INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

ACTIVE SECRETION OF IONS across colonic epithelia serves to produce a driving force for fluid secretion and to modify the composition of secreted fluid (7, 15). Excessive rates of secretion occur in pathophysiological states such as secretory diarrhea and ulcerative colitis. As in other fluid-secreting epithelia, electrogenic Cl secretion is a major mechanism for producing fluid flow (14). Stimulating Cl secretion requires exit of K⁺ entering via the Na⁺-K⁺ pump and Na⁺-K⁺-2Cl cotransport, which can occur via basolateral membrane K⁺ channels (14). In mammalian colon, K⁺ secretion is stimulated together with Cl secretion, contributing to the relatively high luminal K⁺ concentration. Localization studies support the presence of K⁺ and Cl secretory capacity in columnar cells of colonic crypts (18, 19). The cellular mechanism for K⁺ secretion is electrogenic and related to the mechanism for Cl secretion. A key feature is that electrogenic K⁺ secretion measured in vitro is entirely bumetanide sensitive, suggesting an absolute requirement for Na⁺-K⁺-2Cl cotransport (15, 41). In addition, apical and basolateral membrane K⁺ channels allow exit of K⁺ from the cell. Because the rate of K⁺ secretion can vary relative to that of Cl secretion, colonic secretory cells may control K⁺ secretion, in part, by modulating basolateral K⁺ channel activity to alter the amount of K⁺ exiting into the lumen.

Activity of K⁺ channels has been detected in colonic crypts (55), a site for fluid and mucus secretion (7, 16). Channels have been observed both in the crypt base (4, 5, 39) among the first progeny of the crypt stem cell and in the tubular portion of the crypt (32, 44, 45) among the rapidly dividing cells. The predominant cell types of the crypt are columnar cells and goblet cells (16, 19). Goblet cells are distinguished from columnar cells by dense apically located mucin granules that are released during cholinergic stimulation. Cl secretion occurs with either cholinergic activation that increases intracellular Ca²⁺ or secretagogues such as vasoactive intestinal peptide and prostaglandin E₂ (PGE₂) that increase intracellular cAMP (7, 15). An increase in K⁺ conductance would serve to maintain Cl secretion by allowing K⁺ exit and by developing a cell negative membrane electrical potential difference to drive conductive Cl exit through the apical membrane into the lumen. Three major types of K⁺ channels have been observed during secretory activation of isolated colonic crypts as well as colonic tumor cells such as T84 and HT29 (55): large-conductance, Ca²⁺-activated K⁺ channels (slo or BK); intermediate-conductance, Ca²⁺-activated K⁺ channels (IK1); small-conductance, cAMP-activated K⁺ channels (KvLQT/minK). These K⁺ channels belong to the greater group of K⁺ channel proteins that have similar pore-forming domains but distinct regulatory domains and components (10, 27).

Guinea pig distal colon can produce high rates of K⁺ and Cl secretion in response to secretagogues (17, 41). Both epinephrine and low concentrations of PGE₂ stimulate electrogenic K⁺ secretion in the absence of accompanying Cl secretion, whereas high concentrations of PGE₂ or forskolin stimulate electrogenic secretion of both K⁺ and Cl. Thus guinea pig distal colon provides a comparison between these two modes of secretion so that the varied roles of basolateral K⁺ channels can be examined. In particular, increased basolateral membrane K⁺ channel activity would aid Cl secretion by enhancing conductive Cl exit, whereas decreased activity would increase K⁺ secretion by limiting exit of K⁺ into the interstitial space. The present study has indicated that cells in colonic crypts exhibit an inwardly rectified K⁺ channel, ^gpK_ir, that has characteristics distinct from other K⁺ channels previously observed in colonic epithelia. Both cAMP- and Ca²⁺-dependent secretagogues activate ^gpK_ir, whereas K⁺ secretagogues moderate channel activity to lower levels.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Male guinea pigs (400-650 g body wt) received standard guinea pig chow and water ad libitum. Guinea pigs were killed by decapitation in accordance with a protocol approved by the Wright State University Institutional Laboratory Animal Care and Use Committee. Distal colon was removed and defined as the ~20-cm-long segment ending roughly 5 cm from the rectum. Colonic segments were cut open along the mesenteric line and flushed with ice-cold Ringer solution to remove fecal pellets. Epithelium was separated from underlying submucosa and muscle layers by using a glass slide to gently scrape along the length of the colonic segment. The plane of dissection occurred at the base of the crypts such that only components of the mucosa immediately adherent to the epithelium remained. Portions of mucosa were mounted, with the use of cyanoacrylate glue, on to Lucite holders with apertures 1 cm wide and 4 cm long. Mucosal portions on holders were incubated at 38°C in HEPES-buffered solution with indomethacin (1 µM) to reduce spontaneous fluid and mucus secretion (16, 17, 41). Standard HEPES-buffered Ringer solution contained (in mM) 142 Na⁺, 5 K⁺, 2 Ca²⁺, 1.2 Mg²⁺, 143 Cl, 4 H_(3-X)PO, 10 HEPES, and 10 D-glucose. Solutions were continually aerated with room air. Isolation of crypt epithelium from the mucosa followed general procedures developed previously (3, 5, 6, 44, 57). Solutions for separating epithelium from underlying connective tissue contained (in mM) 192 Na⁺, 5 K⁺, 97 Cl, 4 H_(3-X)PO, 10 HEPES, 10 D-glucose, and either 30 mM citrate or EDTA. Isolation solution containing EDTA also had 0.1% bovine serum albumin. Best results were obtained if the EDTA solution was prepared the day of the isolation, as noted previously (3). Mucosal portions were consecutively incubated in 30 mM citrate Ringer with indomethacin (1 µM) for 15-30 min and then 30 mM EDTA Ringer for 15-20 min at 38°C. Holders were then gently agitated in HEPES-buffered Ringer with indomethacin (1 µM) and dithiothreitol (1 mM) to release surface and crypt epithelium. Inclusion of dithiothreitol reduced clumping of epithelium within extruded mucus. Isolated crypts were stored on ice or in the refrigerator until use and were suitable for patch-clamp experiments up to ~36 h.

