miR-16 rescues F508del-CFTR function in native cystic fibrosis epithelial cells.

Cystic fibrosis (CF) is due to mutations in the CFTR gene, which prevents correct folding, trafficking and function of the mutant cystic fibrosis transmembrane conductance regulator (CFTR) protein. The dysfunctional effect of CFTR mutations, principally the F508del-CFTR mutant, is further manifested by hypersecretion of the pro-inflammatory chemokine interleukin-8 into the airway lumen, which further contributes to morbidity and mortality. We have hypothesized that microRNA (miR)-based therapeutics could rescue the dysfunctional consequences of mutant CFTR. Here we report that a miR-16 mimic can effectively rescue F508del-CFTR protein function in airway cell lines and primary cultures, of differentiated human bronchial epithelia from F508del homozygotes, which express mutant CFTR endogenously. We also identify two other miRs, miR-1 and miR-302a, which are also active. Although miR-16 is expressed at basal comparable levels in CF and control cells, miR-1 and miR-302a are undetectable. When miR mimics are expressed in CF lung or pancreatic cells, the expression of the F508del-CFTR protein is significantly increased. Importantly, miR-16 promotes functional rescue of the cyclic AMP-activated apical F508del-CFTR chloride channel in primary lung epithelial cells from CF patients. We interpret these findings to suggest that these miRs may constitute novel targets for CF therapy.

Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the CF transmembrane conductance regulator (CFTR). A single recessive mutation, the deletion of phenylalanine 508 (deltaF508), causes severe CF and resides on 70% of mutant chromosomes. Severe CF is also caused by premature stop mutations, which are found on 5% of CF chromosomes. Here we report that two common, disease-associated stop mutations can be suppressed by treating cells with low doses of the aminoglycoside antibiotic G-418. Aminoglycoside treatment resulted in the expression of full-length CFTR and restored its cyclic AMP-activated chloride channel activity. Another aminoglycoside, gentamicin, also promoted the expression of full-length CFTR. These results suggest that treatment with aminoglycosides may provide a means of restoring CFTR function in patients with this class of mutation.

Transfection of the CD20 cell surface molecule into ectopic cell types generates a Ca2+ conductance found constitutively in B lymphocytes.

CD20 is a plasma membrane phosphoprotein expressed exclusively by B lymphocytes. mAb binding to CD20 alters cell cycle progression and differentiation, indicating that CD20 plays an essential role in B lymphocyte function. Whole-cell patch clamp and fluorescence microscopy measurements of plasma membrane ionic conductance and cytosolic-free Ca2+ activity, respectively, were used to directly examine CD20 function. Transfection of human T and mouse pre-B lymphoblastoid cell lines with CD20 cDNA and subsequent stable expression of CD20 specifically increased transmembrane Ca2+ conductance. Transfection of CD20 cDNA and subsequent expression of CD20 in nonlymphoid cells (human K562 erythroleukemia cells and mouse NIH-3T3 fibroblasts) also induced the expression of an identical transmembrane Ca2+ conductance. The binding of a CD20-specific mAb to CD20+ lymphoblastoid cells also enhanced the transmembrane Ca2+ conductance. The mAb-enhanced Ca2+ currents had the same conductance characteristics as the CD20-associated Ca2+ currents in CD20 cDNA-transfected cells. C20 is structurally similar to several ion channels; each CD20 monomer possesses four membrane spanning domains, and both the amino and carboxy termini reside within the cytoplasm. Biochemical cross-linking of cell-surface molecules with subsequent immunoprecipitation analysis of CD20 suggests that CD20 may be present as a multimeric oligomer within the membrane, as occurs with several known membrane channels. Taken together, these findings indicate that CD20 directly regulates transmembrane Ca2+ conductance in B lymphocytes, and suggest that multimeric complexes of CD20 may form Ca2+ conductive ion channels in the plasma membrane of B lymphoid cells.

Altered regulation of airway epithelial cell chloride channels in cystic fibrosis.

