1Department of Neuroscience, Cell Biology and Physiology, Wright State University, Dayton, Ohio; and 2Division of Molecular Medicine, Harbor-UCLA Medical Center, David Geffen School of Medicine, University of California, Los Angeles, Torrance, California
Submitted 19 November 2004 ; accepted in final form 12 April 2005
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ABSTRACT |
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HMR1556; K+ secretion; epinephrine; prostaglandin E2; cholinergic
The colonic epithelium of mammals is composed of a relatively flat surface epithelium invaginated by numerous crypts of Lieberkühn (12). Within this epithelium, columnar cells and goblet cells are the predominant cell types, with a minor population of enteroendocrine cells. Goblet cells are distinguished from columnar cells by a large, dense cluster of apical mucous granules (59). Other cell types also are present in the colonic mucosa, including myoepithelial cells forming the pericryptal sheath, capillaries, and nerve fibers, as well as various types of leukocytes (8, 9, 48, 51). Previous studies support the concept that crypt columnar cells are major contributors to transepithelial ion secretion and mucus release (28, 29, 31, 32).
The two types of K+ channels proposed as the major components of basolateral membrane K+ conductance that support colonic Cl secretion, Kcnn4 and Kcnq1, have been observed in cells of isolated colonic crypts (24, 63). The intermediate conductance inwardly rectifying K+ channel, Kcnn4, could be the Ca2+-activated basolateral membrane K+ conductance supporting cholinergic stimulation (34, 63). In addition, a Ca2+-dependent inward rectifier that may be Kcnn4 has been observed in human colonic crypts (56). Association of Kcnq1 with minK-related peptide 2 (MiRP2, Kcne3) produces a cAMP-activated basolateral membrane K+ conductance (24, 63) that could support Cl-secretory activation by secretagogues such as vasoactive intestinal peptide or prostaglandin E2 (PGE2) (11, 27, 50), and both of these channel proteins have been localized to lateral membranes of mouse colonic crypts (14, 58). The involvement of Kcnq1/Kcne3 (KVLQT1/MiRP2) K+ channels in colonic Cl secretion is supported further by the inhibition of cAMP-dependent secretion and channel activity by the chromanol 293B (24, 43, 58, 63). In contrast, even though 293B inhibited cAMP-dependent K+ currents in pancreatic acinar cells consistent with Kcnq1/Kcne1 (37, 62), 293B did not inhibit fluid secretion (38). However, the importance of cAMP as an intracellular signal that modulates secretory activity still makes the cellular location of Kcnq1 a useful indicator of possible secretory function.
Secretagogues operating through cAMP-dependent mechanisms produce two distinct modes of electrogenic ion secretion in the colonic epithelium (27, 30, 41). The more familiar mode involves Cl secretion that is also accompanied by electrogenic K+ secretion. This flow of ions into the lumen creates fluid buildup in crypt lumens, producing fluid flow that sweeps along mucus and other material (28), such that the term flushing secretion best summarizes the action of these secretagogues. The second secretory mode produces electrogenic K+ secretion, but without large, sustained Cl secretion. Modulatory secretion is a useful term to conceptualize this secretory function because fluid flow is low, but ion composition would be altered. Rat and guinea pig distal colon both produce these modes of secretion (53, 64), with differences in rates that may serve the specific physiology of an omnivore and a herbivore, respectively (52, 54). Thus a comparison of these two modes of secretion in these species can be used to demonstrate the varied roles of basolateral membrane K+ channels. In particular, increased basolateral membrane K+ channel activity would aid Cl secretion by enhancing the electrochemical driving force for conductive apical Cl exit, whereas decreased activity could increase K+ secretion by limiting basolateral exit of K+ into the interstitial space (40). The focus of this study was to examine the epithelial location of Kcnq1/Kcne3 K+ channels in the colon and to use the chromanol derivative HMR1556 (22) to determine the involvement of this K+ channel type in secretory activation by physiological secretagogues.
