INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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) 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 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 (Cl_vol) and an outwardly rectified Cl channel (Cl_or) 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 () 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  from ~1 pS to >40 pS (33, 52, 69). CLCA is activated by Ca²⁺ with  of 15-30 pS (16). Cell swelling activates Cl_vol, which has outwardly rectified currents with  of uncertain size (12, 47, 61, 62). The outwardly rectified - and depolarization-enhanced open probability (P_o) of Cl_or (20, 26, 43, 61) appears to be distinct from that of Cl_vol (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 P_o, would lead to greater g consistent with an involvement in Cl exit during electrogenic K⁺ secretion.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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, 4 H_(3  X)PO, 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 (V_t) 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 (I_sc) and compensation for solution resistance. Current was passed across the tissue through two Ag-AgCl electrodes connected by Ringer-agar bridges. I_sc is described as positive for current flowing across the epithelium from the mucosal side to the serosal side. Transepithelial conductance (G_t) 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, 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, 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 M 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 G 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.

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 I_sc and G_t to forskolin were fit to Henri-Michaelis-Menten binding curves with a nonlinear least-squares procedure. Two independent binding curves were required (23), I = I_A/[1 + (EC/C)] + I_B/[1 + (EC/C)] or G = G_A/[1 + (EC/C)] + G_B/[1 + (EC/C)], such that total I_sc or G_t was a combination of these two components (I_A and I_B; G_A and G_B) 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.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

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 V_hold, respectively, as determined by the ion concentration gradients (24, 27) together with a cell membrane electrical potential difference (PD) (V_cell) of about 65 mV (42). Thus, at resting V_cell (V_hold = 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 V_hold corresponding to a V_cell of 0 mV. Using high-K⁺ pipette solution (Table 1) has the advantage of shifting the reversal potential for K⁺ channels toward a V_cell of 0 mV. Reversal of K⁺ channel currents near a V_hold of +40 mV (39) indicates that resting V_cell was about 40 mV in these isolated crypts. The shift in reversal potential for K⁺ channels (to V_cell = 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 (^gpK_ir; Ref. 39), supporting a resting V_cell of about 40 mV in these recordings. Other currents consistent with Cl channels also were seen, reversing at V_hold 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).

View larger version (37K): [in this window] [in a new window]   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-) Cl channel of 12 pS. C: current traces are shown for 2 linear- Cl channels of 6 and 17 pS.

Outwardly rectified Cl channels were seen with two sizes of outward current in guinea pig crypts (^gpCl_or); inward currents were similar in size (Fig. 2A). Activity of ^gpCl_or was observed in 16 of 231 patches (7%). Cl currents (Fig. 2B) with linear I-V relations (^gpCl_L) were observed, congregated into three groups (Fig. 2C) on the basis of single-channel : 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 ^gpCl_L 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 ^gpCl_or and ^gpCl_L (Fig. 2, A and B) support outwardly directed Cl flow through these channels at spontaneous V_cell, consistent with the predicted requirements for conductive Cl flow across the basolateral membrane during electrogenic K⁺ secretion.

View larger version (22K): [in this window] [in a new window]   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 (, n = 3). B: I-V relations are shown for linear- 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 (, n = 7; , n = 10; , n = 9). C: single-channel conductance (chord conductance; ) is shown, from I-V relations in A and B. Mean  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 ^gpK_ir (Fig. 1A) and were distinguished by currents exhibiting distinct reversal potentials and rectification. In five patches, ^gpCl_or was observed together with ^gpK_ir. On the basis of the proportion of patches exhibiting each of these channel types (39), four patches would have been expected to contain both ^gpCl_or and ^gpK_ir, 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- Cl channels also was observed with ^gpK_ir (expected number of patches): seven (4) for ^gpCl_L21, seven (4) for ^gpCl_L13, and three (3) for ^gpCl_L8. Appearance of these channels together with ^gpK_ir further supported identification as Cl selective, because nonselective cation currents would have reversed near +40 mV similar to ^gpK_ir. The various Cl channels, as distinguished by conductance, also were observed together. Presence of linear- Cl channels together with ^gpCl_or was observed (expected number of patches): four (1) for ^gpCl_L21, four (1) for ^gpCl_L13, and zero (1) for ^gpCl_L8. The presence together of multiple linear- Cl channel types also was observed (expected number of patches): three (1) for ^gpCl_L21 with ^gpCl_L13, three (1) for ^gpCl_L21 with ^gpCl_L8, and two (1) for ^gpCl_L13 with ^gpCl_L8. The results suggest a clustering of these basolateral channel types.