Isolated crypts were transferred onto a polylysine-coated plastic coverslip in the electrical recording chamber mounted on the stage of an inverted microscope (Diaphot; Nikon, Tokyo, Japan). Bathing solutions were perfused into the chamber by a peristaltic pump (Gilson, Middleton, WI) at room temperature. Pipettes were fabricated from 7052 glass (WPI, Sarasota, FL) by using a two-stage puller (Narishige, Tokyo, Japan), coated with Q-dope (GC Electronics, Rockford, IL), and fire-polished. Pipettes filled with either high-Na⁺ or high-K⁺ solution (Table 1) had resistances of 5-10 M and were connected to the head stage of an EPC-7 patch-clamp amplifier (List Medical, Darmstadt, Germany) via a 150 mM KCl agar salt bridge inside a holder containing a Ag-AgCl electrode (12). The reference electrode was a Ag-AgCl pellet connected to the bath through a 150 mM KCl agar salt bridge. Currents were recorded on videotape at 3-kHz filtering with a pulse code-modulated videocassette recorder (Vetter Instruments, Rebersburg, PA). Seals were made on the central tubular portion of isolated crypts bathed in standard HEPES-buffered Ringer solution. Seals of >1 G were obtained in about one of five attempts. Occasionally, cell depolarization was produced with a high-K⁺ bath made by substituting 135 mM K⁺ for Na⁺ in the standard Ringer solution. Before patches were excised, the bath solution was changed to one containing EGTA (Table 1) to maintain low free Ca²⁺ (~10 µM) that would mimic intracellular conditions. Lower levels of bath free Ca²⁺ (~100 nM and <10 nM free Ca²⁺) were produced by adding only 0.1 mM or 0 mM Ca²⁺, respectively, to these bath solutions. Bath solution pH was adjusted by titration with NaOH or HCl. Solution osmolarity was 292 mosM (290-294 mosM), except for the 300 KCl bath.

Drugs were added in small volumes from concentrated stock solutions. PGE₂ was obtained from Cayman Chemical (Ann Arbor, MI), and epinephrine was from Elkins-Sinn (Cherry Hill, NJ). All other chemicals were obtained from Sigma Chemical (St. Louis, MO). PGE₂ was prepared in an ethanol stock solution that added 0.1% ethanol at 10 µM PGE₂; additions of 1% ethanol alone did not alter transepithelial measures of K⁺ or Cl secretion (17).

Current data were transferred via DigiData-1200 interface to a computer for analysis using pCLAMP6 software (Axon Instruments, Foster City, CA). Currents were filtered at 700 Hz. Junction potentials at pipette tip and bath reference bridge were calculated to correct holding voltages (1, 38). Relative ion permeabilities were calculated by using the Goldman-Hodgkin-Katz potential equation together with the measured reversal potential and solution ion composition. Open probability (P_o) was calculated from all-points histograms of current amplitude. Area under each current peak (A) was determined by a Gaussian fit. P_o was obtained from the relation P_o = [(iA_i)/A_i]/N, with i indicating each peak starting at 0 for the baseline and increasing to N, the number of active channels. A current-voltage relation was constructed from the lowest current peaks to assure that the lowest peak at each voltage indicated the closed state. Records of sufficient length (5-10 min) were obtained for each stimulatory condition to allow a reliable measure of N from the number of observed peaks (22). Currents recorded at holding potentials (V_hold) more positive than about 10 mV were generally too small to allow accurate measurement of P_o. Kinetic analysis was performed on records containing only one channel by producing histograms of open and closed durations from an events list. For this analysis, current records were sampled at a rate of 20 µs/point and then filtered at 1 kHz (Gaussian) to minimize noise but also to maximize bandwidth. Log binning was used to improve fitting and display of exponential curves (24); maximal likelihood estimates were used to obtain time constants from open and closed times. Results are reported as means ± SE. Statistical comparisons were made by using two-tailed Student's t-test for paired comparisons, with significant difference accepted at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Basolateral membranes of isolated crypts were readily accessible to sealing with patch pipettes; all results were from seals on the middle (tubular) section of isolated crypts. Differences between columnar and goblet cells could not be discerned readily to identify the cell type recorded. Spontaneous single-channel currents were observed while cell-attached consistent with K⁺ channel activity (Fig. 1), with both high-Na⁺ and high-K⁺ pipette solutions (Table 1). The reversal potential in cell-attached recordings supported identification of channel types producing currents. For crypt epithelial cells (19, 31), currents from K⁺ and Cl channels recorded with high-Na⁺ pipette solution would be expected to reverse at negative and positive V_hold, respectively, as determined by the ion concentration gradients. Thus, at resting membrane electrical potential difference (V_hold = 0 mV), K⁺ currents would be outward and Cl currents would be inward (net outward Cl flow). In addition, nonselective cation channel currents would reverse at large positive V_hold, corresponding to a cell membrane electrical potential difference of 0 mV. Use of high-K⁺ pipette solution had the advantage of increasing the size of inward K⁺ currents, thus permitting better detection of small-conductance channels. Because the equilibrium potential for K⁺ would be near a membrane electrical potential difference of 0 mV with roughly equal K⁺ concentrations inside and out, cell-attached reversal potentials of K⁺ channel currents with high-K⁺ pipette solution allowed a rough estimate of cell membrane electrical potential difference.

