Cl− Channels in Basolateral TAL Membranes XV. Molecular Heterogeneity Between Cortical and Medullary Channels

We have isolated two new and highly homologous cDNAs, mmClC-Ka from mouse outer medulla and mcClC-Ka from mouse cortex. In both cases, mRNA was obtained from the indicated region and subjected to RT-PCR using primers from the nucleotide sequence of rbClC-Ka, which encodes basolateral Cl⁻ channels (termed rbClC-Ka) in rabbit MTAL. The predicted protein products of mmClC-Ka and mcClC-Ka, mmClC-Ka and mcClC-Ka, respectively, were 85% homologous and had predicted molecular weights of 75 kDa. The predicted protein sequences for mmClC-Ka and rbClC-Ka had three cytosolic sites—threonine 185, threonine 187 and serine 270—which were absent in mcClC-Ka. These three moieties represent potential sites for phosphorylation of mmClC-Ka and rbClC-Ka, but not of mcClC-Ka, and may account for the failure of (ATP + PKA) to increase the open time probability P _o in basolateral CTAL Cl⁻ channels. We prepared antisense oligonucleotides specific for nonhomologous regions of these two cDNAs, mmAntisense for mmClC-Ka and mcAntisense for mcClC-Ka. Using anti-rbClC-Ka, a polyclonal antibody to rbClC-Ka, we found that, when transfected into cultured mouse MTAL and CTAL cells, mmAntisense suppressed the appearance of the 75 kDa band by 50% in vesicles from MTAL but not CTAL cells, while transfection of MTAL and CTAL cells with mcAntisense suppressed appearance of the 75 kDa band in vesicles from CTAL but not MTAL cells. mmAntisense transfection also prolonged the half-time (T_1/2, sec) for ³⁶Cl⁻ efflux in cultured MTAL cells from 82.4 ± 6.8 sec (sem) to 187.8 ± 9.5 sec (n= 5; P= 0.0001) while mcAntisense transfection had no such effect. Conversely, in cultured CTAL cells, mcAntisense transfection prolonged the T_1/2 for ³⁶Cl⁻ efflux from 80.9 ± 6.3 sec to 191.8 ± 6.5 sec (n= 5; P= 0.00005), while mmAntisense had no such effect. We conclude that mmClC-Ka and mcClC-Ka may encode the basolateral Cl⁻ channels mediating net Cl⁻ absorption in mouse MTAL and CTAL, respectively. Received: 16 May 2000/Revised: 30 June 2000     

Cl channels in basolateral TAL membranes. XVII. Kinetic properties of mcClC-Ka,a basolateral CTAL Cl- channel

This paper describes the kinetics of Cl- flux through mcClC-Ka Cl- channels from basolateral membranes of mouse CTAL cells. We have cloned two separate but highly homologous Cl- channels, mmClC-Ka from cultured mouse MTAL cells and mcClC-Ka from cultured mouse CTAL cells. The mmClC-Ka and mcClC-Ka channels appear to mediate net Cl- absorption in the MTAL and CTAL, respectively. The kinetics of Cl- permeation through mmClC-Ka channels exhibit traditional criteria for a first-order process, including saturation kinetics. Thus mmClC-Ka channels operate functionally as if the channels were occupied by a single Cl- ion at any given time. In the present studies, we examined conductance-concentration relations in mcClC-Ka channels, and compared both mole-fraction effects and ion selectivity characteristics in mmClC-Ka and mcClC-Ka channels. In mcClC-Ka channels, we observed both self-block at high external Cl- concentrations and, at constant ionic strength, an anomalous mole-fraction effect using external solutions containing varying F-/Cl- concentrations. Neither effect was obtained in mmClC-Ka channels. These data are consistent with the possibility that Cl- permeation through mcClC-Ka channels involved multi-ion occupancy channels that expressed single-file behavior.     

