Secretory activation of basolateral membrane Clminus channels in guinea pig distal colonic crypts

Yingjun Li, Susan Troutman Halm, and Dan R. Halm

Department of Physiology and Biophysics, Wright State University, Dayton, Ohio 45435


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Cell-attached recordings revealed Cl- channel activity in basolateral membrane of guinea pig distal colonic crypts isolated from basement membrane. Outwardly rectified currents (gpClor) were apparent with a single-channel conductance (gamma ) of 29 pS at resting membrane electrical potential; another outward rectifier with gamma  of 24 pS was also observed (~25% of gpClor). At a holding potential of -80 mV gamma  was 18 pS for both gpClor currents, and at +80 mV gamma  was 67 and 40 pS, respectively. Identity as Cl- channels was confirmed in excised patches by changing bath ion composition. From reversal potentials, relative permeability of K+ over Cl- (PK/PCl) was 0.07 ± 0.03, with relative permeability of Na+ over Cl- (PNa/PCl) = 0.08 ± 0.04. A second type of Cl- channel was seen with linear current-voltage (I-V) relations (gpClL), having subtypes with gamma  of 21, 13, and 8 pS. Epinephrine or forskolin increased the number of open gpClor and gpClL. Open probabilities (Po) of gpClor, gpClL21, and gpClL13 were voltage dependent in cell-attached patches, higher at more positive potentials. Kinetics of gpClor were more rapid with epinephrine activation than with forskolin activation. Epinephrine increased Po at the resting membrane potential for gpClL13. Secretagogue activation of these Cl- channels may contribute to stimulation of electrogenic K+ secretion across colonic epithelium by increasing basolateral membrane Cl- conductance that permits Cl- exit after uptake via Na+-K+-2Cl- cotransport.

potassium ion secretion; chloride secretion; epinephrine; prostaglandin E2; forskolin


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EPITHELIAL ION SECRETION DRIVES fluid secretion by producing transepithelial osmotic gradients (8, 18, 19). Electrogenic Cl- secretion is a common type of transport in these fluid secretory epithelia. The cellular mechanism for this secretion includes Cl- channels in the apical membrane that are activated by various secretagogues through the action of intracellular signaling pathways. Colonic epithelia of mammals also produce electrogenic K+ secretion via a cellular mechanism similar to that for Cl- secretion except that apical Cl- conductance is not activated (17, 19, 23, 50, 51). Secretagogues producing electrogenic K+ secretion with little or no accompanying steady-state Cl- secretion include epinephrine (17, 19, 50, 68), prostaglandin E2 (PGE2) (23, 50), aldosterone (21, 51), and cholinergic agonists such as carbachol (6, 11). Sensitivity of this K+ secretion to bumetanide (17, 50, 51, 68) supports a requirement for basolateral membrane Na+-K+-2Cl- cotransporters. Because Cl- entering together with Na+ and K+ does not exit into the lumen, a second basolateral Cl- transport process is necessary to allow maintenance of intracellular Cl- concentration during steady-state K+ secretion (24, 27). Inhibition of K+ secretion by DIDS further supports that another basolateral Cl- transport pathway is involved (17). Basolateral membrane Cl- channels have been proposed as this Cl- exit step that would contribute to the observed positive charge flow across the epithelium from blood side to lumen (17). Activation of basolateral membrane Cl- channels as well as apical membrane K+ channels presumably would occur during secretagogue stimulation to initiate and sustain steady-state electrogenic K+ secretion.

Basolateral membrane Cl- conductance (g<UP><SUB>b</SUB><SUP>Cl</SUP></UP>) serves multiple functions in epithelial cells. Electrolyte absorption in the thick ascending limb of Henle's loop (52) and in the intestine of teleost fish (25) uses a cellular mechanism in which Cl- enters across the apical membrane via Na+-K+-2Cl- cotransporters and exits, in part, through basolateral membrane Cl- channels. The cochlea and vestibular labyrinth of the inner ear secrete K+ by a mechanism (67) similar to that proposed for the colonic epithelium (17). In addition, g<UP><SUB>b</SUB><SUP>Cl</SUP></UP> is activated during regulatory volume decrease in colonic crypts as well as other cell types (12, 47, 57, 62). Similarities may exist for the control of Cl- channels during transepithelial ion flow and cell volume regulation, but whether the same channels serve both types of function in epithelia has not been determined.

Numerous classes of Cl- channels have been identified that are involved in transepithelial flow and cell volume control as well as contributing to conductances in specific cells to support synaptic signaling and modulation of excitability (15, 33, 47, 60, 62, 69). Some of these Cl- channel types are pertinent to epithelial function, and three have a defined molecular identity: CFTR (33, 60), the CLC family (33, 69), and the Ca2+-activated Cl- channel family CLCA (16). Volume-regulated Cl- channels (Clvol) and an outwardly rectified Cl- channel (Clor) are of uncertain molecular identity but are present in intestinal epithelia (3, 5, 12, 47, 57, 62). CFTR is a Cl- channel with voltage-independent single-channel conductance (gamma ) of ~9 pS activated by cellular protein kinases (33, 60). Members of the CLC family have voltage-dependent currents variously inwardly rectified and outwardly rectified with gamma  from ~1 pS to >40 pS (33, 52, 69). CLCA is activated by Ca2+ with gamma  of 15-30 pS (16). Cell swelling activates Clvol, which has outwardly rectified currents with gamma  of uncertain size (12, 47, 61, 62). The outwardly rectified gamma - and depolarization-enhanced open probability (Po) of Clor (20, 26, 43, 61) appears to be distinct from that of Clvol (47, 61, 62). Blockers have been described for these channel types, but most are relatively nonspecific so that defining channel type by blocker sensitivity is problematic at present (33, 47, 48, 58, 61, 62).

Several of these Cl- channel types have been localized to colonic epithelia. CFTR mRNA is expressed primarily in the lower two-thirds of colonic crypts (63). A basolateral localization of CFTR would be counter to the commonly accepted function as a secretory Cl- channel, but its presence at low levels in the basolateral membrane of sweat gland duct cells may support Cl- absorption from the primary sweat (10, 34). CLCA-1 mRNA is expressed in colon, particularly crypt goblet cells (16). In the CLC family CLC-2, CLC-3, CLC-4, CLC-6, and CLC-7 are broadly distributed (33). CLC-2 appears in the basolateral membrane of surface and crypt epithelium in rat colon and at an intracellular location in human colonic crypts (40); in guinea pig distal colon, CLC-2 was localized only to the basolateral membrane of the surface epithelium (7). An intracellular location also is expected for CLC-3, -4, -5, -6, and -7 (33). A splice variant, CLC-3B, is expressed predominantly in epithelia and alters plasma membrane Cl- conductance (46). CLC-4 colocalizes with CFTR to the apical membrane of rat ileal crypt cells (44); an intracellular localization near the basolateral membrane was apparent for CLC-5 in rat colon (65). CLC-K has been localized to the basolateral membrane in the thick ascending limb of Henle's loop and collecting duct of kidney as well as cochlea and vestibular labyrinth of inner ear (33, 52, 55, 66). Similarly, basolateral Cl- channels in colonic crypt cells would serve to permit exit of Cl- from the cell into the interstitial space and thus support electrogenic K+ secretion. This study provides results indicating that K+ secretagogues activated basolateral membrane Cl- channels in colonic crypt cells. The observed stimulation of several Cl- channel activities, through an increased number of open channels and an increased channel Po, would lead to greater g<UP><SUB>b</SUB><SUP>Cl</SUP></UP> consistent with an involvement in Cl- exit during electrogenic K+ secretion.


