitor, prevented this activation. B, representative blot of PKACI association with the KCNQ1 channel in the female rat distal colonic crypts. Rottlerin (10 µM) prevented this association. The graphs represent densitometric analysis at specific time points of E2 treatment. Values are given as fold changes in F-Kemptide phosphorylation (A) and fold changes in PKACI association with KCNQ1 (B) for female crypts. Values are displayed as mean ± S.E. (n = 3 for A and B). * and ** denotes significance (p < 0.05, p < 0.01) between male and female values.

View larger version (21K): [in this window] [in a new window]   FIGURE 11. The KCNQ1 K+ ion channel is serine-phosphorylated in response to 17-estradiol. The KCNQ1 channel phosphorylation is PKC/PKA dependent. Female colonic crypts were stimulated with E2 (10 nM) for 10 min and the KCNQ1 was immunoprecipitated. Phosphorylation of the channel was detected using an antibody to phosphoserine. Pretreatment with rottlerin (10 µM) and H89 (10 µM) prevented the E2-induced phosphorylation of the channel. The graph represents densitometric analysis of specific treatments. Values are given as fold changes in KCNQ1 serine phosphorylation for female samples. Values are displayed as mean ± S.E. (n = 3). ** denotes significance (p < 0.01) between control and treatments.

It is becoming increasingly evident that non-genomic effects prime latent genomic functions of protein expression and cell differentiation (30). Non-genomic and genomic cross-talk works in both directions and it is untenable to consider rapid and latent responses as unrelated physiological processes.

This is the first report of a gender-specific hormonal modulation of the KCNQ1 channel. Also, this is the first report of modulation of KCNQ1 channels by the novel PKC. The KCNQ1 channel is known to be regulated by multiprotein complexes containing phosphatases, protein kinases, and A Kinase Anchoring Proteins (AKAPs). It has previously been reported that PKA is capable of interacting with and phosphorylating cardiac KCNQ1 at Ser²⁷ via an AKAP, specifically, yotiao (34). The latter study demonstrated PKA phosphorylation of the KCNQ1 channel through the recruitment of a multiprotein complex that included PKA, protein phosphatase I (PPI) and the AKAP yotiao. The requirement for the regulation of the KCNQ1 channel by a macromolecular complex may increase the specificity of the channel targeting by signaling pathways. Our results demonstrate an interaction of PKC and PKACI with the KCNQ1 channel in response to E2 in female rat distal colonic crypts and not in males. Phosphorylation of the KCNQ1 channel by PKA may be the molecular mechanism for E2 inhibition of KCNQ1 currents.

In this study we demonstrated that E2 regulates KCNQ1 channel function indirectly via kinases which results in inhibition of secretagogue stimulated Cl^– secretion. This is an important physiological response, which provides one of the few known examples for a physiological meaning to E2-induced rapid responses. Several studies have shown variability in the response of KCNQ1 to PKA- or PKC-induced phosphorylation (18, 24). A reason for this is the requirement of accessory proteins to transduce protein phosphorylation into channel function. As an example, it has been found in cardiac cells that association of KCNQ1 with its -subunit KCNE1 and to other proteins such as Yotiao is necessary to translate the phosphorylated KCNQ1 subunit into altered channel activity. Therefore, tissue differences in the expression of regulatory proteins could account for the differences reported in the channel response to PKA. In unpublished data from our group we have found gender differences in the expression and association of KCNE subunits in colonic epithelia. Also, PKA regulation of channel activity may be dependent on the activated isoforms of adenylyl cyclase or PKA that are activated and their subcellular localization with cAMP.

An interesting paradox exists in the anti-secretory pathway. The induction of Cl^– secretion by forskolin requires an increase in cAMP activity, however, E2 also increases cAMP activity in rat distal colonic crypts (25). Why, therefore, does activation of PKA by E2 not lead to increased CFTR activity and therefore further stimulate Cl^– secretion rather than exert an anti-secretory effect? It has previously been reported that CFTR activation is via PKA catalytic subunit isoform II (PKACII) which is a membrane-bound insoluble isoform of the kinase (35, 36). The authors have previously shown that E2 does not activate membrane bound PKA in the rat colonic crypt (25). This fraction contains PKACII, the activator of CFTR. Our data, however have shown that it is the PKA isoform, PKACI that is activated by E2 (25), and the current study demonstrates E2 induces association of PKACI with the KCNQ1 channel. Thus CFTR is not a target for estrogen and inhibition of the KCNQ1 channel provides the molecular basis for the anti-secretory response. PKACI has been reported to be recruited by the scaffolding protein AKAP (37) and in our case this may be a mechanism for targeting PKACI to KCNQ1. Other mechanisms for membrane specific targeting of kinases have been well-described involving subcellular compartmentalization of PKA isoforms and their spatial restriction to either the basolateral or apical membrane or indeed a restriction to a particular signaling complex (38). In epithelial cells local pools of cAMP exist which result in the activation of PKA in highly localized areas of the cell (28).