Cl− channels in basolateral renal medullary membranes: VII. Characterization of the intracellular anion binding sites

A unique property of basolateral membrane Cl^– channels from the mTAL is that the Cl^– concentration facing the intracellular aspects of these channels is a determinant of channel open time probability (P ₀ ). The K _1/2 for maximal activation of P ₀ by Cl^– facing intracellular domains of these channels is 10 mm Cl^–. The present experiments evaluated the nature of these Cl^–-interactive sites. First, we found that the impermeant anion isethionate, when exposed to intracellular Cl^– channel faces, could augment P ₀ with a K _1/2 in the range of 10 mm isethionate without affecting conductance (g _Cl, pS). Second, pretreatment of the solutions facing the intracellular aspects of the channels with either 1 mm phenylglyoxal (PGO), an arginine-specific reagent, or the lysine/terminal amine reagent trinitrobenzene sulfonic acid (TNBS, 1 mm), prevented the activation of P ₀ usually seen when the Cl^– concentration of solutions facing intracellular channel domains was raised from 2 to 50 mm. However, when the Cl^– channel activity was increased by first raising the Cl^– concentration bathing intracellular channel faces from 2 to 50 mm, subsequent addition of either PGO or TNBS to solutions bathing intracellular Cl^– channel faces had no effect on P ₀ . We conclude that the intracellular aspects of these Cl^– channels contain Cl^–-interactive loci (termed [Cl] _i ) which are accessible to impermeant anions in intracellular fluids and which contain arginineand lysine-rich domains which can be inactivated, at low ambient Cl^– or isethionate concentrations, by interactions with PGO or TNBS.We acknoeledge the able technical assistance of Anna Grace Stewart. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veteterans Administration Merit Review Grants to T. E.Andreoli and to W. B. Reeves. C. J. Winters is a Veterans Administration Associate Investigator.     

Cl− channels in basolateral renal medullary vesicles: V. Comparison of basolateral mTALH Cl− channels with apical Cl− channels from jejunum and trachea

Summary Cl^– channels from basolaterally-enriched rabbit outer renal medullary membranes are activated either by increases in intracellular Cl^– activity or by intracellular protein kinase A (PKA). Phosphorylation by PKA, however, is not obligatory for channel activity since channels can be activated by intracellular Cl^– in the absence of PKA. The PKA requirement for activation of Cl^– channels in certain secretory epithelia is, in contrast, obligatory. In the present studies, we examined the effects of PKA and intracellular Cl^– concentrations on the properties of Cl^– channels obtained either from basolaterally-enriched vesicles derived from highly purified suspensions of mouse medullary thick ascending limb (mTALH) segments, or from apical membrane vesicles obtained from two secretory epithelia, bovine trachea and rabbit small intestine. Our results indicate that the Cl^– channels from mTALH suspensions were virtually identical to those previously described from rabbit outer renal medulla. In particular, an increase in intracellular (trans) Cl^– concentration from 2 to 50 mm increased both channel activity (P _o) and channel conductance (g _Cl, pS). Likewise, trans PKA increased mTALH Cl^– channel activity by increasing the activity of individual channels when the trans solutions were 2 mm Cl. Under the latter circumstance, PKA did not activate quiescent channels, nor did it affect g _Cl. Moreover, when mTALH Cl^– channels were inactivated by reducing cis Cl^– concentrations to 50 mm, cis PKA addition did not affect P _o. These results are consistent with the view that these Cl^– channels originated from basolateral membranes of the mTALH.Cl^– channels from apical vesicles from trachea and small intestine were completely insensitive to alterations in trans Cl^– concentrations and demonstrated markedly different responses to PKA. In the absence of PKA, tracheal Cl^– channels inactivated spontaneously after a mean time of 8 min; addition of PKA to trans solutions reactivated these channels. The intestinal Cl^– channels did not inactivate with time. Trans PKA addition activated new channels with no effect on basal channel activity. Thus the regulation of Cl^– channel activity by both intracellular Cl^– and by PKA differ in basolateral mTALH Cl^– channels compared to apical Cl^– channels from either the tracheal or small intestine.We acknowledge the able technical assistance of Steven D. Chasteen. Clementine M. Whitman provided her customary excellent secretarial assistance. This work was supported by Veterans Administration Merit Review Grants to T.E. Andreoli and to W.B. Reeves. C.J. Winters is a Veterans Administration Associate Investigator.     