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Male guinea pigs (400- to 700-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. These isolated colonic mucosal sheets were used for measurement of transepithelial current and conductance (23) as well as for further isolation of intact crypts, allowing patch-clamp recording of basolateral membrane currents (39).

Four mucosal sheets from each animal were mounted in Ussing chambers with an aperture of 0.64 cm2. These sheets were supported on the serosal face by Nuclepore filters (Whatman) with a thickness of ~10 µm and a pore diameter of 5 µm. Bathing solutions (10 ml) were circulated by gas lift through water-jacketed reservoirs that were maintained at 38°C. Standard Ringer solution contained (in mM) 145 Na+, 5 K+, 2 Ca2+, 1.2 Mg2+, 125 Cl-, 25 HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, 4 H(3 - X)PO<UP><SUB>4</SUB><SUP>X−</SUP></UP>, and 10 D-glucose. Solutions were continually gassed with 95% O2 and 5% CO2, which maintained solution pH at 7.4. Transepithelial electrical potential difference (Vt) was measured by two calomel electrodes connected to the chambers by Ringer-agar bridges. Chambers were connected to automatic voltage clamps (Physiologic Instruments, San Diego, CA) that permitted continuous measurement of short-circuit current (Isc) and compensation for solution resistance. Current was passed across the tissue through two Ag-AgCl electrodes connected by Ringer-agar bridges. Isc is described as positive for current flowing across the epithelium from the mucosal side to the serosal side. Transepithelial conductance (Gt) was measured by recording currents resulting from bipolar square voltage pulses (±5 mV, 3-s duration) imposed across the mucosa at 1-min intervals.

Portions of mucosa were mounted with cyanoacrylate glue onto Lucite holders with apertures 1 cm wide and 4 cm long to permit isolation of intact crypts. Mucosal portions on holders were incubated at 38°C in HEPES-buffered solution, with indomethacin (1 µM) to reduce spontaneous fluid and mucus secretion (22, 23, 50). Standard HEPES-buffered Ringer solution contained (in mM) 142 Na+, 5 K+, 2 Ca2+, 1.2 Mg2+, 143 Cl-, 4 H(3 - X)PO<UP><SUB>4</SUB><SUP>X-</SUP></UP>, 10 HEPES, and 10 D-glucose. Solutions were continually aerated with room air. Isolation of crypts from the mucosa followed general procedures developed previously (39). Solutions for separating epithelium from underlying connective tissue contained (in mM) 192 Na+, 5 K+, 97 Cl-, 4 H(3 - X)PO<UP><SUB>4</SUB><SUP>X-</SUP></UP>, 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 when the EDTA solution was prepared on the day of the isolation, as noted previously (2). Mucosal portions were consecutively incubated in 30 mM citrate Ringer with indomethacin (1 µM) for 15-30 min and 30 mM EDTA Ringer for 15-20 min at 38°C. Holders then were 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 a refrigerator until use. Patch-clamp recording on these crypts began ~2 h after removal of the colon from the animal and were suitable for patch-clamp experiments up to ~36 h.

Isolated crypts were transferred onto a poly-lysine-coated plastic coverslip in the electrical recording chamber mounted on the stage of an inverted microscope (Diaphot, Nikon). 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) with 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 MOmega and were connected to the head stage of an EPC-7 patch-clamp amplifier (List-Medical) via a 150 mM KCl-agar salt bridge inside a holder containing a Ag/AgCl electrode (14). 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 with 3-kHz filtering using a pulse code-modulated VCR (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 GOmega were obtained in about one of five attempts. Before excision of patches the bath solution was changed to one containing EGTA (Table 1) to maintain low free Ca2+ that would mimic maximal intracellular conditions (~10 µM) but be low enough to avoid activation of nonselective cation channels (5). Lower levels of bath free Ca2+ (~100 nM and <10 nM) were produced by adding only 0.1 mM Ca2+ or no Ca2+, respectively, to these bath solutions. Solution osmolarity was 292 mosM (290-294 mosM), except for the 300 Cl bath.

                              
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Table 1.   Patch-clamp solutions

Drugs were added in small volumes from concentrated stock solutions. PGE2, indomethacin, and NS-398 were obtained from Cayman Chemical (Ann Arbor, MI) and epinephrine from Elkins-Sinn (Cherry Hill, NJ). All other chemicals were obtained from Sigma (St. Louis, MO). PGE2 was prepared in an ethanol stock solution that added 0.1% ethanol at 10 µM of PGE2; additions of 1% ethanol alone did not alter transepithelial measures of K+ or Cl- secretion (23).

Data analysis. Concentration responses of Isc and Gt to forskolin were fit to Henri-Michaelis-Menten binding curves with a nonlinear least-squares procedure. Two independent binding curves were required (23), I = IA/[1 + (EC<UP><SUB>50</SUB><SUP><IT>A</IT></SUP></UP>/C)] + IB/[1 + (EC<UP><SUB>50</SUB><SUP><IT>B</IT></SUP></UP>/C)] or G = GA/[1 + (EC<UP><SUB>50</SUB><SUP><IT>A</IT></SUP></UP>/C)] GB/[1 + (EC<UP><SUB>50</SUB><SUP><IT>B</IT></SUP></UP>/C)], such that total Isc or Gt was a combination of these two components (IA and IB; GA and GB) at each concentration (C). Results are reported as means ± SE. Statistical comparisons were made with a two-tailed Student's t-test for paired responses, with significant difference accepted at P < 0.05.

Patch-clamp current data were transferred via DigiData-1200 interface to a computer for analysis with pCLAMP6 software (Axon Instruments, Foster City, CA). Currents were filtered at 700 Hz. Junction potentials (1, 45) at pipette tip and bath reference bridge were calculated to correct holding potentials (Vhold). Relative ion permeabilities were calculated with the Goldman-Hodgkin-Katz potential equation together with the measured reversal potential and solution ion composition. Po was calculated from all-points histograms of current amplitude. Area (A) under each current peak was determined by a Gaussian fit. Po was obtained from the relation: Po = (Sigma iAi)/Sigma Ai/N with i indicating each peak starting at 0 for baseline and increasing to N, the number of active channels. A I-V relation was constructed from the lowest current peaks to ensure that the lowest peak at each Vhold indicated the closed state. Records of sufficient length (5-10 min) were obtained for each condition to allow a reliable measure of N from the number of observed peaks (29). In records containing only one channel further kinetic analysis was performed 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 to minimize noise but also maximize bandwidth. Log binning was used to improve fitting and display of exponential curves (32); maximal likelihood estimates were used to obtain time constants from open and closed durations.