In summary, the present study has identified the ion transporter target in the anti-secretory response to estrogen in distal colon and has provided a molecular explanation for its female gender-specificity. E2 inhibits KCNQ1 channel current indirectly via PKC and PKACI resulting in the inhibition of Cl^– secretion. The anti-secretory response may also be a contributory factor by which estrogen causes whole body salt and water retention during periods of high circulating estrogen such as certain stages of the menstrual cycle (39), pregnancy (40) and during the use of oral contraceptives (41) and hormone replacement therapy (42). The regulation of the KCNQ1 channel by E2, reported here, provides important leads in the investigation into molecular mechanisms controlling intestinal secretory disorders and drug targeting.

	FOOTNOTES

 
^* This work was supported in part by The Wellcome Trust Programme Grant 06089/Z/00/Z and Higher Education Authority of Ireland (HEA) PRTLI Grants Cycle 1 and 3. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.

² Recipient of a Wellcome Trust Studentship Prize 06379/Z/01/Z.

¹ To whom correspondence should be addressed: Dept. of Molecular Medicine, Royal College of Surgeons in Ireland, Smurfit Bldg., Beaumont Hospital, PO Box 9063, Dublin 9, Ireland. Tel.: 00-353-1-8093824; Fax: 00-353-1-8093778; E-mail: fomahony{at}rcsi.ie.

³ The abbreviations used are: CFTR, cystic fibrosis transmembrane conductance regulator; PKC, protein kinase C; PKA, cAMP-dependent protein kinase A.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