Ion selectivity. Activity of ^gpCl_or 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 (P_K/P_Cl) = 0.07 ± 0.03 (n = 7) and relative permeability of Na⁺ over Cl (P_Na/P_Cl) = 0.08 ± 0.04 (n = 4). Ion selectivity of ^gpCl_L 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 ^gpCl_L; however, activity did not persist in enough cases to resolve the excised I-V relations completely.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Ion selectivity of gpClor . A: after excision into inside-out (I/O) configuration, bath solution (Table 1) was changed among NaCl (, ; n = 7, 2), KCl (; n = 7), K-gluconate (; n = 5), Na-gluconate (; n = 2), 300 KCl (; n = 2), and 300 NaCl (; 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.

View larger version (17K): [in this window] [in a new window]   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; , 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 (, 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 I_sc across guinea pig distal colonic mucosa (Fig. 5, A and B), consistent with cation secretion. Identification of this forskolin-stimulated I_sc 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 I_sc and G_t (data not shown), similar to the action on epinephrine-stimulated electrogenic K⁺ secretion (17). Forskolin stimulated (Fig. 5, C and D) negative I_sc consistent with K⁺ secretion at low concentrations and additionally at higher concentrations positive I_sc consistent with Cl secretion (50), which mimicked the response to PGE₂ (23, 50) and cAMP (17).

View larger version (15K): [in this window] [in a new window]   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 = Isc/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 ().

View larger version (37K): [in this window] [in a new window]   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).

View larger version (52K): [in this window] [in a new window]   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.

View larger version (21K): [in this window] [in a new window]   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.

View larger version (21K): [in this window] [in a new window]   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 (; n = 3) or 5 µM epinephrine (; n = 4). Po of gpClL21 for basal condition (; 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 (; n = 3) and gpClL13 (; n = 3) is shown. Stimulation of gpClL13 with either epinephrine (5 µM) or low-concentration PGE2 (100 nM) altered Po (; 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).

View larger version (32K): [in this window] [in a new window]   Fig. 10.   Excision of gpClor . A: 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 () and epinephrine () from Fig. 9A, are also shown in relation to Vm after correction for apparent Vcell of 40 mV (39).

View larger version (15K): [in this window] [in a new window]   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 (; from Fig. 2A). Also shown are time-averaged currents, iClPo, for basal/forskolin state () and for epinephrine state (). 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] (), gpClL13[basal] () 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 ^gpCl_or. Patches with only one ^gpCl_or 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 P_o (Fig. 9A). Thus the powerful K⁺ secretagogue epinephrine induced a kinetic mode for ^gpCl_or distinct from the basal/forskolin kinetic mode.

View larger version (27K): [in this window] [in a new window]   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).

View larger version (16K): [in this window] [in a new window]   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 (; n = 4). Activity during epinephrine (; n = 3) and after excision (; n = 5) also are shown. Asterisk indicates voltage-dependent time constant; value at +60 mV is shown.

View larger version (21K): [in this window] [in a new window]   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 (O) are shown for basal/forskolin (; n = 4), epinephrine (; n = 3), and excised (; n = 4) conditions. Also shown is voltage dependence of C2 during epinephrine stimulation (; 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 (V_t) 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 (V_a) and basolateral (V_b) 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.

View larger version (16K): [in this window] [in a new window]   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.]

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 secr