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Isolated crypts had basolateral membranes that were accessible to sealing with patch pipettes; seals were obtained on the middle section of the crypt cylinder. Identity of cells as either columnar or goblet (22) was not readily discernable. Reversal potentials of ionic currents while cell attached aided identification of the channel types producing those currents (39). For crypt epithelial cells, currents from Cl- and K+ channels recorded with high-Na+ pipette solution were expected to reverse at positive and negative Vhold, respectively, as determined by the ion concentration gradients (24, 27) together with a cell membrane electrical potential difference (PD) (Vcell) of about -65 mV (42). Thus, at resting Vcell (Vhold = 0 mV), Cl- currents would be inward (net outward Cl- flow) and K+ currents would be outward. In addition, nonselective cation channel currents would reverse at large positive Vhold corresponding to a Vcell of 0 mV. Using high-K+ pipette solution (Table 1) has the advantage of shifting the reversal potential for K+ channels toward a Vcell of 0 mV. Reversal of K+ channel currents near a Vhold of +40 mV (39) indicates that resting Vcell was about -40 mV in these isolated crypts. The shift in reversal potential for K+ channels (to Vcell = 0 mV) also provided greater separation from the expected Cl- channel reversal potential.

Spontaneous single-channel currents consistent with Cl- channel activity were observed while cell attached (Fig. 1) with both high-Na+ and high-K+ pipette solutions. Currents from outwardly rectified Cl- channels (Fig. 1A) occasionally were seen together with inwardly rectified guinea pig K+ channels (gpKir; Ref. 39), supporting a resting Vcell of about -40 mV in these recordings. Other currents consistent with Cl- channels also were seen, reversing at Vhold of about +5 mV. Several sizes of current events were apparent (Fig. 1, B and C), generally smaller than the outwardly rectified Cl- currents. Nonselective cation channels rarely were observed in guinea pig crypts, as noted previously (39).


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Fig. 1.   Cl- channel currents in crypt cells. Current traces are shown of cell-attached patches from basolateral membrane of isolated colonic crypts. Pipette solution was high-K+ containing 140 mM K+ (Table 1). Dashed lines indicate closed state. A: an outwardly rectified Cl- channel (gpClor) is shown together with an inwardly rectifying K+ channel (gpKir). Currents from gpKir reverse near +40 mV, and those for gpClor reverse near +5 mV; trace at +80 mV was chosen to show only gpClor, and traces at negative holding potential (Vhold) were chosen to show only gpKir. B: current traces are shown for a voltage-independent (linear-gamma ) Cl- channel of 12 pS. C: current traces are shown for 2 linear-gamma Cl- channels of 6 and 17 pS.

Outwardly rectified Cl- channels were seen with two sizes of outward current in guinea pig crypts (gpClor); inward currents were similar in size (Fig. 2A). Activity of gpClor was observed in 16 of 231 patches (7%). Cl- currents (Fig. 2B) with linear I-V relations (gpClL) were observed, congregated into three groups (Fig. 2C) on the basis of single-channel gamma : 17-25 pS (17 of 231; 7%), 11-15 pS (15 of 231; 6%) and 5-9 pS (10 of 231; 4%). Although these distinctions are somewhat arbitrary, the three groups were clearly separable as indicated by the small variability within each group. The presence of multiple Cl- channel classes also was supported by appearance of two or more types of gpClL in some patches (Fig. 1C), so that differing cell composition alone could not account for the distinct conductances. Cl- channels were observed in 47 of 231 patches (20%), with more than one Cl- channel type occasionally present in a single patch (Fig. 1C). Positive reversal potentials for gpClor and gpClL (Fig. 2, A and B) support outwardly directed Cl- flow through these channels at spontaneous Vcell, consistent with the predicted requirements for conductive Cl- flow across the basolateral membrane during electrogenic K+ secretion.


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Fig. 2.   Cl- channel conductive properties. A: current-voltage (I-V) relations are shown (means ± SE) for gpClor activity observed in cell-attached patches of crypt basolateral membrane. Two distinct groups were observed, with larger outward currents (, n = 9) and with smaller outward currents (open circle , n = 3). B: I-V relations are shown for linear-gamma Cl- channel (gpClL) activity, exhibiting both inward and outward currents, observed in cell-attached patches of crypt basolateral membrane. Three distinct sizes of activity were observed (black-lozenge , n = 7; black-down-triangle , n = 10; black-triangle, n = 9). C: single-channel conductance (chord conductance; gamma ) is shown, from I-V relations in A and B. Mean gamma  for gpClL21, gpClL13, and gpClL8 was 21.4 ± 1.0, 13.2 ± 0.4, and 8.3 ± 0.5 pS, respectively; each group was significantly different from the other 2 (P < 0.05).

Cl- channels were seen together with gpKir (Fig. 1A) and were distinguished by currents exhibiting distinct reversal potentials and rectification. In five patches, gpClor was observed together with gpKir. On the basis of the proportion of patches exhibiting each of these channel types (39), four patches would have been expected to contain both gpClor and gpKir, assuming uniform channel distribution among patched cells. Preferential localization, of the channel types being compared, to separate cell types would reduce the expected number of joint occurrences. Each of the linear-gamma Cl- channels also was observed with gpKir (expected number of patches): seven (4) for gpClL21, seven (4) for gpClL13, and three (3) for gpClL8. Appearance of these channels together with gpKir further supported identification as Cl selective, because nonselective cation currents would have reversed near +40 mV similar to gpKir. The various Cl- channels, as distinguished by conductance, also were observed together. Presence of linear-gamma Cl- channels together with gpClor was observed (expected number of patches): four (1) for gpClL21, four (1) for gpClL13, and zero (1) for gpClL8. The presence together of multiple linear-gamma Cl- channel types also was observed (expected number of patches): three (1) for gpClL21 with gpClL13, three (1) for gpClL21 with gpClL8, and two (1) for gpClL13 with gpClL8. The results suggest a clustering of these basolateral channel types.

Ion selectivity. Activity of gpClor often persisted after excision into an inside-out (I/O) configuration (8 of 12 patches; 67%), which permitted ion selectivity to be determined more precisely. Increasing or decreasing bathing solution Cl- concentration (Table 1) shifted the reversal potential as expected for a Cl--selective channel (Fig. 3A). Relative ion permeability was calculated for Cl- with respect to K+ and Na+: relative permeability of K+ over Cl- (PK/PCl) = 0.07 ± 0.03 (n = 7) and relative permeability of Na+ over Cl- (PNa/PCl) = 0.08 ± 0.04 (n = 4). Ion selectivity of gpClL was more difficult to determine precisely because these channels generally inactivated on excision; but ion substitution in a few cases (n = 3) supported preference for Cl- over K+ (data not shown). In addition, excision into symmetrical Cl- concentrations could be expected to produce inward rectification for gpClL; however, activity did not persist in enough cases to resolve the excised I-V relations completely.