Cl− channels in basolateral renal medullary memnbranes: III. Determinants of single-channel activity

Summary We evaluated the effects of vawrying aqueous Cl^– concentrations, and of the arginyl- and lysyl-specific reagent phenylglyoxal (PGO), on the properties of Cl^– channels fused from basolaterally enriched renal medullary vesicles into planar lipid bilayers. The major channel properties studied were the anion selectivity sequence, anionic requirements for, channel activity. and the efects of varying Cl^– concentrations and/or PGO on the relation between holding voltageV _H -mV) and open-time probability (P _o).Reducingcis Cl^– concentrations, in the range 50–320mm, produced a linear reduction in fractional open time (P _v) with a half-maximal reduction inP _o atcis Cl^–170mM. Channel activity was sustained by equimolar replacement ofcis Cl^– with F^–, but not with impermeant isethionate. Fortrans solutions, the relation between Cl^– concentration andP ₀ at 10mm Cl^–. Reducingcis Cl^– had no effect on the gating charge (Z) for channel opening, but altered significantly the voltage-independent, energy (G) for channel opening.Phenylglyoxal (PGO) reducedZ and altered G for Cl^– channel activity when added tocis, but nottrans solutions, Furthermore, in the presence ofcis PGO, reducing thecis Cl^– concentration had no effect onZ but altered G. Thus we propose thatcis PGO and,cis Cl^– concentrations affect separate sites determining channel activity at the extracellular faces of, these Cl^– channels.     

Identification and regulation of whole-cell Cl− and Ca2+-activated K+ currents in cultured medullary thick ascending limb cells

The whole-cell patch-clamp technique has been used to study membrane currents in cultured rabbit medullary thick ascending limb (MTAL) epithelial cells. A Ca²⁺-activated K⁺ current was characterized by its voltage-dependent and Ca²⁺-dependent properties. When the extracellular K⁺ ion concentration was increased from 2 to 140 mm, the rereversal potential (E_k) was shifted from –85 to 0 mV with a slope of 46 mV per e-fold change. The Ca²⁺-activated K⁺ current is blocked by charybdotoxin (CTX) in a manner similar to the apical membrane Ca²⁺-activated K⁺ channel studied with the single channel patch-clamp technique. The results suggest that the Ca²⁺-activated K⁺ current is the predominant, large conductance and Ca²⁺-dependent K⁺ pathway in the cultured MTAL cell apical membrane. The biophysical properties and physiological regulation of a Cl^– current were also investigated. This current was activated by stimulation of intracellular cAMP using forskolin and isobutyl-1-methylxanthine (IBMX). The current-voltage (I–V) relationship of the Cl^– current showed an outward-rectifying pattern in symmetrical Cl^– solution. The Cl^– selectivity of the whole-cell current was confirmed by tail current analysis in different Cl^– concentration bath solutions. Several Cl^– channel blockers were found to be effective in blocking the outward-rectifying Cl^– current in MTAL cells. The cAMP-dependent Cl^– transport in MTAL cells was further confirmed by measuring changes in the intensity of Cl^– sensitive dye using fluorescence microscopy. These results suggest that the Cl^– channel in the apical or basolateral membrane of MTAL cells may be regulated by cAMP-dependent protein-kinase-induced phosphorylation.This study was supported by the National Institutes of Health grants GM46834 to L.L. and DK32753 to W.B.G., and by a Grant-in-Aid from the American Heart Association of Ohio to L.L.     

Single chloride channels in colon mucosa and isolated colonic enterocytes of the rat

Summary Chloride channels from rat colonic enterocytes were studied using the patch-clamp technique. After removal of mucus, inside-out patches were excised from the apical membrane of intact epithelium located at the luminal surface. They contained spontaneously switching Cl^– channels with a conductance of 35–40 pS. The channels were blocked reversibly by anthracene-9-carboxylic acid (1mm).In excised patches from single enterocytes, isolated by calcium removal, the Cl^– channels were studied in more detail. TheI–V relation was linear between ±80 mV. The selectivity was I^–>Br^–>Cl^–=NO ₃ ^– >F^–=HCO ₃ ^– .Thirty pS Cl^– channels were also found on the basolateral membrane of crypts isolated by brief calcium removal. TheI–V curve of these Cl^– channels was also linear.The results provide direct evidence for the existence of Cl^– channels in the apical membrane of surface cells in colonic mucosa. The properties of these channels are similar to those previously observed when incorporating membrane vesicles into planar lipid bilayers. Both results support the validity of the theoretical models describing intestinal secretion.     