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METHODS |
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Tissue fixation. Colonic tissues were fixed either by perfusion of fixative or after isolation of the mucosa. For perfusion-fixation, animals were perfused transcardially (20) with a vascular rinse solution (4°C), followed by 4% paraformaldehyde in phosphate buffer (PB). The colon was removed, cut into annuli, and postfixed with 4% paraformaldehyde in PB for 1 h. Perfusion-fixation did not produce satisfactory structural preservation in guinea pig colonic mucosa. Fixation also was accomplished by pinning isolated mucosal sheets in a Sylgard-coated dish for immersion in fixation solutions. Each mucosal specimen was fixed in PB containing 1% paraformaldehyde and 0.125% glutaraldehyde (15 min at room temperature). The mucosal specimens were fixed further in PB with 4% paraformaldehyde (20 min at room temperature). Chemicals used for the preparation of solutions were obtained from Sigma Chemical (St. Louis, MO). Vascular rinse solution contained (in mM) 161 Na+, 3.4 K+, 140 Cl, 6.0 HCO3, 1.9 H2PO4, and 8.1 HPO42. PB contained (in mM) 181 Na+, 19 H2PO4, and 81 HPO42. Phosphate-buffered saline (PBS) contained (in mM) 168 Na+, 2.7 K+, 153 Cl, 1.9 H2PO4, and 8.1 HPO42. Tris-buffered saline (TBS) contained (in mM) 137 Na+, 155 Cl, and 20 Tris.
Immunolocalization.
Mucosal tissues were prepared for immunofluorescence (2, 20) by dehydration in PB (4°C) with sucrose (15% wt/vol) and then frozen with optimal cutting temperature compound. Sections were cut (6 µm) on a cryostat and thaw mounted on gelatin-coated slides. Sections were permeabilized with PBST (PBS with 0.1% Triton X-100; 30 min), blocked in PBST with normal horse serum (10%, 1 h, room temperature), and then incubated (4°C) overnight with primary antibody in PBST. The following antibodies for K+ channel and auxiliary subunits were obtained from commercial suppliers (Chemicon International, Temecula, CA; Santa Cruz Biotechnology, Santa Cruz, CA; Jackson ImmunoResearch, West Grove PA): polyclonal anti-KVLQT1 (6.5 ng/µl; Chemicon, COOH-terminal residues of human Kcnq1), two polyclonal anti-Kcne3 [4.0 ng/µl, Santa Cruz Biotechnology, internal domain (L-20) and NH2-terminal residues (N-18) of human Kcne3], and polyclonal anti-metabotropic glutamate receptor (1.0 ng/µl, Chemicon; residues 11801191 of rat mGluR1-, Grm1). After being washed three times in PBS, sections were incubated in the dark with the appropriate secondary antibodies (Jackson ImmunoResearch) and donkey-anti-rabbit or donkey-anti-goat IgG antibody conjugated to fluorescein isothiocyanate (FITC; 15 ng/µl for 2 h at room temperature). Sections were washed and mounted in Vectashield (Vector Laboratories, Burlingame, CA). Absorption controls were preformed by preincubation of primary antibody with the antigenic peptide in PBS (6090 min at room temperature) before addition to sections. Fluorescence was visualized using an Olympus BX60 epifluorescence microscope.
Detection of ion channel proteins also was accomplished using immunoblot analysis. Isolated colonic mucosa was disrupted by performing sonication in a buffered solution containing protease inhibitors (on ice). The isolation solution contained (in mM) 178 Na+, 1.5 Mg2+, 153 Cl, 50 HEPES, 10 EDTA, 10% glycerol, 1% Triton X-100, and 1.0 4-(2-aminoethyl)benzenesulfonyl fluoride, as well as (in µM) 1.54 aprotinin, 23.5 leupeptin, and 14.6 pepstatin A. Samples were centrifuged at 6,000 g (for 10 min at 4°C) followed by centrifugation of the resulting supernatant at 100,000 g (for 60 min at 4°C) to obtain a membrane sample; protein content was determined using the Bradford method (7). Proteins were electrophoresed by performing SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. These membranes were blocked with 10% nonfat dry milk in TBST (TBS with 0.1% Tween 20), followed by incubation with specific primary antibody and then with horseradish peroxidase-conjugated secondary antibody. Membranes were developed (90 s) with LumiGLO (Cell Signaling Technology, Beverly, MA) before film was exposed to detect the product.
Transepithelial current measurement.