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Fig. 3.   Ion selectivity of gpClorA: after excision into inside-out (I/O) configuration, bath solution (Table 1) was changed among NaCl (, open circle ; n = 7, 2), KCl (black-lozenge ; n = 7), K-gluconate (; n = 5), Na-gluconate (; n = 2), 300 KCl (black-triangle; n = 2), and 300 NaCl (black-down-triangle ; n = 2). Pipette solution was high-K+ (filled symbols) or high-Na+ (open symbols). Bath solutions containing gluconate would have lower Ca2+ activities than those with EGTA alone, because of Ca2+ binding by gluconate, but gpClor activity was not noticeably altered by reduction in Ca2+ activity (data not shown). B: single-channel conductance (chord conductance) is shown from I-V relations in A.

Increasing Cl- concentration at the cytoplasmic face of the patch increased gamma  for gpClor at negative Vm (Fig. 3B), as expected for conductive Cl- exit. The dependence of gamma  on intracellular Cl- activity (Vm = -80 mV) conformed to a Henri-Michaelis-Menten binding curve with gamma max of 59 pS and K1/2 of 87 mM. This apparent Cl- affinity for conduction was approximately threefold higher than for T84Clor (20). The presence of Na+ in the pipette rather than K+ (Fig. 3B) yielded lower gamma  at negative Vm when the intracellular Cl- concentration was 160 mM, but not at 50 mM Cl-. Comparison of cell-attached currents with those after excision into a low Cl- concentration that mimics intracellular values (Fig. 4A) suggests that the small-gamma form of gpClor predominated in the excised condition, even though the larger form was more common when cell attached (75%). For the large-gamma form of gpClor, cell-attached gamma  at negative Vm was similar to the excised gamma  in 50 mM Cl- but at positive Vm cell-attached gamma  was similar to excised gamma  in 160 mM Cl- (Fig. 4B), further suggesting that gamma  may be controlled by cytosolic components.


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Fig. 4.   Conductive properties of gpClor after excision. A: I-V relations are shown for I/O condition with K-gluconate bath () together with those for cell-attached condition (Fig. 2A) after correction (, large gpClor; black-lozenge , small gpClor) for apparent cell membrane potential difference (Vcell) of -40 mV (39), Vm = Vhold + Vcell. Apparent intracellular Cl- concentration for cell-attached condition was 36 mM, obtained with reversal potentials of corrected currents. B: single-channel conductance (chord conductance) is shown from I-V relations in A together with conductance from Fig. 3B (diamond , symmetrical KCl).

Secretagogue activation of Cl- channels. Distinct groups of secretagogues stimulate various amounts of electrogenic K+ and Cl- secretion across distal colonic epithelia (19, 23). Epinephrine or PGE2 stimulates electrogenic K+ secretion, and at higher concentrations PGE2 also stimulates Cl- secretion (23, 50). In addition, cholinergic agonists such as carbachol (CCh) stimulate K+ secretion when added alone but also stimulate Cl- secretion when added together with other secretagogues such as PGE2 (6, 11). Forskolin, which increases intracellular cAMP through activation of adenylyl cyclase (4, 64), stimulated a negative Isc across guinea pig distal colonic mucosa (Fig. 5, A and B), consistent with cation secretion. Identification of this forskolin-stimulated Isc as electrogenic K+ secretion was supported (Fig. 5) by sensitivity to bumetanide as well as a transepithelial equivalent electromotive force similar to that with other K+ secretagogues, such as aldosterone, epinephrine, and PGE2 (21, 23, 50). Also, Ba2+ (10 mM) added to the mucosal solution or DIDS (100 µM) added to the serosal solution inhibited forskolin-stimulated Isc and Gt (data not shown), similar to the action on epinephrine-stimulated electrogenic K+ secretion (17). Forskolin stimulated (Fig. 5, C and D) negative Isc consistent with K+ secretion at low concentrations and additionally at higher concentrations positive Isc consistent with Cl- secretion (50), which mimicked the response to PGE2 (23, 50) and cAMP (17).


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Fig. 5.   Forskolin stimulation of K+ secretion. Short-circuit current (Isc) and transepithelial conductance (Gt) were recorded from guinea pig distal colonic mucosa. Spontaneous rates of ion secretion due to apparent autocrine stimulation (23) were suppressed by repeated (3×) replacement of bathing solutions and addition of cyclooxygenase inhibitors indomethacin (2 µM) and NS-398 (2 µM). Amiloride (100 µM) added to mucosal solution inhibited electrogenic Na+ absorption. Forskolin (0.3 µM) addition to mucosal and serosal solutions (0 min) stimulated both Isc (A) and Gt (B). The equivalent electromotive force (EMF = Delta Isc/Delta Gt) was -24 mV, similar to other K+ secretagogues (21, 23, 50). Subsequent (35.8 min) serosal addition of bumetanide (100 µM) inhibited both Isc and Gt. Cumulative forskolin concentration responses (n = 7) for Isc (C) and Gt (D) exhibited 2 saturable components. Solid lines indicate fits for a 2-component model (see METHODS), with high-affinity EC50 values of 64 (Isc) and 74 (Gt) nM and low-affinity EC50 values of 4.0 (Isc) and 2.3 (Gt) µM. Dashed lines indicate individual fit components; EMF (Imax/Gmax) for high-affinity response was -23 mV and for lower-affinity response was +24 mV. Forskolin-stimulated values after bumetanide (100 µM) addition are also shown (triangle ).

Stimulation of Cl channel activity was measured as increases in number (N) and Po. The conductance (g) contributed by each channel type can be calculated from these values together with single-channel conductance, g = NPogamma . Spontaneous activity of gpClor occurred in 10 of 16 patches with detectable gpClor (62%). Linear-gamma Cl- channels occurred spontaneously in 25 of 36 patches with discernable gpClL (69%). These incidence rates overestimate spontaneous activity by ignoring channels that remained resistant to activation because of experimental conditions. This relatively high level of apparently spontaneous Cl- channel activity in the basolateral membrane is consistent with stimulation of K+ secretion by low concentrations of PGE2 and other possible endogenously produced lipid mediators (23).

Epinephrine (5 µM) activated gpClor (increased N) in 4 of 57 quiescent cell-attached patches (7%), and all were the larger-gamma form. Onset was abrupt, with rapid opening and closing kinetics (Fig. 6). PGE2 (100 nM) failed to activate gpClor in 18 quiescent patches (a rate similar to epinephrine would have predicted 1 activated gpClor). Addition of forskolin (1 µM) to the bath during cell-attached recording (Fig. 7A) led to gpClor activation (3 of 58 quiescent patches; 5%). Subsequent addition (Fig. 7B) of epinephrine (5 µM) activated another gpClor, PGE2 (10 µM) addition led to a single gpClor, and CCh (10 µM) produced erratic current amplitudes together with rapid kinetics. Currents at the beginning of bursts were near the pre-CCh size but declined during the burst. These CCh-attenuated currents at positive Vhold had a gamma  of roughly 60% of the preceding conditions (data not shown). In two other cell-attached patches with active gpClor, CCh (10 µM) addition in combination with PGE2 (10 µM) also converted channel kinetics from flickering closures to short openings and closings, but gamma  was not decreased (data not shown). Reversal potentials for gpClor or gpClL were not significantly different among spontaneous and secretagogue-induced conditions (data not shown), indicating an unaltered net driving force for Cl-. In the combined presence of epinephrine and forskolin (3 patches), the rapid kinetic mode was replaced by the flickering closures characteristic of spontaneous activity (Fig. 7B).