Cl− channels in basolateral renal medullary membrane vesicles: IV. Analogous channel activation by Cl− or cAMP-dependent protein kinase

Summary We examined the interactions of cAMP-dependent protein kinase and varying aqueous Cl^– concentrations in modulating the activity of Cl^– channels obtained by fusing basolaterally enriched renal outer medullary vesicles into planar lipid bilayers. Under the present experimental conditions, thecis andtrans solutions face the extracellular and intracellular aspects of these Cl^– channels, respectively. Raising thetrans Cl^– concentration from 2 to 50mm increased the channel open-time probability, raised the unit channel conductance, and affected the voltage-independent determinant (G) of channel activity but not the gating charge (Winters, C.J., Reeves, W.B., Andreoli, T.E. 1990.J. Membrane Biol. 118:269–278). With 2mm trans KCl,trans addition of the catalytic subunit of PKA (C-PKA) plus ATP increased channel open-time probability and altered the voltage-independent determinant of channel activity without affecting either unit channel conductance or gating charge. The effect was ATP specific, did not occur with (C-PKA plus ATP) addition tocis solutions, and was abolished by denaturing C-PKA. Finally, (C-PKA plus ATP) activation of channel activity was not detected with relatively high (50mm)trans Cl^– concentrations. These data indicate that (C-PKA plus ATP) might modulate Cl^– channel activity by phosphorylation at or near the Cl^–-sensitive site on the intracellular face of these channels.     

Chloride transport activation by plasma osmolarity during rapid adaptation to high salinity of Fundulus heteroclitus

Transition from low salt water to sea water of the euryhaline fish, Fundulus heteroclitus, involves a rapid signal that induces salt secretion by the gill chloride cells. An increase of 65 mOsm in plasma osmolarity was found during the transition. The isolated, chloridecell-rich opercular epithelium of sea-water-adapted Fundulus exposed to 50 mOsm mannitol on the basolateral side showed a 100% increase in chloride secretion, which was inhibited by bumetanide 10^–4 m and 10^–4 m DPC (N-Phenylanthranilic acid). No effect of these drugs was found on apical side exposure. A Na⁺/H⁺ exchanger, demonstrated by NH₄Cl exposure, was inhibited by amiloride and its analogues and stimulated by IBMX, phorbol esters, and epithelial growth factor (EGF). Inhibition of the Na⁺/H⁺ exchanger blocks the chloride secretion increase due to basolateral hypertonicity. A Cl^–/HCO ₃ ^– exchanger was also found in the chloride cells, inhibited by 10^–4 m DIDS but not involved in the hyperosmotic response. Ca²⁺ concentration in the medium was critical for the stimulation of Cl^– secretion to occur. Chloride cell volume shrinks in response to hypertonicity of the basolateral side in sea-water-adapted operculi; no effect was found on the apical side. Freshwater-adapted fish chloride cells show increased water permeability of the apical side. It is concluded that the rapid signal for adaptation to higher salinities is an increased tonicity of the plasma that induces chloride cell shrinkage, increased chloride secretion with activation of the Na⁺K⁺2Cl^– cotransporter, the Na⁺/H⁺ exchanger and opening of Cl^– channels.The work was supported by the National Institutes of Health, Research Grant EYO1340 to J.A.Z. Part of this research was performed while Dr. Zadunaisky was a Scholar In Residence at the Fogarty International Center of The National Institutes of Health in Bethesda, Maryland. Ms. Dawn Roberts was a fellow of the Grass Foundation and Pew Foundation during this work. Grants from the National Science Foundation and the National Institutes of Health to the Mount Desert Island Biological Laboratory also provided assistance for this research.     