1. Barrett, K. E., and Keely, S. J. (2000) Annu. Rev. Physiol. 62, 535–572[CrossRef][Medline] [Order article via Infotrieve]
2. Field, M., and Semrad, C. E. (1993) Annu. Rev. Physiol. 55, 631–655[CrossRef][Medline] [Order article via Infotrieve]
3. Crocker, A. D. (1971) J. Physiol. 214, 257–264[Abstract/Free Full Text]
4. Harvey, B. J., Condliffe, S., and Doolan, C. M. (2001) News Physiol. Sci. 16, 174–177[Abstract/Free Full Text]
5. Thomas, M. L., Xu, X., Norfleet, A. M., and Watson, C. S. (1993) Endocrinology 132, 426–430[Abstract/Free Full Text]
6. Condliffe, S. B., Doolan, C. M., and Harvey, B. J. (2001) J. Physiol. 530, 47–54[Abstract/Free Full Text]
7. McNamara, B., Winter, D. C., Cuffe, J. E., O'Sullivan, G. C., and Harvey, B. J. (1999) J. Physiol. 519, 251–260[Abstract/Free Full Text]
8. Liao, T., Wang, L., Halm, S. T., Lu, L., Fyffe, R. E., and Halm, D. R. (2005) Am. J. Physiol. Cell Physiol. 289, C564–C575[Abstract/Free Full Text]
9. Kunzelmann, K., Hubner, M., Schreiber, R., Levy-Holzman, R., Garty, H., Bleich, M., Warth, R., Slavik, M., von Hahn, T., and Greger, R. (2001) J. Membr. Biol. 179, 155–164[CrossRef][Medline] [Order article via Infotrieve]
10. Halm, S. T., Liao, T., and Halm, D. R. (2006) Am. J. Physiol. Cell Physiol. 291, C636–C648[Abstract/Free Full Text]
11. Jespersen, T., Grunnet, M., and Olesen, S. P. (2005) Physiology (Bethesda) 20, 408–416[CrossRef][Medline] [Order article via Infotrieve]
12. Warth, R., and Bleich, M. (2000) Rev. Physiol. Biochem. Pharmacol. 140, 1–62[Medline] [Order article via Infotrieve]
13. Moller, C., and Netzer, R. (2006) Eur. J. Pharmacol. 532, 44–49[CrossRef][Medline] [Order article via Infotrieve]
14. Steinberg, S. F. (2004) Biochem. J. 384, 449–459[CrossRef][Medline] [Order article via Infotrieve]
15. Di Mari, J. F., Mifflin, R. C., and Powell, D. W. (2005) Gastroenterology 128, 2131–2146[CrossRef][Medline] [Order article via Infotrieve]
16. Song, J. C., Hanson, C. M., Tsai, V., Farokhzad, O. C., Lotz, M., and Matthews, J. B. (2001) Am. J. Physiol. Cell Physiol. 281, C649–C661[Abstract/Free Full Text]
17. Doolan, C. M., and Harvey, B. J. (1996) J. Biol. Chem. 271, 8763–8767[Abstract/Free Full Text]
18. Kurokawa, J., Motoike, H. K., Rao, J., and Kass, R. S. (2004) Proc. Natl. Acad. Sci. U. S. A. 101, 16374–16378[Abstract/Free Full Text]
19. Hubscher, C. H., Brooks, D. L., and Johnson, J. R. (2005) Biotech. Histochem. 80, 79–87[CrossRef][Medline] [Order article via Infotrieve]
20. Nequin, L. G., Alvarez, J., and Schwartz, N. B. (1979) Biol. Reprod. 20, 659–670[Abstract]
21. Schultheiss, G., Lan Kocks, S., and Diener, M. (2002) Biol. Proced. Online 3, 70–78[Medline] [Order article via Infotrieve]
22. Hamill, O. P., Marty, A., Neher, E., Sakmann, B., and Sigworth, F. J. (1981) Pflugers Arch. 391, 85–100[CrossRef][Medline] [Order article via Infotrieve]
23. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254[CrossRef][Medline] [Order article via Infotrieve]
24. Kurokawa, J., Chen, L., and Kass, R. S. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2122–2127[Abstract/Free Full Text]
25. Doolan, C. M., Condliffe, S. B., and Harvey, B. J. (2000) Br. J. Pharmacol. 129, 1375–1386[CrossRef][Medline] [Order article via Infotrieve]
26. Gschwendt, M., Muller, H. J., Kielbassa, K., Zang, R., Kittstein, W., Rincke, G., and Marks, F. (1994) Biochem. Biophys. Res. Commun. 199, 93–98[CrossRef][Medline] [Order article via Infotrieve]
27. Unsold, B., Kerst, G., Brousos, H., Hubner, M., Schreiber, R., Nitschke, R., Greger, R., and Bleich, M. (2000) Pflugers Arch. 441, 368–378[CrossRef][Medline] [Order article via Infotrieve]
28. Tasken, K., and Aandahl, E. M. (2004) Physiol. Rev. 84, 137–167[Abstract/Free Full Text]
29. Reenstra, W. W. (1993) Am. J. Physiol. 264, C161–C168[Medline] [Order article via Infotrieve]
30. Falkenstein, E., Tillmann, H. C., Christ, M., Feuring, M., and Wehling, M. (2000) Pharmacol. Rev. 52, 513–556[Abstract/Free Full Text]
31. Zangar, R. C., Reiners, J. J., Jr., and Novak, R. F. (1995) Carcinogenesis 16, 2593–2597[Abstract/Free Full Text]
32. Cutler, R. E., Jr., Maizels, E. T., Brooks, E. J., Mizuno, K., Ohno, S., and Hunzicker-Dunn, M. (1993) Biochim. Biophys. Acta 1179, 260–270[Medline] [Order article via Infotrieve]
33. Shanmugam, M., Krett, N. L., Maizels, E. T., Cutler, R. E., Jr., Peters, C. A., Smith, L. M., O'Brien, M. L., Park-Sarge, O. K., Rosen, S. T., and Hunzicker-Dunn, M. (1999) Mol. Cell. Endocrinol. 148, 109–118[CrossRef][Medline] [Order article via Infotrieve]
34. Marx, S. O., Kurokawa, J., Reiken, S., Motoike, H., D'Armiento, J., Marks, A. R., and Kass, R. S. (2002) Science 295, 496–499[Abstract/Free Full Text]
35. Steagall, W. K., Kelley, T. J., Marsick, R. J., and Drumm, M. L. (1998) Am. J. Physiol. 274, C819–C826[Medline] [Order article via Infotrieve]
36. Singh, A. K., Tasken, K., Walker, W., Frizzell, R. A., Watkins, S. C., Bridges, R. J., and Bradbury, N. A. (1998) Am. J. Physiol. 275, C562–C570[Medline] [Order article via Infotrieve]
37. Smith, F. D., Langeberg, L. K., and Scott, J. D. (2006) Trends Biochem. Sci 31, 316–323[CrossRef][Medline] [Order article via Infotrieve]
38. Cooper, D. M. (2005) Biochem. Soc. Trans. 33, 1319–1322[CrossRef][Medline] [Order article via Infotrieve]
39. Michell, A. R. (1981) Q. J. Exp. Physiol. 66, 515–524[Abstract/Free Full Text]
40. Atherton, J. C., Dark, J. M., Garland, H. O., Morgan, M. R., Pidgeon, J., and Soni, S. (1982) J. Physiol. 330, 81–93[Abstract/Free Full Text]
41. Blahd, W. H., Lederer, M. A., and Tyler, E. T. (1974) J. Reprod. Med. 13, 223–225[Medline] [Order article via Infotrieve]
42. Stevenson, J. C. (2006) J. Br. Menopause Soc. 12, 8–10[CrossRef][Medline] [Order article via Infotrieve]

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