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Fig. 6.   Epinephrine activation of gpClor. Currents from gpClor were recorded while cell attached. Pipette solution was high-K+. Dashed lines indicate closed state. A: epinephrine (5 µM) was added to the bath solution just before the start of the current trace. Within 1 min gpClor became active, after having been quiescent during 17 min of recording in basal condition. Vhold was +30 mV. B: current traces are shown for gpClor activated by epinephrine (A).



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Fig. 7.   Forskolin activation of gpClor. Currents from gpClor were recorded while cell attached. Pipette solution was high-K+. Dashed lines indicate closed state. A: gpClor became active ~9 min after forskolin (1 µM) addition; Vhold was +40 mV. B: subsequent addition of epinephrine (5 µM) together with forskolin activated a second gpClor; cumulative PGE2 addition (10 µM) led to a single gpClor; cumulative carbachol (CCh; 10 µM) addition reduced single-channel current amplitude. Vhold was +60 mV.

Linear-gamma Cl- channels also were activated by K+ secretagogues. Epinephrine (5 µM) activated (in 57 quiescent patches) gpClL21 (2; 4%), gpClL13 (1; 2%), and gpClL8 (1; 2%). PGE2 (100 nM) activated (in 18 quiescent patches) gpClL21 (1; 6%) and gpClL13 (1; 6%), but not gpClL8. Forskolin (1 µM) activated (in 58 quiescent patches) gpClL21 (1; 2%), gpClL13 (2; 3%), and gpClL8 (2; 3%). CCh did not have a discernable action on any gpClL. Addition of epinephrine or PGE2 to patches with active gpClL13 increased channel Po by reducing the apparent number of long closures (Fig. 8).


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Fig. 8.   Epinephrine stimulation of gpClL13. Currents from gpClL13 were recorded while cell attached for the 13 min preceding epinephrine (5 µM) addition. The epinephrine-stimulated trace was recorded 6 min after addition, and similar activity was recorded for an additional 8 min during epinephrine stimulation. Pipette solution was high-K+. Vhold was -60 mV. Dashed lines indicate closed state.

The total incidence of Cl- channel activation (all channel types) by epinephrine (5 µM) was 8 of 57 quiescent cell-attached patches (14%). For PGE2 (100 nM), the total incidence of Cl- channel activation was 2 of 18 quiescent patches (11%). The total incidence of Cl- channel activation with forskolin (1 µM) was 8 of 58 quiescent cell-attached patches (14%). These incidence rates likely underestimate secretagogue sensitivity because many nonresponsive patches may not have contained Cl- channels. These activation results do support that secretagogues significantly increased g<UP><SUB>b</SUB><SUP>Cl</SUP></UP> but indicate that no particular Cl- channel type was solely responsible.

Po of these Cl- channels was secretagogue dependent. For spontaneously active gpClor, Po was lower at negative than at positive Vhold (Fig. 9A). In the physiological range of Vcell (near Vhold = 0 mV), Po was ~0.4. Po in the forskolin-stimulated state was not detectably different from spontaneous activity, but only values at positive Vhold were measurable. Epinephrine activation produced a lower Po than in the basal or forskolin conditions (Fig. 9A), consistent with the briefer open times (Figs. 1A and 6B); at physiological Vcell, Po of gpClor in the epinephrine condition was ~0.2. Spontaneously active gpClL21 and gpClL13 also had voltage-dependent Po, lower at negative Vhold with the steepest slope near the spontaneous Vcell (Fig. 9B). Neither 1 µM forskolin (n = 2) nor 100 nM PGE2 (n = 1) altered Po for gpClL21 (data not shown), but only values at positive Vhold were measurable. Stimulation with epinephrine (Fig. 8) or PGE2 (100 nM) removed the voltage dependence for gpClL13, such that Po was ~0.5 (Fig. 9B). Spontaneously active gpClL8 had voltage-independent Po with a value of either 0.54 ± 0.07 (n = 3) or 0.19 ± 0.04 (n = 3), suggesting the existence of two kinetic modes.


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Fig. 9.   Cell-attached open probability (Po). Currents from Cl- channels were recorded while cell attached before and after secretagogue addition. Po was calculated from amplitude histograms of current records (see METHODS). Pipette solution was high-K+. A: dependence of gpClor Po on Vhold is shown for basal condition (; n = 6) and in the presence of 1 µM forskolin (black-lozenge ; n = 3) or 5 µM epinephrine (black-down-triangle ; n = 4). Po of gpClL21 for basal condition (open circle ; n = 3) is also shown. Fits to Boltzmann distribution (dashed lines) allowed estimates (28) of equivalent gating charge (zg) and holding voltage at half-activation (V1/2) for gpClor[basal] (zg = 1.6, V1/2 = +22 mV) and gpClor[epi] (zg = 1.0, V1/2 = +63 mV). B: dependence of Po on Vhold for spontaneously active gpClL21 (open circle ; n = 3) and gpClL13 (; n = 3) is shown. Stimulation of gpClL13 with either epinephrine (5 µM) or low-concentration PGE2 (100 nM) altered Po (black-down-triangle ; n = 3). Fits to Boltzmann distribution (dashed lines) gave estimates of zg and V1/2 for gpClL21 (zg = 1.6, V1/2 = +6 mV) and gpClL13[basal] (zg = 2.5, V1/2 = +9 mV).

Excision into I/O configuration altered kinetic behavior for gpClor, perhaps as cytosolic components were lost into the bath solution. Rapid closing kinetics due to epinephrine activation were slowed such that the channels spent more time open (Fig. 10A). In addition, Po became generally voltage independent after excision with a value of ~0.8 (Fig. 10B). Reducing bath solution free Ca2+ did not alter Po of excised gpClor (data not shown), similar to previous reports for Clor (5, 37, 43). Excision of quiescent patches rarely (2 of 231 patches; 1%) led to activation of gpClor. Activity of all gpClL types generally was lost after excision.


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Fig. 10.   Excision of gpClorA: a cell-attached patch with an epinephrine-activated gpClor was excised into NaCl bath solution. Vhold was +50 mV. Pipette solution was high-K+. Dashed lines indicate closed state. Openings of 2 gpClor were apparent after excision. B: Po of gpClor is shown for excised condition (; n = 10). Cell-attached conditions, basal/forskolin (open circle ) and epinephrine (down-triangle) from Fig. 9A, are also shown in relation to Vm after correction for apparent Vcell of -40 mV (39).

Voltage dependence of gamma  (Fig. 2C) and Po (Fig. 9A) for gpClor was used to calculate a time-averaged current that would predict the voltage dependence for a steady-state whole cell current due to gpClor (Fig. 11A). The combination of voltage dependence from gamma  and Po results in a sharp outward rectification; simply multiplying by the number of active channels would reproduce exact whole cell current amplitudes. Voltage dependence of Po for gpClL21 and gpClL13 (Fig. 9B) also resulted in time-averaged currents with outward rectification (Fig. 11B). Although time-averaged currents from gpClor and gpClL13 are difficult to distinguish, activation of K+ secretion with epinephrine or low-concentration PGE2 would evoke a nearly linear time-averaged current from gpClL13.