Vasopressin alters the mechanism of apical Cl− entry from Na+:Cl− to Na+:K+:2Cl− cotransport in mouse medullary thick ascending limb

Summary Experiments were performed usingin vitro perfused medullary thick ascending limbs of Henle (MTAL) and in suspensions of MTAL tubules isolated from mouse kidney to evaluate the effects of arginine vasopressin (AVP) on the K⁺ dependence of the apical, furosemide-sensitive Na⁺:Cl^– cotransporter and on transport-related oxygen consumption (QO₂). In isolated perfused MTAL segments, the rate of cell swelling induced by removing K⁺ from, and adding onemm ouabain to, the basolateral solution [ouabain(zero-K⁺)] provided an index to apical cotransporter activity and was used to evaluated the ionic requirements of the apical cotransporter in the presence and absence of AVP. In the absence of AVP cotransporter activity required Na⁺ and Cl^–, but not K⁺, while in the presence of AVP the apical cotransporter required all three ions.⁸⁶Rb⁺ uptake into MTAL tubules in suspension was significant only after exposure of tubules to AVP. Moreover,²²Na⁺ uptake was unaffected by extracellular K⁺ in the absence of AVP while after AVP exposure²²Na⁺ uptake was strictly K⁺-dependent. The AVP-induced coupling of K⁺ to the Na⁺:Cl^– cotransporter resulted in a doubling in the rate of NaCl absorption without a parallel increase in the rate of cellular²²Na⁺ uptake or transport-related oxygen consumption. These results indicate that arginine vasopressin alters the mode of a loop diuretic-sensitive transporter from Na⁺:Cl^– cotransport to Na⁺:K⁺:2Cl^– cotransport in the mouse MTAL with the latter providing a distinct metabolic advantage for sodium transport. A model for AVP action on NaCl absorption by the MTAL is presented and the physiological significance of the coupling of K⁺ to the apical Na⁺:Cl^– cotransporter in the MTAL and of the enhanced metabolic efficiency are discussed.     

pHi regulation in Ehrlich mouse ascites tumor cells: Role of sodium-dependent and sodium-independent chloride-bicarbonate exchange

pH _i recovery in acid-loaded Ehrlich ascites tumor cells and pH _i maintenance at steady-state were studied using the fluorescent probe BCECF.Both in nominally HCO ₃ ^– -free media and at 25 mm HCO ₃ ^– , the measured pH _i (7.26 and 7.82, respectively) was significantly more alkaline than the pH _i . value calculated assuming the transmembrane HCO ₃ ^– gradient to be equal to the Cl^– gradient. Thus, pH _i in these cells is not determined by the Cl^– gradient and by Cl^–/HCO ₃ ^– exchange.pH _i recovery following acid loading by propionate exposure, NH ₄ ⁺ withdrawal, or CO₂ exposure is mediated by amiloride-sensitive Na⁺/H⁺ exchange in HCO₃ ^– free media, and in the presence of HCO ₃ ^– (25 mm) by DIDS-sensitive, Na⁺-dependent Cl^–/HCO ₃ ^– exchange. A significant residual pH _i recovery in the presence of both amiloride and DIDS suggests an additional role for a primary active H⁺ pump in pH _i regulation. pH _i maintenance at steady-state involves both Na⁺/H⁺ exchange and Na⁺-dependent Cl^–/HCO ₃ ^– exchange.Acute removal of external Cl^– induces a DIDS-sensitive, Na⁺-dependent alkalinization, taken to represent HCO ₃ ^– influx in exchange for cellular Cl^–. Measurements of ³⁶Cl^– efflux into Cl^–-free gluconate media with and without Na⁺ and/or HCO ₃ ^– (10 mm) directly demonstrate a DIDS-sensitive, Na⁺ dependent Cl^–/HCO ₃ ^– exchange operating at slightly acidic pH _i (pH_o 6.8), and a DIDS-sensitive, Na⁺-independent Cl^–/HCO ₃ ^– exchange operating at alkaline pH _i (pH _o 8.2).The excellent technical assistance of Marianne Schiødt and Birgit B. Jørgensen is gratefully acknowledged. The work was supported by the Carlsberg Foundation (B.K.) and by a grant from the Danish Natural Science Foundation (E.K.H. and L.O.S.).     