In many epithelial cells the chloride conductance of the apical membrane increases during the stimulation of electrolyte secretion. Single-channel recordings from human airway epithelial cells showed that beta-adrenergic stimulation evoked apical membrane chloride channel activity, but this response was absent in cells from patients with cystic fibrosis (CF). However, when membrane patches were excised from CF cells into media containing sufficient free calcium (approximately 180 nanomolar), chloride channels were activated. The chloride channels of CF cells were similar to those of normal cells as judged by their current-voltage relations, ion selectivity, and kinetic behavior. These findings demonstrate the presence of chloride channels in the apical membranes of CF airway cells. Their regulation by calcium appears to be intact, but cyclic adenosine monophosphate (cAMP)-dependent control of their activity is defective.

Cell cycle dependence of chloride permeability in normal and cystic fibrosis lymphocytes.

Cystic fibrosis (CF) is a genetic disease characterized by abnormal regulation of epithelial cell chloride channels. Nonepithelial cells, including lymphocytes and fibroblasts, may exhibit a similar defect. Two independent techniques were used to assess the macroscopic chloride permeability (PCl) of freshly isolated B lymphocytes and of B and T lymphocyte cell lines. Values for PCl increased specifically during the G1 phase of the cell cycle and could be further enhanced by increasing intracellular adenosine 3',5'-monophosphate (cAMP) or calcium. In lymphocytes from CF patients, regulation of PCl during the cell cycle and by second messengers was absent. Characterization of the cell cycle-dependent expression of the chloride permeability defect in lymphocytes from CF patients increases the utility of these cells in the analysis of the functional consequences of mutations in the CF gene.

Crypts are the site of intestinal fluid and electrolyte secretion.

The site of adenosine 3',5'-monophosphate-mediated fluid and electrolyte secretion across mammalian large intestine was found to be the crypts of Lieberkühn by means of two techniques. First, the formation of fluid droplets was visualized on the oil-covered mucosal surface directly over crypt duct openings when secretion was stimulated. Second, microelectrode impalement of individual surface and crypt cells revealed that only crypts cells produced a pattern of secretagogue induced alterations in membrane potential and resistance that was characteristic of secretory epithelia.

Correction of lethal intestinal defect in a mouse model of cystic fibrosis by human CFTR.

Cystic fibrosis (CF) is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). A potential animal model of CF, the CFTR-/- mouse, has had limited utility because most mice die from intestinal obstruction during the first month of life. Human CFTR (hCFTR) was expressed in CFTR-/- mice under the control of the rat intestinal fatty acid-binding protein gene promoter. The mice survived and showed functional correction of ileal goblet cell and crypt cell hyperplasia and cyclic adenosine monophosphate-stimulated chloride secretion. These results support the concept that transfer of the hCFTR gene may be a useful strategy for correcting physiologic defects in patients with CF.

Chloride conductance expressed by delta F508 and other mutant CFTRs in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR) is associated with expression of a chloride conductance that is defective in cystic fibrosis (CF). Xenopus oocytes injected with RNA coding for CFTR that contained mutations in the first nucleotide binding fold (NBF1) expressed chloride currents in response to raising adenosine 3',5'-monophosphate (cAMP) with forskolin and 3-isobutyl-1-methylxanthine (IBMX). The mutant CFTRs were less sensitive than wild-type CFTR to this activating stimulus, and the reduction in sensitivity correlated with the severity of cystic fibrosis in patients carrying the corresponding mutations. This demonstration provides the basis for detailed analyses of NBF1 function and suggests potential pharmacologic treatments for cystic fibrosis.

Taurodeoxycholate activates potassium and chloride conductances via an IP3-mediated release of calcium from intracellular stores in a colonic cell line (T84)