Isolated mucosal sheets were used for measurement of transepithelial current and conductance (30, 53). Four mucosal sheets from each animal were mounted in Ussing chambers (0.64-cm2 aperture) and supported on the serosal face by Nuclepore filters (10 µm thick, 5-µm pore diameter; Whatman, Clifton NJ). Bathing solutions (10 ml) were circulated by gas lift through water-jacketed reservoirs (38°C). Standard Ringer solution contained (in mM) 145 Na+, 5.0 K+, 2.0 Ca2+, 1.2 Mg2+, 125 Cl, 25 HCO3, 4.0 H(3X)PO4X, and 10 D-glucose. Solutions were continually gassed with 95% O2-5% CO2, which maintained the solution at pH 7.4. Chambers were connected to automatic voltage clamps (Physiologic Instruments, San Diego, CA) that permitted compensation for solution resistance and continuous measurement of short-circuit current (Isc). Transepithelial electrical potential difference was measured using paired calomel electrodes connected to the chambers by Ringer-agar bridges. Current was passed across the tissue through two Ag-AgCl electrodes connected by Ringer-agar bridges. Isc was referred to as positive for flow across the epithelium from the mucosal to the serosal side. Transepithelial conductance (Gt) was calculated on the basis of currents produced by bipolar square voltage pulses imposed across the mucosa (±5 mV, 3-s duration, 1-min intervals).
PGE2, indomethacin, and NS398 were obtained from Cayman Chemical (Ann Arbor, MI), and epinephrine (EPI) was purchased from Elkins-Sinn (Cherry Hill, NJ). K+ channel blockers HMR1556 {(3R,4S)-(+)-N-[3-hydroxy-2,2-dimethyl-6-(4,4,4-trifluorobutoxy)chroman-4-yl]-N-methylethanesulfonamide} and 293B [trans-6-cyano-4-(N-ethylsulfonyl-N-methylamino)-3-hydroxy-2,2-dimethylchromane] were provided by Dr. Uwe Gerlach (Aventis Pharma Deutschland, Frankfurt-am-Main, Germany). All other chemicals were obtained from Sigma Chemical. Drugs were added in small volumes from concentrated stock solutions. PGE2 was prepared in an ethanol stock solution that added 0.03% ethanol at 3 µM PGE2. Stock solutions of HMR1556 (10 mM) and 293B (100 mM) were made with DMSO. Additions of 1% ethanol or DMSO alone did not alter transepithelial measures of K+ or Cl secretion (30).
Inhibitor-sensitive components of Isc and Gt were calculated using the paired responses of adjacent mucosal tissues. Stripchart recordings of Isc were digitized at 10-s intervals to examine secretory onset. Concentration responses of Isc to inhibitors were fit to Henri-Michaelis-Menten binding curves using a nonlinear least-squares procedure (30). Results are reported as means ± SE. Statistical comparisons were performed using a two-tailed Students t-test for paired responses, with statistically significant differences accepted at P < 0.05.
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RESULTS |
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The use of the secondary antibody alone eliminated all membrane labeling of epithelial cells observed in rat (Fig. 1B) and guinea pig colon (data not shown), indicating that the Kcnq1 antibody was necessary for the observed labeling. The antigenic peptide was not available for preabsorption of the Kcnq1 antibody as a further control for nonspecific reactions. An antibody against the metabotropic glutamate receptor was used as an additional control for nonspecific labeling of membrane proteins, but no mucosal labeling was detected with this antibody (data not shown). In perfusion-fixed specimens, the surface epithelium had a diffuse, low-level labeling (Fig. 1A), predominantly in the cytoplasm (darker nuclei), which also was evident in the absence of the primary antibody (Fig. 1B). The bright cells in the interstitium were probably leukocytes as reported previously, with visibility likely due to autofluorescence of granule contents and nonspecific binding of the secondary antibody (8, 48).
Immunoreactivity for the K+ channel regulatory protein MiRP2, Kcne3, was detected in the lateral membrane of guinea pig colonic crypt epithelia (Fig. 3A), consistent with previous reports for mouse colon using immunolocalization and patch-clamp recording of channel activity (14, 58). Lateral membranes of surface cells also labeled for Kcne3 (Fig. 3, C and D). All of the membrane labeling was eliminated by preabsorption of the primary antibody with the antigenic peptide (Fig. 3B). The labeling in crypt and surface epithelial cells occurred with a beaded appearance, suggesting that Kcne3 clustered at sites along the lateral membrane. Neither apical nor basal membranes in surface cells showed detectable labeling.