ATP-stimulated electrolyte and mucin secretion in the human intestinal goblet cell line HT29-C1.16E

The response of confluent monolayers of HT29-Cl.16E cells to stimulation by extracellular ATP and ATP analogues was investigated in terms of mucin and electrolyte secretion. Mucin secretion was measured as release of glucosamine-labeled macromolecules trapped at the stacking/running gel interface of polyacrylamide gels and electrolyte secretion as shortcircuit current (I_sc). Luminal ATP stimulated a transient increase in the release of mucins and of I _sc corresponding to a secretory Cl^– current. Both secretions peaked at 3 to 5 min after addition of ATP. Maximal ATP-stimulated mucin secretion over 15 min was up to 18-fold above control with an apparent ED₅₀ of approximately 40 m. Maximal peak I _sc after stimulation with ATP was approximately 35 A/cm² with an apparent ED₅₀ of about 0.4 mm. ATP-dependent I _sc was at least in part due to Cl^– secretion since removal of Cl^– from the medium reduced the peak I _sc by 40% and the I _sc integrated over 40 min by 80%. The secretory responses were not associated with cell damage as assessed by failure of ethidium bromide to enter into the cells, absence of release of lactate dehydrogenase, maintenance of monolayer conductance, viability, and responses to repeated applications of ATP. The order of efficacy of nucleotide agonists was similar for both processes with ATP>ADP>AMPadenosine. Luminal ATP was much more effective than basolateral addition of this compound. These results suggest involvement of a luminal P₂-type receptor which can initiate signaling pathways for granule fusion and mucin release as well as for activation of Cl^– channels. P₂-receptor-stimulated mucin and I _sc release was strongly inhibited by a 30 min preincubation with the classical K⁺ channel blockers quinine (1 mm), quinidine (1 mm), and Ba²⁺ (3 mm). Experiments with amphotericin B to measure separately the conductance changes of either luminal or basolateral plasma membrane revealed that quinidine did not directly block the ATP-induced basolateral K⁺ or the luminal anion channels. The quinidine inhibition after preincubation is therefore most easily explained by interference with granule fusion and location of anion channels in granule membranes. Luminal P₂ receptors may play a role in intestinal defense mechanisms with both fluid and mucin secretion aiding in the removal of noxious agents from the mucosal surface.Supported by grants from the National Institutes of Health (DK 39658) to U.H., the Philippe Foundation to D.M., the French Cystic Fibrosis Foundation (AFLM) and L'Association Pour La Recherche Sur Le Cancer to C.L. The authors thank Mr. J. Polack for his efforts and skill with electron microscopy and Dr. George Dubyak for helpful discussions. We also acknowledge the Cystic Fibrosis Center Core grant (DK-27651) for its support of electron and light microscopy.     

Cl− transport in basolateral renal medullary vesicles: II. Cl− channels in planar lipid bilayers

Summary The present studies examined some of the properties of Cl^– channels in renal outer medullary membrane vesicles incorporated into planar lipid bilayers. The predominant channel was anion selective having aP _Cl/P _K ratio of 10 and a unit conductance of 93 pS in symmetric 320mm KCl. In asymmetric KCl solutions, theI-V relations conformed to the Goldman-Hodgkin-Katz equation. Channel activity was voltage-dependent with a gating charge of unity. This voltage dependence of channel activity may account, at least in part, for the striking voltage dependence of the basolateral membrane Cl^– conductance of isolated medullary thick ascending limb segments. The Cl^– channels incorporated into the planar bilayers were asymmetrical: thetrans surface was sensitive to changes in ionized Ca²⁺ concentrations and insensitive to reducing KCl concentrations to 10mm, while thecis side was insensitive to changes in ionized Ca²⁺ concentrations, but was inactivated by reducing KCl concentrations to 50mm.     