View larger version (51K): [in this window] [in a new window]   Fig. 1.   K+ channel currents in crypt cells. Current traces are shown for K+ channels from cell-attached patches of basolateral membrane in isolated colonic crypts. Closed states are indicated by dashed lines. A: currents at several holding potentials (Vhold) are shown for an ~9-pS K+ channel with high-Na+ pipette solution, containing 5 mM K+ (see Table 1). B: currents for an inwardly rectified ~9-pS K+ channel with high-K+ pipette solution, containing 140 mM K+ (see Table 1). C: currents for a larger conductance (~19 pS) inwardly rectified K+ channel with high-K+ pipette solution. D: currents for an ~85-pS K+ channel with high-K+ pipette solution.

Currents consistent with Cl channels, as distinguished by reversal potentials, also were observed (30). Nonselective cation channels (52) reversing at positive V_hold were not observed in cell-attached patches. Rarely (5 of 304 patches, 1.6%), nonselective cation channels were observed reversing at V_hold of 0 mV, suggesting a depolarization of membrane electrical potential difference for these cells. Changing the bath solution to a high-K⁺ solution, which presumably depolarized cells, resulted occasionally (9 of 57 patches, 16%) in the appearance of nonselective cation channels in cell-attached patches. For patches with nonselective cation channels, two to six channels were present with a voltage-independent single-channel conductance of ~25 pS.

The most common K⁺ channel activity with high-Na⁺ pipette solution (10 of 73 patches, 14%) had a linear current-voltage relation with a single-channel conductance () of 9 pS (Figs. 1A and 2). During recording with high-K⁺ pipette solution, inwardly rectified current-voltage relations were observed (58 of 231 patches, 25%) with  of 9 and 19 pS at V_hold = 0 mV (Figs. 1, B and C, and 2). These two channel behaviors with high-K⁺ pipette solution may be specific conductance states of the same channel, because they were observed together (3 of 6 patches with large-conductance ^gpK_ir, 50%). Cell-attached currents consistent with a K⁺ channel of ~85 pS also were seen in one patch with the standard bath solution (Figs. 1D and 2A) and in one other patch with high-K⁺ bath solution that presumably depolarized cells. Apparent higher incidence of K⁺ channels with high-K⁺ pipette solution may only reflect the generally greater ease of identifying the resulting larger inward K⁺ currents.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   K+ channel conductive properties. A: current-voltage relations are shown for 4 types of K+ channel activity observed in cell-attached patches of crypt basolateral membrane (means ± SE). With high-Na+ pipette solution, single-channel currents were linearly dependent on voltage (, n = 6). With high-K+ pipette solution, currents were inwardly rectified, and those exhibiting both inward and outward currents were averaged (, n = 15). Larger inwardly rectified currents forming a distinct group were averaged (, n = 6). A larger conductance (~85 pS) K+ channel recorded with high-K+ pipette solution also is shown (, n = 1). Symbols often obscure small error bars. I, current. B: single-channel conductance (chord conductance, ) is shown from current-voltage relations in A.