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Fig. 11.   Time-averaged conductive properties of basolateral membrane Cl- channels. A: I-V relation is shown (means ± SE) for gpClor activity in cell-attached condition (open circle ; from Fig. 2A). Also shown are time-averaged currents, iClPo, for basal/forskolin state (black-lozenge ) and for epinephrine state (black-down-triangle ). Cell-attached Po from Fig. 9A was used to calculate iClPo. Vm was calculated from Vhold and apparent Vcell, -40 mV (39). B: time-averaged currents (iClPo, calculated from Figs. 2 and 9) are shown for gpClor[epi] (), gpClL21[basal] (black-triangle), gpClL13[basal] (black-down-triangle ) producing outwardly rectified currents. Currents were normalized to an inward conductance of 10; the dashed line indicates the case for voltage-independent conductance.

Kinetic analysis of gpClor. Patches with only one gpClor present allowed detailed analysis of kinetic behaviors induced by secretagogue activation. Open duration distributions (Fig. 12, A and B) exhibited two exponentials during spontaneous and forskolin activity, supporting the presence of two open states, but during epinephrine activation distributions had only a single exponential with a shorter time constant (Table 2). Closed duration distributions had three distinct exponentials (Fig. 12, C and D, and Table 2), consistent with three closed states, and a few long-duration closures (>1 s), suggesting an additional longer-lived closed state of rare occurrence. Closed duration distributions differed during epinephrine activation by a shift in events from the short-duration C1 closed state to the longer-duration C2 state (Fig. 13 and Table 2). Both the shorter-duration open time and longer-duration closed time during epinephrine activation would contribute to lower Po (Fig. 9A). Thus the powerful K+ secretagogue epinephrine induced a kinetic mode for gpClor distinct from the basal/forskolin kinetic mode.


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Fig. 12.   Opening and closing kinetics for gpClor . Histograms are shown of open durations (A and B) and closed durations (C and D) with log binning (see METHODS) for single gpClor during secretagogue stimulation while cell attached. Vhold was +60 mV. Each current record was ~20 s in length, with a large number of events in each condition: forskolin (1,512) and epinephrine (4,041). Open time histograms for forskolin condition had a peak fit well by a mixture of 2 exponentials; open time histograms for epinephrine condition had a peak fit best by a single exponential. 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 forskolin, 1.6 ms (0.04) and 11.9 ms (0.96); epinephrine, 2.2 ms. Closed time 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 forskolin, 0.6 ms (0.82), 3.6 ms (0.17), 67.9 ms (0.01); epinephrine, 1.0 ms (0.21), 2.8 ms (0.785), 171.6 ms (0.005).


                              
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Table 2.   Time constants for gpClor



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Fig. 13.   Closed states for gpClor. Time constants and proportions of events for kinetic states obtained from fits of duration histograms to mixtures of exponentials were averaged from patches containing single gpClor (Table 2). Basal and forskolin-stimulated cell-attached activity were similar and combined for averaging (black-lozenge ; n = 4). Activity during epinephrine (black-down-triangle ; n = 3) and after excision (open circle ; n = 5) also are shown. Asterisk indicates voltage-dependent time constant; value at +60 mV is shown.

The time constant (tau ) of the most common open events was voltage dependent during spontaneous activity, forskolin activation, and epinephrine activation (Fig. 14). Closed tau  were voltage independent, except during epinephrine activation, when one closed time constant (C2) was voltage dependent (Fig. 14). This extra voltage dependence contributed to the epinephrine-induced shift in the dependence of Po on voltage (Fig. 9A), such that the voltage at half-activation (V1/2) was ~40 mV more positive than in the basal/forskolin kinetic mode. The proportion of events making up each open and closed duration exponential was voltage independent (data not shown), within the variations of the measurement (~10%). After excision, the dominant open tau  (for long-duration open state) became twofold longer and voltage independent (Table 2 and Fig. 14). Closed tau  were similar to basal spontaneous activity (Table 2 and Fig. 13) after excision, supporting the presence of a diffusible mediator controlling the epinephrine-induced closed state distribution.


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Fig. 14.   Voltage dependence of time constants for gpClor. Open and closed kinetics were examined for patches with single gpClor (as in Fig. 12). Dependence on Vhold of open time constants (tau O) are shown for basal/forskolin (black-lozenge ; n = 4), epinephrine (black-down-triangle ; n = 3), and excised (; n = 4) conditions. Also shown is voltage dependence of tau C2 during epinephrine stimulation (triangle ; n = 3).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Electrogenic secretion of K+ across colonic epithelia can occur without accompanying Cl- secretion or Na+ absorption and produces a lumen-positive transepithelial electrical potential difference (Vt) via a cellular mechanism (Fig. 15), apparently employing apical membrane K+ channels and basolateral membrane Cl- channels (17, 19, 21, 50). Together these conductive pathways contribute to a cell-negative electrical PD at apical (Va) and basolateral (Vb) membranes that aids in driving basolateral Cl- exit. Secretory exit of K+ into the lumen is dependent on the relative K+ conductance of apical and basolateral membranes as well as the electrochemical driving forces (39, 42, 50). Basolateral exit of Cl- is essential during this type of K+ secretion because basolateral Na+/K+ pumps drive K+ uptake via continual extrusion of Na+ entering through basolateral Na+-K+-2Cl- cotransporters, which results in Cl- entry (19). Maintenance of cell volume during sustained secretion requires a balance between these basolateral influx and efflux pathways for Cl- (Fig. 15). Activation by K+ secretagogues indicates that the Cl- channels observed in colonic crypts (Table 3) contributed to the exit pathway for Cl-.


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Fig. 15.   Cellular transport model for electrogenic K+ secretion. The proposed mechanism for electrogenic K+ secretion across distal colonic epithelium includes 4 types of transport proteins (17, 50). Prevailing electrochemical gradients and phosphorylation potential determine the directions of net flow (arrows). The model schematic shows apical membrane K+ channels acting in concert with a combination of basolateral membrane transporters (Na+/K+-ATPases, Na+-K+-2Cl- cotransporters, Cl- channels, K+ channels) that permit net K+ uptake from the serosal interstitium into the cell and K+ exit from the cell into the lumen. Positive charge flows across these epithelial cells from serosa to lumen carried by K+ through apical membrane channels and by Cl- through basolateral membrane channels. [The standard Cl- secretory model (15, 18, 19) is similar, except that Cl- channels dominate the apical membrane conductance and basolateral membrane Cl- conductance would be assumed minimal.]


                              
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Table 3.   Properties of gpClor and gpClL

Conversion of transport function to active Cl- secretion transforms cellular ion flow by redirecting Cl- exit to apical membrane Cl- channels. Colonic epithelia may contain separate cell types producing these two modes of ion secretion, but crypts respond to secretagogues with changes in cell composition, suggesting that both the electrogenic K+ secretory mode and the electrogenic KCl secretory mode occur in columnar cells (24, 27). Cl- permeability of colonic crypts is distinct between these modes, with a larger Cl- efflux capacity in the K+ secretory mode (24). For epithelial cells capable of producing both secretory modes, coordinated regulation of Cl- channels in apical and basolateral membranes permits Cl- exit to support either of these secretory modes. Although opening apical Cl- channels is a key event for initiating Cl- secretion, managing basolateral membrane Cl- conductance (g<UP><SUB>b</SUB><SUP>Cl</SUP></UP>) also would be necessary for the sustained ion secretion observed in colonic epithelia. Presumably, closing basolateral Cl- channels would best serve promotion of Cl- secretion, but cell volume control may necessitate specific amounts of g<UP><SUB>b</SUB><SUP>Cl</SUP></UP>.