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: Lack of evidence for a cAMP-activated basolateral K+ conductance

Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments.Changes in electrophysiological parameters provoked by serosal addition of 10^–5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 · cm² from a mean value of 138±13 · cm² before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of –50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl^– in the medium. Subsequent addition of bumetanide (10^–4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba²⁺ (1mm), a known inhibitor of basolateral K⁺ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K⁺ conductance.The results suggest that forskolin-induced Cl^– secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl^– conductance of the apical membrane but does not affect K⁺ or Cl^– conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl^– and the basal Ba²⁺-sensitive K⁺ conductance are sufficiently large to maintain the Cl^– efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl^– channel blockers NPPB and DPC on basolateral conductance rather than apical Cl^– conductance.     

Potassium channel in rabbit corneal endothelium activated by external anions

Summary The apical membrane of the rabbit corneal endothelium contains a potassium-selective ionic channel. In patch-clamp recordings, the probability of finding the channel in the open state (P _o) depends on the presence of either HCO ₃ ^– or Cl^– in the bathing medium. In a methane sulfonate-containing bath,P _o is <0.05 at all physiologically relevant transmembrane voltages. With 0mm [HCO ₃ ^– ] _o at +60 mV,P _o was 0.085 and increased to 0.40 when [HCO ₃ ^– ] _o was 15mm. With 4mm [Cl^–] _o at +60 mV,P _o was 0.083 and with 150mm Cl^–,P _o increased to 0.36. LowP _o's are also found when propionate, sulphate, bromide, and nitrate are the primary bath anions. The mechanism of action of the anion-stimulated K⁺ channel gating is not yet known, but a direct action of pH seems unlikely. The alkalinization of cytoplasm associated with the addition of 10mm (NH₄)₂SO₄ to the bath and the acidification accompanying its removal do not result in channel activation nor does the use of Nigericin to equilibrate intracellular pH with that of the bath over the pH range of 6.8 to 7.8. Channel gating also is not affected by bathing the internal surface of the patch with cAMP, cGMP, GTP--s, Mg²⁺ or ATP. Blockers of Na/H⁺ exchange, Na⁺–HCO ₃ ^– cotransport, Na⁺–K⁺ ATPase and carbonic anhydrase do not block the HCO ₃ ^– stimulation ofP _o. Several of the properties of the channel could explain some of the previously reported voltage changes that occur in corneal endothelial cells stimulated by extracellular anions.     

Modulation of Calcium-dependent Chloride Secretion by Basolateral SK4-like Channels in a Human Bronchial Cell Line

The human bronchial cell line16HBE14o– was used as a model of airway epithelial cells to study the Ca²⁺-dependent Cl^– secretion and the identity of K_Ca channels involved in the generation of a favorable driving force for Cl^– exit. After ionomycin application, a calcium-activated short-circuit current (I _sc) developed, presenting a transient peak followed by a plateau phase. Both phases were inhibited to different degrees by NFA, glybenclamide and NPPB but DIDS was only effective on the peak phase. ⁸⁶Rb effluxes through both apical and basolateral membranes were stimulated by calcium, blocked by charybdotoxin, clotrimazole and TPA. 1-EBIO, a SK-channel opener, stimulated ⁸⁶Rb effluxes. Block of basolateral K_Ca channels resulted in I _sc inhibition but, while reduced, I _sc was still observed if mucosal Cl^– was lowered. Among SK family members, only SK4 and SK1 mRNAs were detected by RT-PCR. KCNQ1 mRNAs were also identified, but involvement of K_cAMP channels in Cl^– secretion was unlikely, since cAMP application had no effect on ⁸⁶Rb effluxes. Moreover, chromanol 293B or clofilium, specific inhibitors of KCNQ1 channels, had no effect on cAMP-dependent I _sc. In conclusion, two distinct components of Cl^– secretion were identified by a pharmacological approach after a Ca _i ²⁺ rise. K_Ca channels presenting the pharmacology of SK4 channels are present on both apical and basolateral membranes, but it is the basolateral SK4-like channels that play a major role in calcium-dependent chloride secretion in 16HBE14o– cells.     