Ion selectivity of ^gpK_ir. Activity of ^gpK_ir generally persisted after excision into an inside-out (I/O) configuration (17 of 18 patches, 94%), which allowed ion selectivity to be determined more precisely. With a high-K⁺ pipette solution and a K-gluconate bath solution (containing 50 mM Cl, Table 1), the reversal potential was near 0 mV (Fig. 3A), as expected for a cation channel. Increasing bath solution KCl concentration shifted the reversal potential to negative voltages (Fig. 3A) consistent with cation selectivity. Relative anion permeability (P_Cl/P_K) was <0.03 (n = 9). Lowering bath solution K⁺ concentration by substitution with Na⁺ shifted the reversal potential to positive voltages, as expected for a K⁺-selective channel (Fig. 3A). Relative Na⁺ permeability (P_Na/P_K) was 0.02 ± 0.02 (n = 6). Substituting bath K⁺ completely with Na⁺ produced steep inwardly rectified currents without outward currents (Fig. 3C), also consistent with high selectivity for K⁺ over Na⁺. Similarly (Fig. 3A), relative Rb⁺ permeability (P_Rb/P_K) was 1.12 (n = 2, range 1.05-1.19). Currents from the 9-pS channel with high-Na⁺ pipette and Na-gluconate bath solution also were completely inwardly rectified (Fig. 3C), supporting identification as a K⁺ channel. Ion selectivity of the 19-pS ^gpK_ir also supported high preference for K⁺ over Na⁺ or Cl (data not shown). These current measurements indicate that the observed channels were K⁺ selective with significant Rb⁺ permeability, suggesting a divergence from other inwardly rectified K⁺ channels (8, 49).

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Ion selectivity of inwardly rectified K+ channel. Current-voltage relations are shown with various bath ion compositions (means ± SE). A: after excision into inside-out (I/O) configuration, bath solution (see Table 1) was changed among K-gluconate (, n = 7), 70 mM K+-85 mM Na+ (70K/85Na; , n = 6), 300 mM KCl (, n = 2), and RbCl (, n = 2). Pipette solution was high-K+. Symbols often obscure small error bars. B: single-channel conductance is shown from current-voltage relations in A. C: only inward currents were observed for I/O patches with Na-gluconate (Na-glcn) bath solution with either high-K+ pipette solution (, n = 2) or high-Na+ pipette solution (, n = 5). Currents from A with K-gluconate () and 70K/85Na () also are shown for comparison of variations in K+ and Na+ concentration. D: single-channel conductance is shown from current-voltage relations in C. Chord-conductance  for Na-gluconate conditions with high-K+ pipette solution was calculated with a reversal potential (Erev) of +90 mV, obtained from Na+/K+ selectivity exhibited in A;  at membrane potentials (Vm) = 0 mV ranged over ~1 pS when Erev changed from +80 mV to +100 mV. For high-Na+ pipette solution, Erev of +70 mV was estimated by extrapolation. E: single-channel conductance is shown for I/O condition with K-gluconate bath () together with those for cell-attached conditions ( and ) from Fig. 2B with inclusion of apparent cell membrane electrical potential differences (Vcell) exhibited in Fig. 2A (40 mV): Vm = Vhold + Vcell. Pipette solution was high-K+.

Concentration dependence of ^gpK_ir. Increasing K⁺ concentration at the cytoplasmic face of the patch (0, 70, and 143 mM; with constant ionic strength) increased  of ^gpK_ir at positive membrane potentials (V_m) (Fig. 3D), consistent with saturation of outward flow at <70 mM K⁺. Further increase of K⁺ concentration to 300 mM (with increased ionic strength) led to ~45% larger  at positive V_m (Fig. 3B). For inward K⁺ flow from the pipette, decreasing K⁺ concentration in the bath (with constant ionic strength) did not alter  at large negative V_m (Fig. 3D), but high bath K⁺ concentration (300 mM; with higher ionic strength) increased  at negative V_m (Fig. 3B). One possible explanation for this apparent influence of cytoplasmic K⁺ on K⁺ influx, and efflux, is that the small-conductance state of ^gpK_ir was converted to the large-conductance state by increased ionic strength, since  at negative V_m with 300 mM bath K⁺ (Fig. 3B) was similar to  of the large-conductance ^gpK_ir (Fig. 2B).

Voltage dependence of  for ^gpK_ir. Excision into an I/O configuration can lead to altered channel behavior as cytoplasmic components are lost. Single-channel conductance of ^gpK_ir was not changed dramatically by excision into a high-K⁺ solution that mimicked intracellular ion composition (Figs. 2B and 3B). Generally the small-conductance state was present, although the large-conductance state was observed (2 of 18 patches, 11%). However, a direct comparison of conductance-voltage dependence for cell-attached and excised conditions requires an estimate of resting cell membrane electrical potential difference (V_cell). Assuming that cell-attached reversal potentials of K⁺ channels with high-K⁺ pipette solution represented a V_cell of 0 mV, cell-attached V_hold then can be adjusted to indicate actual V_m (Fig. 3E). Excised  appeared to conform best in size to the small-conductance state seen with cell-attached recordings. The ~50 mV rightward shift in voltage dependence for  indicates that cytoplasmic factors controlling  may have been lost upon excision. One possibility is that Mg²⁺ in the bath solution (Table 1) during I/O conditions blocks ^gpK_ir with different voltage dependence than the native cytoplasmic components (27).