Whole-cell patch-clamp techniques and fluorescence measurements of intracellular Ca2+ concentration, (Ca2+)i, were used to investigate the mechanism of taurodeoxycholate (TDC) stimulation of Cl- secretion in the T84 colonic cell line. During perforated whole-cell recordings, the cell membrane voltage was alternately clamped to EK and ECl. Initially, TDC (0.75 mM) stimulated inward nonselective cation currents that were composed of discrete large conductance single-channel events. This initial response was followed by activation of K+ and Cl- currents with peak values of 385 +/- 41 pA and 98 +/- 28 pA, respectively (n = 12). The K+ and Cl- currents oscillated while TDC was present and returned to baseline levels upon its removal. The threshold for activation of the oscillatory currents was 0.1 mM TDC. Taurocholate, a bile acid that does not stimulate colonic Cl- secretion, induced no current response. The TDC-induced currents could be activated in Ca(2+)-free bathing solutions. Preincubation of cells with the Ca2+ chelator, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethy)-ester (20 microM), (BAPTA-AM), eliminated the K+ and Cl- current responses, although the nonselective cation channel events were still present. Replacement of bath Na+ with NMDG+ inhibited the TDC-induced nonselective cation current but did not affect the K+ or Cl- currents. TDC induced a transient (Ca2+)i rise of 575 +/- 70 nM from a baseline of 71 +/- 5 nM (n = 15); thereafter, (Ca2+)i either plateaued or oscillated. TDC-induced (Ca2+)i oscillations were observed in the absence of bath Ca2+; however, removal of bath Ca2+ during the TDC response caused (Ca2+)i to return to near baseline values. Simultaneous K+ current and (Ca2+)i measurements confirmed that the initial nonselective cation current was independent of (Ca2+)i, while K+ current oscillations were in phase with the (Ca2+)i oscillations. TDC induced inositol monophosphate (IP) accumulation, reflecting production of inositol 1,4,5-trisphosphate (IP3) during TDC stimulation. The response to TDC during standard whole-cell patch-clamp was similar to that observed with perforated whole-cell recordings, except the nonselective cation current was prolonged. When heparin (1 mg/ml) was added to the pipette under these conditions, the Ca(2+)-activated currents were inhibited, but the nonselective cation currents were unaffected. These data suggest that TDC induces a Ca(2+)-independent nonselective cation conductance, perhaps by directly permeabilizing the plasma membrane. TDC stimulates Cl- secretion by activating K+ and Cl- conductances via an IP3-mediated release of Ca2+ from intracellular stores.

Ionic permeability of epithelial tissues.

The overall permeability of epithelial tissues to solutes is generally determined by analyzing net or unidirectional transepithelial fluxes in response to transepithelial differences of concentration and/or electrical potential using relations that describe diffusional movements across a single membrane. If the solute is uncharged and diffusional movements are transcellular, the overall transepithelial permeability coefficient is determined by the permeabilities of the two limiting cell membranes combinded in series. However, if the solute is charged and the pathway for transepithelial movement involves diffusional flows across at least two membranes arranged in series (i.e. transcellular transport), the value of the overall transepithelial permeability coefficient determined using relations that describe ionic diffusion across a single membrane is not an accurate measure of the permeabilities of the two limiting membranes combined in series. Further, if ionic diffusion is transcellular, permeability coefficients determined from studies of transepithelial fluxes are not only quantitatively incorrect but can also result in grossly erroneous interpretations of changes in transepithelial permeabilities and faulty inferences regarding the route of transepithelial ionic diffusion.

Single chloride channel currents from canine tracheal epithelial cells.

Patch-clamp techniques were used to characterize the properties of anion-selective channels in canine tracheal epithelial cells that had been maintained in primary culture. Gigaohm seals (10-30 G omega) were obtained in single isolated cells or cells at the edge of a confluent sheet, and channels were studied in the cell attached or the inside-out, excised patch configuration. Pretreatment with isotonic KCl caused the cells to round-up and allowed us to have better success in obtaining good seals. Based on conductance, anion-cation selectivity and voltage-dependent kinetic properties, four anion channel types could be detected in symmetrical solutions of 0.15 M NaCl: (i) a 30-50 pS Cl- channel of high selectivity, active at negative potentials and inactivated by large positive potentials; (ii) an approx. 20 pS Cl- channel of high selectivity, active at positive potentials and inactivated at negative potentials; (iii) an approx. 250 pS channel of moderate selectivity (PCl/PNa = 4) that was not voltage-dependent, and (iv) an approx. 10 pS Cl- channel with characteristics similar to (iii) above, but remaining somewhat active at large negative voltages. All excised patches were exposed to relatively high calcium concentrations on the intracellular side. Channel activity was increased in tracheal cells treated with 1 mM cAMP, suggesting that at least one of these channels plays a role in the increase of the apical membrane Cl- conductance that is mediated by cAMP and elicited by agonists of active Cl- secretion.