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Distinct secretory states were produced after attaining the basal condition by adding specific secretagogues (30, 53). Sustained electrogenic K+ secretion of the modulatory type was stimulated by addition of either EPI (5 µM) or PGE2 at low concentration (5 nM). Addition of PGE2 at high concentration (3 µM) stimulated flushing-type secretion consisting of sustained Cl secretion together with K+ secretion. Adding carbachol (CCh; 10 µM) cumulatively with PGE2 (3 µM) produced a further large synergistic increase in Cl secretion with a transient component lasting 1020 min. Each of these distinct secretory modes was examined for sensitivity to Kcnq1 inhibitors, including 1) modulatory-type K+ secretion, 2) flushing-type Cl and K+ secretion, and 3) synergistic Cl secretion.
Inhibition of secretory modes.
The chromanol 293B has been shown to inhibit the Kcnq1 K+ channel as well as Cl secretion (21, 43, 45, 62, 63). A higher-affinity inhibition of Kcnq1 is obtained with the 293B derivative HMR1556 (22); from different cell types, the IC50 for HMR1556 ranges from 7 to 170 nM (5, 23, 37, 39, 61). In rat colonic mucosa (Fig. 5), HMR1556 (10 µM) inhibited a portion of the flushing-type Cl-secretory Isc and Gt stimulated by PGE2 but did not alter the modulatory-type K+-secretory Isc and Gt stimulated by EPI. Inhibition was rapid for both Isc and Gt. The HMR1556-sensitive component of the PGE2 response reached a maximum within 10 min and remained stable with only a slight decline over 15 min (Fig. 5C). The HMR1556-sensitive and HMR1556-resistant components of the PGE2-secretory response were similar in size.
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The secretory responses in the rat were dependent on the position along the colon (Fig. 7), with a larger EPI-stimulated, modulatory K+ secretion at more distal sites and a larger PGE2-stimulated, flushing Cl secretion at more proximal sites. The Isc and Gt in basal and PGE2 conditions were similar to earlier results found using mucosal preparations of rat distal colon (3, 15). In addition, Gt tended to be larger at proximal sites, similar to findings in earlier studies (33, 57). The PGE2-stimulated Cl-secretory response was calculated as the difference between the Isc after PGE2 addition and the prior EPI-stimulated Isc to include the full range of secretory capacity (Fig. 7B). The HMR1556-sensitive and HMR1556-resistant components of this PGE2-stimulated Cl-secretory response in rat colon were not different in size at any position examined (Fig. 7, B and C). The IC50 was 55 ± 11 nM for this HMR1556 inhibition of the PGE2-stimulated Cl-secretory response (Fig. 8). For the guinea pig distal colon, the EPI-stimulated, modulatory Isc, and PGE2-stimulated flushing Isc were approximately 100 µA/cm2 and +120 µA/cm2, respectively, in both early and late portions, and the lack of inhibition by HMR1556 also was similar along the length of the distal colon.
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DISCUSSION |
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Voltage-sensitive K+ channels (KV) are not obvious choices as components of epithelial transport mechanisms, because changes in membrane electrical potential differences are relatively modest compared with other cell types, such as neurons and muscle cells. However, in addition to voltage-dependent gating, other signaling cascades regulate many of the KV channels. In particular, KVLQT1 (Kcnq1) is activated by cAMP-dependent mechanisms (36, 55, 58, 65), possibly acting through protein kinase A, and has been shown by several means to contribute to the basolateral membrane K+ conductance necessary for electrogenic Cl secretion. The presence in the colon of the mRNA encoding Kcnq1 was shown using Northern blot analysis (14, 65), and in situ hybridization confirmed that colonic crypt epithelial cells contained this message (58). With the use of rat crypt epithelial cells, previous investigators obtained a full-length cDNA (669 amino acids) of Kcnq1 that was 90% identical to the amino acid sequence for human Kcnq1 (36). The involvement of Kcnq1 in Cl secretion was demonstrated using the chromanol 293B that inhibits both Kcnq1-dependent currents (6, 36, 58), as well as a current component in rat colonic crypt cells and transepithelial electrogenic Cl secretion in rat and mouse colon (36, 43, 45, 58, 62).