Kinetic modes of ^gpK_ir. P_o of ^gpK_ir during cell-attached recording was voltage independent but occurred in two distinct modes (Fig. 4A), moderate activity (P_o = 0.41 ± 0.01, n = 8) and low activity (P_o = 0.09 ± 0.01, n = 6). Abrupt transitions between low and moderate states were observed (4 of 58 patches with ^gpK_ir, 7%) but were not reversed, suggesting that a voltage-independent regulatory process controlled transitions between these two kinetic modes of ^gpK_ir. Excision into an I/O configuration increased P_o of low-activity ^gpK_ir, producing a kinetic mode with brief open and closed events (Fig. 4B); transition to moderate P_o occasionally occurred only several minutes after excision. In the excised I/O condition, P_o was similar to the moderate-activity state (P_o = 0.42 ± 0.01, n = 5) regardless of whether cell-attached activity had been low or moderate (Fig. 4C), indicating that excision may remove a cytoplasmic component that acts to limit P_o.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Open probability (Po) of guinea pig inward rectifier K+ channel (gpKir). Currents from gpKir were recorded before and after excision into I/O configuration. Po was calculated from current records (see METHODS). Pipette solution was high-K+. A: dependence of Po on Vhold is shown for cell-attached condition (means ± SE). Spontaneous activity was observed in either of 2 states, moderate (, n = 8) or low (, n = 6). B: a cell-attached patch was excised into K-gluconate bath solution. Current traces are shown for Vhold of 50 mV. Closed states are indicated by dashed lines. Openings for 2 gpKir were apparent after excision. C: Po is shown for those patches (n = 5) in which gpKir from basal conditions were recorded in both cell-attached (open symbols) and excised I/O (filled symbols) configurations. Cell-attached Po is shown with inclusion of apparent Vcell, 40 mV (see Fig. 2A).

Secretagogue modulation of ^gpK_ir activity. Numerous secretagogues stimulate electrogenic K⁺ and Cl secretion across distal colonic epithelia (7, 15). Electrogenic K⁺ secretion is stimulated by epinephrine or PGE₂, and at higher concentrations PGE₂ stimulates Cl and K⁺ secretion (17, 41). In addition, the cholinergic agonist carbachol (CCh) stimulates Cl secretion. Forskolin, which increases intracellular cAMP through activation of adenylate cyclase, also stimulates electrogenic Cl secretion. Spontaneous activity of ^gpK_ir was observed with both high-Na⁺ (7 of 10 patches with ^gpK_ir, 70%) and high-K⁺ pipettes (42 of 58 patches with ^gpK_ir, 72%). Addition of forskolin to the bath during cell-attached recording increased both the number of open ^gpK_ir (N) and apparent P_o in quiescent patches (Figs. 5A and 7A) and in patches with spontaneous activity (Figs. 6A and 7B). CCh added to the bath during cell-attached recording also activated ^gpK_ir in a quiescent patch (Fig. 5B).

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Secretagogue activation of gpKir. Currents from gpKir were recorded during cumulative addition of secretagogues to spontaneously quiescent patches. Activity generally was recorded at 10-mV increments of Vhold. Pipette solution was high-K+. Closed states are indicated by dashed lines. A: a cell-attached patch was sequentially treated with forskolin (1 µM) and epinephrine (5 µM). Currents are shown 10.8 min after forskolin addition for Vhold of 60 mV and 6.5 min after epinephrine addition for Vhold of 50 mV; because Po was voltage independent (see Fig. 4A), kinetic changes can be assessed from these current traces. The number of open gpKir increased to 3 with forskolin. B: a cell-attached patch was treated with carbachol (CCh; 10 µM) for Vhold of 50 mV. Time after addition of CCh is noted. The number of open gpKir increased to 7 with CCh.

View larger version (36K): [in this window] [in a new window]   Fig. 6.   Secretagogue modulation of gpKir. Currents from spontaneously active gpKir were recorded during cumulative addition of secretagogues. Pipette solution was high-K+. Closed states are indicated by dashed lines. A: a cell-attached patch was sequentially treated with forskolin (10 µM) and PGE2 (10 µM) for Vhold of 50 mV. Currents are shown 13.0 min after forskolin addition and 11.7 min after PGE2 addition. The number of open gpKir increased from 1 in basal to 4 with forskolin and PGE2. B: a cell-attached patch was sequentially treated with epinephrine (5 µM), PGE2 (10 µ), and forskolin (10 µM) for Vhold of 60 mV. Currents are shown 12.8 min after epinephrine addition, 9.3 min after PGE2 addition, and 8.7 min after forskolin addition. The apparent number of open gpKir in basal was 5.

View larger version (26K): [in this window] [in a new window]   Fig. 7.   Secretagogues reduce activity of gpKir. Currents from gpKir were recorded during cumulative addition of secretagogues. Pipette solution was high-K+. Closed states are indicated by dashed lines. A: a cell-attached patch in which CCh (10 µM) had failed to activate gpKir after ~20 min was sequentially treated with forskolin (10 µM) and PGE2 (10 µM) (upper traces). Currents are shown ~19 min after forskolin addition for Vhold of 80 mV and ~20 s after PGE2 addition for Vhold of 40 mV. Po was 0.01 ± 0.001 during forskolin treatment. Open gpKir inactivated after ~1 min with PGE2 (lower trace) such that activity at other Vhold was not obtained. B: a cell-attached patch was treated sequentially with forskolin (1 µM), epinephrine (5 µM), and PGE2 (100 nM) for Vhold of 50 mV. Currents are shown 18.5 min after forskolin addition, 17.4 min after epinephrine addition, and 17.6 min after PGE2 addition. Only 1 gpKir was open throughout treatment; Po was 0.05 ± 0.02 (basal), 0.71 ± 0.02 (forskolin), 0.46 ± 0.02 (epinephrine), and 0.34 ± 0.01 (PGE2).