Basolateral membrane Cl- channels. Two major Cl- channel types were observed while cell attached on basolateral membrane of guinea pig distal colonic crypts (Figs. 1 and 2 and Table 3): outwardly rectified (gpClor) and linear (gpClL) I-V relations, with multiple conductance forms. Two conductance states likely exist within a single gpClor type because patches with the larger-gamma form of gpClor had the smaller-gamma form after excision (Fig. 4), even though outward currents were carried by 160 mM Cl- from the pipette in both cases. Whether the three sizes of gpClL (Fig. 2B) represent distinct channel types or simply were conductance states of a particular Cl- channel cannot be resolved conclusively from the present data. However, voltage dependence of Po also was different among the three gpClL sizes (Fig. 9B and Table 3), supporting functionally distinct channel activities. Single-channel conductance (Fig. 2B) and voltage dependence of Po (Fig. 9) can be used to compare these Cl- channel activity patterns (Fig. 11B) with those of other Cl- channel types.

Clor has been observed in basolateral membranes of rat and mouse colonic crypts (3, 43) as well as mouse jejunal crypts (5). Those studies focused primarily on the cells at the base of the crypt near the stem cell, whereas the results reported here were from cells making up the cylindrical portion of the crypt where rapid cell division and secretion occur. The gamma  of mClor in basolateral membranes of mouse colonic crypts (43) is similar to gpClor at negative Vhold and at positive Vhold is intermediate to the two forms of gpClor (Fig. 2B). Excision permits comparison of Clor under well-defined conditions of Cl- concentration and Vm; at Vm = 0 mV and 160 mM Cl-, gamma  is similar for gpClor, mClor, and T84Clor: 47 (Fig. 3B), 36 (5, 43), and 41 (20, 26) pS, respectively. On the basis of outward rectification and ion selectivity, CLC-3 appears at present the most likely candidate for producing Clor (30, 46, 69) and therefore also gpClor.

Po for Clor generally is larger at more positive Vhold in cell-attached recording (26, 37, 43), similar to gpClor (Fig. 9A). Consequently, the time-averaged I-V relations for gpClor were steeply rectified (Fig. 11). After excision this voltage dependence of Po often is retained (26, 37, 43) but sometimes is lost (5, 48), as with gpClor (Fig. 10B). Clearly, time-averaged currents would be less rectified with voltage-independent Po. Po for CLC-1 is largest at more positive Vm, with an equivalent gating charge of ~1 (33) similar to gpClor (Fig. 9A); however, gamma  for CLC-1 is only ~2 pS (33). Po for CLC-2 is largest at more negative Vm and for CLC-3 is relatively voltage independent (9, 33). This range of voltage sensitivities for CLC members suggests that this general channel structure could produce the characteristics of gpClor, but the exact features of gpClor cannot be assigned conclusively to any of the CLC members.

Both gpClL21 and gpClL13 had voltage-independent gamma  with Po that was larger at more positive Vhold (Figs. 2C and 9B). These conduction characteristics produce outwardly rectified time-averaged currents (Fig. 11B) that are similar to many Cl- currents recorded in whole cell mode (16, 33, 47, 57, 62). The ~15-pS gamma  of CLCA is comparable to that of gpClL13 (16). A basolateral membrane Cl- channel, with linear gamma  of 28 pS, activated by cell swelling in rat colonic crypts (12, 13), may correspond to gpClL21. Basolateral membrane Cl- channels in kidney tubular cells have linear gamma  of 20-28 pS, and those from the thick ascending limb of Henle's loop have Po that is larger at more positive Vm (53, 56). Although expression of CLC-K has only been demonstrated in the kidney and inner ear (33, 52, 55, 66), modest outward rectification of CLC-Kb/barttin whole cell current (66) is consistent with the behavior of gpClL21 (Fig. 11B).

The Cl- channel formed by CFTR has voltage-independent gamma  of 6-10 pS (33, 60) as well as voltage-independent Po (59). Both of these characteristics are consistent with gpClL8 behavior (Fig. 2C). However, CFTR has modestly outwardly rectified currents when recorded while cell attached compared with the linear I-V relation for gpClL8. As with the other gpClL, gamma  was voltage independent either because the low intracellular Cl- concentration was saturating for conduction (consistent with relatively high-affinity Cl- interaction in the pore) or currents would be modestly inwardly rectified with equal Cl- concentrations on both sides. For CFTR the outward rectification appears to include influences from phosphorylation and permeation by other intracellular anions (60). If gpClL8 were produced by CFTR, then other regulatory influences would be necessary to account for these differences in ion conduction. The voltage-independent steady-state conductance of CLC-Ka/barttin (66) also is consistent with the behavior of gpClL8.

Uncertainty concerning the molecular identity of gpClor and gpClL exists, in part, because the functional properties differ somewhat from those of presently defined Cl- channel families. Channels observed in native tissue may occur as heteromultimeric assemblies of channel subunits together with regulatory components that produce distinct behavior. Combinations of CLC-1 and CLC-2 result in biophysical properties that are not a simple sum of the homomultimeric channels; in particular, voltage dependence is largely lost (41). The presence of the beta -subunit barttin also alters the properties of CLC-K (66). Isolation of crypts may initiate a progressive alteration in channel subunits present such that biophysical and regulatory properties might change. However, colonic mucosa retains secretagogue sensitivity for K secretion for >8 h after isolation (51), and channel responses were similar over the ~36-h patch-clamp study period. Species-dependent differences in channel function also may contribute to difficulty in precisely identifying these guinea pig Cl- channels. Although the identities of the Cl- channels observed in this study remain undetermined, a basolateral location does allow contributions to cell functions ranging from cell volume control to transepithelial ion flow.

Regulation of K+ secretion. Control of g<UP><SUB>b</SUB><SUP>Cl</SUP></UP> contributes to determining the rate of electrogenic K+ secretion by providing an exit path for Cl- entering via Na+-K+-2Cl- cotransporters (Fig. 15) and by influencing the electrical driving force for apical K+ exit. The Cl- channels gpClor and gpClL (Table 3) are likely involved in augmenting this g<UP><SUB>b</SUB><SUP>Cl</SUP></UP> needed for K+ secretion. Stimulation of electrogenic K+ secretion occurs through beta -adrenergic receptors (17) and prostaglandin EP2 receptors (23), both of which act through increasing intracellular cAMP. Activation of prostaglandin DP receptors, which also increase cAMP production, stimulates electrogenic KCl secretion (23), suggesting a functional compartmentalization of cAMP allowing selective induction of Cl- secretion. The concentration dependence of the forskolin response supports the existence of two interactions with distinct affinity (Fig. 5B) similar to adenylyl cyclase activation in human colonic crypts (4). This divergence of sensitivities to forskolin may represent distinct forms of adenylyl cyclase, because activation with the G protein GSalpha increases forskolin affinity of adenylyl cyclase from an EC50 of >10 µM to ~100 nM (64). Specific activation of Cl- channels to support each ion secretory mode may occur via adenylyl cyclases that are functionally compartmentalized through the action of other intracellular signaling cascades.