Immunofluorescence labeling of mouse colon for Kcnq1 supports a presence of this K+ channel in the lateral membrane of crypt epithelial cells (14, 62). Similarly, guinea pig and rat colon showed lateral membrane localization of Kcnq1 immunoreactivity (Figs. 1 and 2). Although nonspecific staining obscured detection of Kcnq1 in surface epithelial cells of perfusion-fixed rat colon, the low background staining in isolation-fixed guinea pig and rat mucosa allowed a clear determination that Kcnq1 also was localized to the lateral membrane of surface epithelial cells (Figs. 1 and 2). Further support for the presence of Kcnq1 in surface epithelial cells was indicated using RT-PCR to detect Kcnq1 mRNA in rat colonic surface cells (36). Similar to results in other studies of colonic mucosa (14, 36, 58), Kcnq1 was present together with MiRP2 (Kcne3) (Fig. 3), an auxiliary subunit that modifies gating kinetics and inhibitor sensitivity (6, 36, 49, 55, 58). Of course the mere localization of both Kcnq1 and Kcne3 to the same membrane using immunofluorescence does not prove a functional connection. However, 293B-sensitive currents have been measured in rat colonic crypt cells with properties similar to defined Kcnq1/Kcne3 currents (36, 63), such that these two K+ channel subunits likely combine to form a component of lateral membrane K+ conductance. Also, the low IC50 for HMR1556 (Fig. 8) supports the involvement of Kcnq1/Kcne3 rather than Kcnq1 alone (39). The additional finding of both Kcnq1 and Kcne3 in lateral membranes of colonic surface epithelial cells (Figs. 2 and 3) suggests that these two subunits also combine at this location to produce the weakly voltage-dependent K+ channel characteristic of Kcnq1/Kcne3. Thus the presence of this K+ channel type does not distinguish surface cells from crypt cells, but perhaps instead a distinction between these cell types occurs at the level at which signaling pathways activate these channels to augment K+ flow.
Ion transport characteristics vary along the length of the colon with amiloride-sensitive Na+ absorption occurring predominantly at distal sites (18, 52, 54). A similar gradient of amiloride-sensitive Isc was detected in this study (Fig. 7B). The response to physiological secretagogues also was examined and showed a gradient along the length of the rat colon. The Isc stimulated by the flushing secretagogue PGE2 was 40% higher at proximal positions compared with more distal positions. For stimulation by the modulatory secretagogue EPI, a sustained Isc was apparent only at distal positions. These results are consistent with an earlier study in which the investigators used a mucosal-submucosal preparation (33), except that the basal Isc was generally higher (by 2050 µA/cm2) than in the present study (Fig. 7A), suggesting a difference in secretory status.
The modulatory mode of secretion, characterized by sustained electrogenic K+ secretion without sustained Cl secretion, may be considered the most fundamental secretory mode in the distal colon. This concept is supported by experiments in which colonic mucosa were stimulated after the secretory influences from nerves and endogenous production of paracrine factors were first reduced. Stimulation from a quiescent basal state by -adrenergic (27, 53, 64) (Figs. 5 and 6), prostanoid EP2 subtype (30, 53), and cholinergic agonists (10, 46) (Figs. 10 and 11) all result in modulatory K+ secretion. In addition, stimulation with a low concentration of forskolin, which activates adenylyl cyclase to produce cAMP, also leads to modulatory secretion (41), consistent with the action of
-adrenergic and EP2 prostanoid receptors to increase cellular cAMP. The cholinergic activation of modulatory secretion from a quiescent state (Fig. 10) indicates that other second messengers also are capable of activating this secretory mode and indicates that a distinct secretory state exists compared with the cholinergically induced reduction in flushing-mode K+ secretion of rabbit distal colon (16).
Addition of the lipid-soluble 293B or HMR1556 could block either apical or basolateral K+ channels, but the localization of Kcnq1 to the basolateral membrane (Figs. 1 and 2) suggests that these inhibitors would act to enhance modulatory K+ secretion by diverting K+ exit to the apical membrane. Because neither HMR1556 nor 293B altered the modulatory response (Figs. 5, 6, and 11) similarly to results with 293B in human and cystic fibrosis mouse colon (45, 47), Kcnq1 appears to be largely inactive during the modulatory mode of secretion.