View larger version (33K): [in this window] [in a new window]   Fig. 8.   Secretagogue dependence of Po for gpKir. Po and number of active channels (N) for gpKir (12 patches) were determined from current records (see METHODS). Values for Po and N before and during each secretagogue treatment are connected and plotted in pairs for comparison: solid lines connect changes from patches that had spontaneous activity, and dashed lines connect changes from patches that were quiescent spontaneously. A: secretagogue-induced changes of Po are shown for previously untreated patches: forskolin (; f), CCh (; C), PGE2 (; P), and epinephrine (; e). Subsequent secretagogue responses of these treated patches also are shown (open symbols), with a label at the "before" value indicating the order of prior cumulative secretagogue additions. Average Po for each patch was calculated from determinations at several Vhold (means ± SE), because Po was voltage independent (C). Symbols often obscure small error bars. *Significant changes (P < 0.05). B: secretagogue-induced changes of N are shown for experiments in A. Counting current levels provides an accurate measure of N for values of 4 or smaller (20). N was >4 in only two patches, but both had Po of ~0.4 so that N probably was not seriously underestimated. C: dependence of Po on Vhold is shown for the cell-attached condition. Activity was averaged for periods after addition of each secretagogue (means ± SE): forskolin (, n = 6), CCh (, n = 3), PGE2 (, n = 9), and epinephrine (, n = 5); averages include those experiments that had Po measured for 3 or more Vhold.

Kinetic mechanism of ^gpK_ir. Open and closed durations of ^gpK_ir activity were examined to obtain a preliminary mechanism controlling P_o changes by secretagogues. Histograms of open durations from a patch containing a single ^gpK_ir (Fig. 7B) exhibited one peak. In the basal and forskolin condition, the peak was fit well by one exponential, and after K⁺ secretagogue additions, two exponentials were required for an adequate fit (Fig. 9, A-D) consistent with at least two open states for ^gpK_ir. Histograms of closed durations exhibited several peaks, indicating multiple closed states for ^gpK_ir (Fig. 9, E-H). Closed durations for the basal condition were fit well by four exponentials (Fig. 9E) with widely separated time constants, indicating at least four closed states for spontaneously active ^gpK_ir. Closed durations during forskolin, epinephrine, and PGE₂ addition also were fit best by four exponentials (Fig. 9, F-H), indicating at least four closed states for secretagogue-stimulated conditions. Forskolin stimulation of P_o (Fig. 8A) occurred through elimination of closed events longer than ~400 ms with a small relative increase in closed events of intermediate duration (Fig. 9F). Reduction of P_o by epinephrine and PGE₂ occurred through a further increase in the number of closed events of intermediate duration (Fig. 9, G and H) and a decrease of time constants for open events (Fig. 9, C and D). Activation of ^gpK_ir appears to have occurred largely by altering residence in various closed states, which can be seen qualitatively in the current records (Figs. 5-7). Modulation of P_o at intermediate levels also occurred through changes of relative residence in closed states, together with shortening of the longer open time constant.

View larger version (73K): [in this window] [in a new window]   Fig. 9.   Secretagogue dependence of kinetics for gpKir. Histograms of open (A-D) and closed durations (E-H) are shown in which log binning was used (see METHODS) for a single gpKir during cumulative addition of several secretagogues while cell-attached (see Fig. 7B). Vhold was 70 mV. Each current record was ~50 s in length with a various number of events in each condition: basal (740), forskolin (5,479), epinephrine (8,252), and PGE2 (8,380). Open-duration histograms for basal and forskolin conditions had a peak fit well by a single exponential; open-duration histograms for epinephrine and PGE2 conditions had a peak fit best by a mixture of 2 exponentials. Relative frequency was obtained by normalizing to the number of open events, estimated from the fitted exponentials. Individual fit components are shown as dotted lines. Fitted open time constants and proportions of events (in parentheses) were basal, 9.4 ms; forskolin, 7.7 ms; epinephrine, 1.1 ms (0.38) and 2.4 ms (0.62); and PGE2, 0.7 ms (0.40) and 1.5 ms (0.60). Closed-duration histograms exhibited multiple peaks requiring a mixture of exponentials for an adequate fit; relative frequency was obtained by normalizing to the number of open events for that condition. Fitted closed time constants and proportions of events (in parentheses) were basal, 0.3 ms (0.94), 8.6 ms (0.03), 187 ms (0.02), and 14.5 s (0.01); forskolin, 0.3 ms (0.87), 3.7 ms (0.06), 10.8 ms (0.06), and 101 ms (0.01); epinephrine, 0.3 ms (0.45), 2.3 ms (0.30), 6.6 ms (0.24), and 59.3 ms (0.01); and PGE2, 0.3 ms (0.54), 3.4 ms (0.30), 8.3 ms (0.14), and 50.4 ms (0.01). Kinetic parameters from currents at Vhold of 80 mV were similar.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   Open and closed states of gpKir. Time constants and proportions of events for kinetic states obtained from fits of duration histograms to mixtures of exponentials were averaged from patches containing only 1 gpKir (means ± SE, range is shown for n = 2). Spontaneous cell-attached activity with low Po (n = 3) and medium Po (n = 3) together with excised I/O activity (n = 3) are shown. Activity during PGE2 (n = 2) and epinephrine (n = 1) treatments also is shown. A: open states of shorter (OS) and longer (OL) time constants are connected for each condition. Burst analysis (1-ms delimiter) yielded burst-duration histograms fit by a mixture of 2 exponentials. B: closed time constants clustered into groups. For those gpKir that did not exhibit a fit in a particular closed-duration group, a proportion of zero was included in the group average; n is indicated on plots for those cases. *Significantly different from medium-Po or excised conditions (P < 0.05). Vertical dotted lines demarcate apparent ranges of observed time constants; average values (from spontaneous activity) for each group were C1, 0.3 ± 0.01 ms, n = 10; C2, 3.2 ± 0.1 ms, n = 9; C3, 12.7 ± 1.5 ms, n = 8; C4, 148.4 ± 35.2 ms, n = 8; C5, 1.31 ± 54 s, n = 4; and C6, 25.2 ± 10.7 s, n = 2.