Anion permeation in an apical membrane chloride channel of a secretory epithelial cell.

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.

Ionic conductances of extracellular shunt pathway in rabbit ileum. Influence of shunt on transmural sodium transport and electrical potential differences.

The unidirectional influxes of Na, K, and Cl into isolated strips of rabbit ileum are comprised of movements across the mucosal membrane of the epithelial cells and ionic diffusion into an extracellular shunt pathway. A large fraction of the Na influx across the mucosal membrane alone is inhibited by Li, suggesting the participation of a carrier mechanism in the influx process. The partial ionic shunt conductances of Na, K, and Cl account for at least 82% of the total tissue conductance. The calculated shunt permeabilities (P) are (in centimeters per hour) P(K) = 0.040, P(Na) = 0.035, and P(Cl) = 0.019, so that P(K):P(Na):P(Cl) = 1.14:1.00:0.55. Diffusion potentials across the tissue resulting from isotonic replacement of NaCl in the mucosal solution with mannitol or KCl are described by the Goldman constant-field equation together with the above permeabilities of the shunt pathway. These observations are not consistent with permeation through a fixed-charge pore but can be explained by a model featuring constant ionic partition into a neutral-polar pore that traverses the tight junction. Such a pore may be lined with either fixed dipoles or fixed dipolar ions oriented such that electronegative groups influence the permselective properties of the diffusion pathway. The essential feature of both models is that electroneutrality is maintained by means of fixed membrane components and does not depend upon the presence of mobile counterions.

Effects of monovalent cations on the sodium-alanine interaction in rabbit ileum. Implication of anionic groups in sodium binding.

H, K, Rb, and Li inhibit Na-dependent alanine influx across the brush border of rabbit ileum. Kinetic analysis indicates that H and K behave as competitive inhibitors of influx so that increasing the concentration of H or K in the mucosal solution is kinetically indistinguishable from decreasing the Na concentration. In addition the coupling between alanine and Na influxes is markedly reduced at pH 2.5. With the exception of H and Li, none of these monovalent cations significantly affects carrier-mediated alanine influx in the absence of Na indicating that their inhibitory effects are largely restricted to the Na-dependent fraction of influx. Increasing H concentration from 0.03 to 3 mM does not affect influx in the absence of Na but markedly inhibits influx in the presence of Na. Li significantly enhances alanine influx in the absence of Na. Ag, UO(2), and La also inhibit the Na-dependent fraction of alanine influx. These findings suggest that anionic groups having a pK(a) of approximately 4 are involved in the interaction between Na and the alanine-carrier complex; present evidence implicates carboxylate groups however, phosphoryl residues cannot be ruled out. The previously proposed kinetic model for the Na-alanine interaction has been extended to accommodate these effects of H and other monovalent cations. The mechanistic and physiological implications of these findings are discussed.

ATP alters current fluctuations of cystic fibrosis transmembrane conductance regulator: evidence for a three-state activation mechanism.

The cystic fibrosis gene product cystic fibrosis transmembrane conductance regulator (CFTR) is a low conductance, cAMP-regulated Cl- channel. Removal of cytosolic ATP causes a cessation of cAMP-dependent kinase-phosphorylated CFTR channel activity that resumes upon ATP addition. (Anderson, M. P., H. A. Berger, D. R. Rich, R. J. Gregory, A. E. Smith, and M. J. Welsh. 1991. Cell. 67:775-784). The aim of this study was to quantify possible effects of ATP on CFTR gating. We analyzed multichannel records since only 1 of 64 patches contained a single channel. ATP increased the channel open probability (Po) as a simple Michaelis-Menten function of concentration; the effect was half maximal at 24 microM, reached a maximum of 0.44, and had a Hill coefficient of 1.13. Since the maximum Po was not 1, the simplest description of the effect of ATP on CFTR gating is the noncooperative three-state mechanism of del Castillo and Katz (1957. Proceedings of the Royal Society of London. B. 146:369-381). We analyzed current fluctuations to quantify possible changes in CFTR gating. The power density spectra appeared to contain a single Lorentzian in the range of 0.096-31 Hz. Analysis of the corner frequency (fc) of this Lorentzian revealed that ATP increased 2 pi fc as a Michaelis-Menten function with a Hill coefficient of 1.08, and it provided estimates of the ATP dissociation constant (44 tau open (154 ms), and the ATP-sensitive tau close [(185 ms) (44 microM/[ATP] + 1)]. These results suggest that the binding reaction is rapid compared to the opening and closing rates. Assuming that there is a single set of closed-to-open transitions, it is possible to verify the outcome of fluctuation analysis by comparing fluctuation-derived estimates of Po with measures of Po from current records. The two values were nearly identical. Thus, noise analysis provides a quantitative description of the effect of ATP on CFTR opening. The noncooperative three-state model should serve as a basis to understand possible alterations in CFTR gating resulting from regulators or point mutations.