Secretagogues for electrogenic K+ secretion, epinephrine and either PGE2 or forskolin at low concentration, increased the number of active gpClor and gpClL (Table 3), consistent with higher g<UP><SUB>b</SUB><SUP>Cl</SUP></UP>. The epinephrine secretory state of gpClor had lower Po due to a shift in the Po voltage dependence to more positive Vhold (Fig. 9A). gpClL13 also supported secretagogue activation of g<UP><SUB>b</SUB><SUP>Cl</SUP></UP> with increased Po at the spontaneous Vcell (Fig. 9B). In addition, high-concentration PGE2 that stimulates Cl- secretion (23, 50) appeared to decrease N for gpClor (Fig. 7B) such that g<UP><SUB>b</SUB><SUP>Cl</SUP></UP> would be reduced. Cholinergic stimulation of a Cl- secretory state increased flickering of gpClor, consistent with reduced Po (Fig. 7B). GTP induced a similar flickery state with reduced current amplitude in mClor excised from colonic crypts (43). Overall, K+ secretagogues increased N for gpClor and gpClL, but epinephrine produced a distinct secretory mode for gpClor with lower Po than with forskolin (Fig. 9A). Therefore, other intracellular signals in addition to cAMP may be needed to regulate the basolateral membrane Cl- channels involved in electrogenic K+ secretion.

Kinetic analysis of gpClor activity (Figs. 12, 13, and 14) provided further insight into regulation of K+ secretion. Epinephrine activation of gpClor occurred with flickery kinetics distinct from basally active or forskolin-activated channels (Figs. 6 and 7). Open durations were shorter (Fig. 12 and Table 2), and closed durations were dominated by events with a medium-length tau  (Figs. 12 and 13 and Table 2), leading to a shift in the voltage dependence of Po (Fig. 9B). Absence of the flickery epinephrine mode in the presence of forskolin (Fig. 7B) suggested that a basal/forskolin-induced factor can suppress entry into this flickery kinetic mode. Because excision of patches with gpClor in the flickery mode led to slower kinetics similar to the basal/forskolin mode (Fig. 10 and Table 2), a component of this kinetic control factor apparently was readily exchangeable. Excision of patches also led to a loss of voltage dependence for Po (Fig. 10) and reduction in gamma  at positive Vm (Fig. 4), consistent with readily exchangeable regulators.

Activation of Clor after excision is a general observation in many cell types (3, 5, 26, 30, 33, 43, 61), supporting the existence of cytosolic inhibitory regulators (35, 36, 38). The exact nature of these regulators is presently unknown, but several classes of compounds have been implicated. Extracts of cytosol appear to contain both a high-molecular-mass (10-300 kDa) substance, likely a protein, and a lower-molecular-mass (<1 kDa) substance similar to steroids (36). Addition of cytosol to excised HT29Clor reduces tau O from 32 to 7 ms (49), similar to the difference between excised and basal/forskolin modes for gpClor (Table 2). Closed tau  for HT29Clor are essentially unchanged by cytosol (49) similar to excised and basal/forskolin modes for gpClor (Figs. 13 and 16). Fatty acids such as arachidonic acid can reduce Po of Clor, but the mode of action and molecular size suggest that the inhibitor present in cytosol is distinct (31, 36, 38).


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Fig. 16.   Regulation of closed states for Clor. Time constants and proportions of events for kinetic states are shown for gpClor (Fig. 13) and Clor recorded from the human colonic tumor cell line HT29 (49). All kinetic constants for HT29Clor are from excised I/O patches; aldosterone (100 µM) and cytosol were added to the bath solution.

The flickery nature of the epinephrine-induced mode for gpClor can be mimicked in excised HT29Clor by aldosterone or glibenclamide (48, 49); tau O is reduced to ~1 ms, and events with tau C of ~2 ms dominate closures (Fig. 16). The high EC50 for aldosterone action (~20 µM) led Rabe and Frömter (49) to suggest that another lipid component may be the actual endogenous control component. Both aldosterone and glibenclamide are proposed to act by moving HT29Clor into a specific kinetic mode rather than by blocking (48, 49), which was supported by the action of the secretagogue epinephrine to induce a nearly identical kinetic mode in gpClor (Fig. 16 and Table 2). Furthermore, such a steroidlike lipid may be the specific mediator of epinephrine activation.

Secretory control of gpClor and gpClL included increased N and Po such that a range of g<UP><SUB>b</SUB><SUP>Cl</SUP></UP> was possible. Each of these Cl- channel activities may contribute a unique set of regulatory responses to adjust for demands on secretory rate and cell volume control. Regulatory subunits associating with the observed Cl- channels may be necessary to produce the specific types of secretagogue sensitivities. The CLC-K beta -subunit barttin promotes channel activity (66), and subunits alter responsiveness of K+ channels to cytoplasmic signals. In particular, CFTR belongs to a protein family that includes the sulfonylurea receptor that confers ATP sensitivity to K+ channels, and CFTR has been shown to associate with K+ channels (54). The coincident presence of gpKir with gpClor (Fig. 1A) and gpClL suggests that a tight regulatory connection potentially could exist between g<UP><SUB>b</SUB><SUP>K</SUP></UP> and g<UP><SUB>b</SUB><SUP>Cl</SUP></UP> in colonic crypts. Similarly, association of gpClL with gpClor might confer control like that between CFTR and Clor (30).

A basolateral membrane location of Cl- channels in epithelia presumably serves both volume-regulatory needs and transepithelial flow requirements. Whether volume-activated channels open during K+ secretion was not determined in the present study, but conductive Cl- exit would aid maintenance of cell volume during secretory stimulation of Cl- influx through Na+-K+-2Cl- cotransporters. As noted numerous times, transepithelial solute flow combines aspects of regulatory volume increase (RVI) and regulatory volume decrease (RVD). The distinct situation in epithelia, as during electrogenic K+ secretion (Fig. 15), is that cell volume is maintained near control values while transport processes resembling RVI and RVD remain active. Simultaneous and continual operation of influx and efflux pathways is what produces sustained transepithelial flow. Thus a complete control scheme for basolateral Cl- channels will include not only the initiating events but also the modulating signals that ensure stable cellular conditions during ongoing secretion.


    ACKNOWLEDGEMENTS

This study was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-39007) and the Wright State University Research Challenge Program.


    FOOTNOTES

Address for reprint requests and other correspondence: D. R. Halm, Dept. of Physiology and Biophysics, Wright State Univ., 3640 Colonel Glenn Hwy., Dayton OH 45435 (E-mail: dan.halm{at}wright.edu).

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.

First published December 27, 2002;10.1152/ajpcell.00464.2002

Received 3 October 2002; accepted in final form 5 December 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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