Cytoplasmic regulators. Distinctions between known inwardly rectified K⁺ channels can be made, in part, from sensitivities to solution composition at the cytoplasmic face of the channel. Notably, dependence on Ca²⁺ and pH can be used to aid in distinguishing among intermediate-conductance Ca²⁺-activated K⁺ channels (IK1; KCNN4), inward rectifier K⁺ channels (Kir; KCNJ), and so-called background K⁺ channels (TWIK; KCNK1) (10). Activity of ^gpK_ir was similar with bath pH of 7.2 and 6.6 (Fig. 11A), whereas increasing bath pH to 8.1 reduced ^gpK_ir activity modestly (n = 3). Because renal epithelial K⁺ channel (ROMK, Kir1.1; KCNJ1) activity decreases with acidification (9, 36), another channel type apparently was responsible for this ^gpK_ir observed in crypts. Reduction in bath solution free Ca²⁺ (Fig. 11B) did not lead to lower activity for ^gpK_ir (n = 4), indicating that IK1 (13, 23) alone could not be responsible for producing these currents.

View larger version (38K): [in this window] [in a new window]   Fig. 11.   Control of gpKir. Currents from gpKir were recorded in excised I/O configuration during changes in bath solution. Pipette solution was high-K+. Closed states are indicated by dashed lines. A: currents are shown for bath solution pH (see methods) of 7.2, 8.1, or 6.6 for Vhold of 50 mV. Po was voltage independent: 0.39 ± 0.02 (pH 7.2), 0.39 ± 0.03 (pH 6.6), and 0.28 ± 0.03 (pH 8.1); the number of active gpKir was 4. B: currents are shown for bath solution free Ca2+ concentration (see METHODS) of ~10 µM (basal), ~100 nM (low Ca2+), or <10 nM (0 Ca2+) for Vhold of 50 mV. Po was voltage independent: 0.46 ± 0.01 (basal), 0.45 ± 0.01 (low Ca2+), and 0.42 ± 0.01 (0 Ca2+); the number of active gpKir was 5.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Active ion secretion across epithelia produces an osmotic gradient that drives fluid secretion (14, 15). The lumen negative transepithelial electrical potential difference produced by Cl secretion results from a cellular mechanism employing apical membrane Cl channels and basolateral membrane K⁺ channels. Together with the K⁺ concentration gradient developed by operation of the Na⁺-K⁺ pump, basolateral membrane K⁺ conductance serves to generate a cell negative basolateral membrane electrical potential difference (V_b). Apical membrane Cl channels allow Cl exit into the lumen, driven by the electrochemical gradient. This gradient is directed outward because of the influence of basolateral membrane K⁺ channels on apical membrane electrical potential difference (V_a). Continued Cl secretion, and thus fluid secretion, depends on a basolateral membrane K⁺ conductance large enough to maintain Cl exit across the apical membrane by ensuring that V_a exceeds the size of the inwardly directed concentration gradient.

Colonic Cl secretion in mammals is accompanied by electrogenic K⁺ secretion, apparently by inclusion of apical membrane K⁺ channels in Cl secretory cells (15, 41). For colonic secretory cells, therefore, K⁺ channels in both apical and basolateral membranes contribute to maintaining a cell negative V_a that promotes Cl secretion. In addition, K⁺ conductance of apical membrane relative to basolateral membrane contributes to determining the proportion of intracellular K⁺ exiting into the lumen. Elevated luminal K⁺ concentration (19) would lower the K⁺ concentration gradient across the apical membrane and thereby reduce the ability of apical K⁺ channels to ensure conductive Cl exit. Relative rates of Cl and K⁺ secretion vary among mammalian species (15), but in guinea pig distal colon these electrogenic flows are roughly equal when stimulated by high concentrations (>100 nM) of PGE₂ (17, 41). Activation by epinephrine (15, 41) or low concentrations (<100 nM) of PGE₂ (17, 41) produces electrogenic K⁺ secretion without accompanying Cl secretion. Cl entering via basolateral membrane Na⁺-K⁺-2Cl cotransporters during this type of sustained electrogenic K⁺ secretion apparently exits across the basolateral membrane through Cl channels (15, 30, 41). Control of basolateral K⁺ channels during primary electrogenic K⁺ secretion, thus, also would contribute to maintaining a driving force for Cl exit from the cell as well as to determining the proportion of K⁺ exiting into the lumen.