Electrogenic Cl− absorption byAmphiuma small intestine: Dependence on serosal Na+ from tracer and Cl− microelectrode studies

Summary The Na⁺ requirement for active, electrogenic Cl^– absorption byAmphiuma small intestine was studied by tracer techniques and double-barreled Cl^–-sensitive microelectrodes. Addition of Cl^– to a Cl^–-free medium bathingin vitro intestinal segments produced a saturable (K _m =5.4mm) increase in shortcircuit current (I _sc) which was inhibitable by 1mm SITS. The selectivity sequence for the anion-evoked current was Cl^–=Br^–>SCN^–>NO ₃ ^– >F^–=I^–. Current evoked by Cl^– reached a maximum with increasing medium Na concentration (K _m =12.4mm). Addition of Na⁺, as Na gluconate (10mm), to mucosal and serosal Na⁺-free media stimulated the Cl^– current and simultaneously increased the absorptive Cl^– flux (J _ms ^Cl ) and net flux (J _net ^Cl ) without changing the secretory Cl^– flux (J _sm ^Cl ). Addition of Na⁺ only to the serosal fluid stimulatedJ _ms ^Cl much more than Na⁺ addition only to the mucosal fluid in paired tissues. Serosal DIDS (1mm) blocked the stimulation. Serosal 10mm Tris gluconate or choline gluconate failed to stimulateJ _ms ^Cl . Intracellular Cl^– activity (a _Cl ⁱ ) in villus epithelial cells was above electrochemical equilibrium indicating active Cl^– uptake. Ouabain (1mm) eliminated Cl^– accumulation and reduced the mucosal membrane potential _m over 2 to 3 hr. In contrast, SITS had no effect on Cl^– accumulation and hyperpolarized the mucosal membrane. Replacement of serosal Na⁺ with choline eliminated Cl^– accumulation while replacement of mucosal Na⁺ had no effect. In conclusion by two independent methods active electrogenic Cl^– absorption depends on serosal rather than mucosal Na⁺. It is concluded that Cl^– enters the cell via a primary (rheogenic) transport mechanism. At the serosal membrane the Na⁺ gradient most likely energizes H⁺ export and regulates mucosal Cl^– accumulation perhaps by influencing cell pH or HCO ₃ ^– concentration.     

Outwardly rectifying chloride channels in lymphocytes

Summary Outwardly rectifying Cl^– channels in cultured human Jurkat T-lymphocytes were activated by excising a patch of membrane using the inside-out (i/o) patch-clamp configuration and holding at depolarized voltages for prolonged periods of time (1–6 min at +80 mV, 20°C). The single-channel current at +80 mV was 4.5 ± 0.3 pA and at –80 mV, it was 1.0 ± 0.4 pA. After activation, the probability of being open (P ₀)for the lymphocyte channel was voltage independent. Activation of the Cl^– channel in lymphocytes was temperature dependent. Nineteen percent of i/o recordings from lymphocytes made at 20°C exhibited Cl^– channel activity. In contrast, 49% of recordings made at 30°C showed channel activity. The number of channels in an active patch was not significantly different at the two temperatures. Channel activation in excised, depolarized patches also occurred 20-fold faster at 30°C than at 20°C. There was no marked change in the single-channel conductance at 30°C. Open-channel conductance was blocked by 200 m indanyloxyacetic acid (IAA) or 1 mm SITS when applied to the intracellular side of the patch. The characteristics of this channel are similar to epithelial outwardly rectifying Cl^– channels thought to be involved in fluid secretion     

Response of chloride efflux from skeletal muscle ofRana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl^– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl^– efflux. The pH-dependent Cl^– efflux can be described by the relationu=1/(1+10n(pK _a -pH)), whereu is the Cl^– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK _a is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK _a at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK _a suggests that the critical pH-sensitive moiety producing the pH-dependent Cl^– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl^– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl^– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k ^–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q₁₀ of 2.5. The Cl^– efflux inhibitor SCN^– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl^– efflux by DEPC. The findings that the apparent pK _a is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl^– efflux, and that this action is retarded by SCN^– supports the notion that protonation of histidine groups associated with Cl^– channels is the controlling reaction for the pH-dependent Cl^– efflux.