Flushing secretion is driven by electrogenic Cl secretion, together with an accompanying electrogenic K+ secretion, and is elicited by several types of secretagogues. In particular, PGE2 is produced within the mucosa and at high concentration activates the flushing mode via receptors distinct from the EP prostanoid type (30). The regulatory mechanism stimulating flushing secretion likely involves cAMP because at high concentration forskolin produces large Cl-secretory Isc (11, 50). In the guinea pig distal colon, increasing forskolin concentration reverses a negative Isc to a positive Isc, which is indicative of conversion from modulatory secretion to flushing secretion (41). The flushing-type Cl-secretory Isc stimulated in colonic mucosa from human (46), mouse (45, 62), rabbit (43), and rat (36, 64) by either forskolin or inhibitors of phosphodiesterase was inhibited from 60 to 90% with the chromanol 293B. In rabbit distal colon, flushing secretion produced by the secretagogues PGE2, adenosine, and vasoactive intestinal peptide was inhibited 7080% by 293B (43).
A difficulty with assigning a quantitatively specific role for Kcnq1 on the basis of 293B inhibition is that 293B also inhibits the Cl channel CFTR with an IC50 of 2030 µM (4). Because CFTR is a component of the apical Cl conductance needed for Cl secretion (50), the potency of 293B may result from action at both secretory K+ and Cl conductance. The chromanol derivative HMR1556 inhibits Kcnq1 with 100-fold higher affinity than 293B (22, 23), such that any similar nonspecificity would not be encountered at concentrations sufficient to inhibit Kcnq1. In addition, the dose-response curve of Cl-secretory Isc in rat colon (Fig. 8) did not include an inflection that would be consistent with such a high concentration inhibitory effect on CFTR. Thus the
50% inhibition of flushing-type secretion by HMR1556 (Figs. 5 and 7) in rat colon strongly supports a limited requirement for Kcnq1 K+ channels and the need for at least one other K+ channel type. The lack of inhibition by HMR1556 in guinea pig distal colon (Fig. 6) was not due to the insensitivity of guinea pig Kcnq1, because HMR1556 blocks Kcnq1 currents in guinea pig cardiomyocytes (5, 23). This failure further indicates that flushing secretion could occur without the involvement of Kcnq1 as part of the basolateral membrane K+ conductance.
The activation time course for flushing secretion in rat colon (Fig. 12, A and B) supports the concept that Kcnq1 was needed to produce the slower-onset, secondary phase of secretory capacity. In contrast, the flushing response in guinea pig colon (Fig. 6) had a rapid onset resembling the HMR-resistant component observed in rat colon. Because Kcnq1 was present in the epithelial cells of guinea pig colonic mucosa, the signaling elicited by PGE2 in these cells apparently lacked regulatory pathways to produce this secondary phase of flushing secretion. Another major difference with rat colon is the higher relative rate of K+ secretion during PGE2 stimulation in the guinea pig colon (53), such that apical membrane K+ channels may satisfy the requirements for additional K+ conductance needed to support high rates of Cl secretion.
The present results using the Kcnq1/Kcne3 inhibitor HMR1556 support the concept that distinct K+ channels are needed to produce the secretory modes activated by different types of secretagogues (24). However, the results also indicate that a single type of K+ channel would not be sufficient to produce Cl secretion via the ubiquitous flushing secretagogue PGE2. Other studies have indicated that the possible involvement of Kcnq1 may not be apparent until additional K+ channel types are inhibited (44). However, those observations still suggest that Kcnq1 apparently is not the preferred K+ channel for activation by those secretagogues. This concept that the secretory cells have a reserve capacity for the activation of K+ channels able to support secretion is underscored by the large Cl-secretory currents (300500 µA/cm2) that are possible without the need for Kcnq1 (Figs. 11 and 12). The presence of Kcnq1 in colonic epithelial cells could serve requirements for cell volume regulation (25) as well as secretory needs. The early oscillatory behavior of the HMR-resistant response (Fig. 12A) may represent a volume instability of these secretory cells in attempting to initiate the Kcnq1-dependent secondary phase of secretion. Thus the choices that epithelial cells make with regard to which type of K+ channel to activate during secretion may depend on advantages conveyed by the specific activation and kinetic details of each channel type.
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GRANTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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