K+ channel types. Several classes of K⁺ channels have been identified by amino acid sequence homology (10, 27). Although all seem to conserve a general pore-forming component, wide variation occurs in other portions of the sequence that presumably control channel activation and kinetic behavior. Three of these K⁺ channel types exhibit distinct inwardly rectified : Kir (KCNJ), IK1 (KCNN4), and TWIK (KCNK1) (10). With symmetrical 150 mM K⁺ concentration,  at V_m = 0 mV is 30, 16, and 28 pS for Kir1.1 (9), IK1 (23, 25), and TWIK (29), respectively. Specific sensitivities to pH, Ca²⁺, and ATP also contribute to a functional identification of these channel types (10). In particular, the ROMK channel (Kir1.1; KCNJ1) is inhibited by acidification at the cytoplasmic side of the channel (9, 36). Increasing cytoplasmic ATP also inactivates ROMK (Kir1.1; KCNJ1) (9, 36); association with a regulatory subunit, the sulfonylurea receptor, confers even greater ATP sensitivity to Kir6 (10). Activation by cytoplasmic Ca²⁺ is a key feature of IK1 shared with BK (slo; KCNMA) (10). The TWIK channel has been termed a background channel because it does not have any direct dependence on pH, Ca²⁺, or V_m (10, 29). Behavior of K⁺ channels in native circumstances may vary from characteristics of overexpressed versions, however, because K⁺ channels may exist as heteromultimeric assemblies of channel and regulatory subunits that result in divergent properties (10, 27, 43, 51).

Colonic K+ channels. Three major types of K⁺ channels have been observed in cell-attached patches on basolateral membrane of colonic and small intestinal crypts (55): large-conductance K⁺ channels (5, 6, 26, 32, 33, 35, 42), intermediate-conductance Ca²⁺-activated K⁺ channels (4, 5, 20, 35, 42, 44), and very small conductance K⁺ channels (57). Nonselective cation channels, with  of 20-40 pS, also have been observed in basolateral membranes of colonic epithelial cells (4, 6, 42, 47), similar in size to those observed in guinea pig crypts. Large-conductance K⁺ channels in crypts have  ranging from ~100 pS to >200 pS, and some are activated by increases in Ca²⁺ activity at the cytoplasmic face of the channel (5, 26, 32, 33, 35). Kinetic behavior and large conductance suggest that these K⁺ channels, and those observed in guinea pig crypts (Figs. 1D and 2), are intestinal assemblies of the BK (slo; KCNMA) channel (10). Colonic intermediate-conductance Ca²⁺-activated K⁺ channels often are inwardly rectified (5, 20, 39, 42) with  of 30-35 pS in symmetrical 150 mM K⁺ concentrations at V_m = 0 mV. Inwardly rectified Ca²⁺-activated K⁺ channels with  of ~20 pS have been observed as well in the colonic tumor cell line T84 (11, 49). Identification of these K⁺ channels as IK1 (KCNN4) (10) is supported further by detection of mRNA for IK1 in T84 cells and colonic epithelial cells (56). Another K⁺ channel with very small conductance (<4 pS) also has been detected in colonic crypts, with the use of noise analysis (57), similar in size to KvLQT1 (KCNQ1) assembled with the regulatory subunit minK⁺ (KCNE) (58). Identity of this crypt K⁺ channel as KvLQT1/minK⁺ (KCNQ1/KCNE) is supported by in situ hybridization localizing mRNA for both subunits in colonic crypt cells (46).

Regulation of Cl and K+ secretion. Control of ^gpK_ir in the basolateral membrane participates in production of Cl and K⁺ secretion by contributing to maintenance of V_a that drives Cl exit into the lumen while also adjusting the proportion of K⁺ exit into the lumen. Activation of ^gpK_ir occurred with two Cl secretagogues, forskolin and CCh (Figs. 5-7), consistent with this channel being used to augment basolateral membrane K⁺ conductance. Both the number of active ^gpK_ir (N_K) and P_o increased (Fig. 8) such that basolateral membrane K⁺ conductance (g^K) would increase: g^K = N_KP_o^K. The K⁺ secretagogues PGE₂ and epinephrine led to decreased g^K primarily by reducing P_o (Fig. 8) and occasionally by deactivating ^gpK_ir (N_K). This type of control would suit the requirements of balancing K⁺ and Cl secretory rates by moderating basolateral membrane K⁺ conductance within a precise range.