Regulation of CFTR Cl- channel gating by ADP and ATP analogues.

The cystic fibrosis gene product (CFTR) is a chloride channel which, once phosphorylated, is regulated by nucleotide phosphates (Anderson, M. P., and M. J. Welsh. 1992. Science. 257:1701-1704; Venglarik, C. J., B. D. Schultz, R. A. Frizzell, and R. J. Bridges. 1994. Journal of General Physiology. 104:123-146). Nucleotide triphosphates initiate channel activity, while nucleotide diphosphates and nonhydrolyzable ATP analogues do not. To further characterize the role of these compounds on CFTR channel activity we examined their effects on chloride channel currents in excised inside-out membrane patches from CFTR transfected mouse L cells. ADP competitively inhibited ATP-dependent CFTR channel gating with a Ki of 16 +/- 9 microM. AMP neither initiated CFTR channel gating nor inhibited ATP-dependent CFTR channel gating. Similarly, ATP analogues with substitutions in the phosphate chain, including AMPCPP, AMPPCP, AMPPNP, and ATP gamma S failed to support CFTR channel activity when present at the cytoplasmic face of the membrane and none of these analogues, when present at three to 10-fold excess of ATP, detectably altered ATP-dependent CFTR channel gating. These data suggest that none of these ATP analogues interact with the ATP regulatory site of CFTR which we previously characterized and, therefore, no inference regarding a requirement for ATP hydrolysis in CFTR channel gating can be made from their failure to support channel activity. Furthermore, the data indicate that this nucleotide regulatory site is exquisitely sensitive to alterations in the phosphate chain of the nucleotide; only a nonsubstituted nucleotide di- or triphosphate interacts with this regulatory site. Alternative recording conditions, such as the presence of kinase and a reduction in temperature to 25 degrees C, result in a previously uncharacterized kinetic state of CFTR which may exhibit distinctly different nucleotide dependencies.

Sodium chloride transport by rabbit gallbladder. Direct evidence for a coupled NaCl influx process.

The results of the present study that NaCl transport by in vitro rabbit gallbladder must be a consequence of a neutral coupled carrier-mediated mechanism that ultimately results in the active absorption of both ions; pure electrical coupling between the movements of Na and Cl can be excluded on the grounds of electrphysiologic considerations. Studies on the unidirectional influxes of Na and Cl have localized the site of this coupled mechanism to the mucosal membranes. Studies on the intracellular ion concentrations and the intracellular electrical potential are consistent with the notion that (a) the coupled NaCl influx process results in the movement of Cl from the mucosal solution into the cell against an apparent electrochemical potential difference; (b) the energy for the uphill movement of Cl is derived from the Na gradient across the mucosal membrane which is maintained by an active Na extrusion mechanism located at the basolateral membranes; and (c) Cl exit from the cell across the basolateral membranes is directed down an electrochemical potential gradient and may be diffusional. Finally, as for the case of rabbit ileum, the coupled NaCl influx process is inhibited by elevated intracellular levels of cyclic 3',5'-adenosine monophosphate. A working model for transcellular and paracellular NaCl transport by in vitro rabbit gallbladder is proposed.

Electrophysiology of flounder intestinal mucosa. II. Relation of the electrical potential profile to coupled NaCl absorption.

We characterized the hyperpolarization of the electrical potential profile of flounder intestinal cells that accompanies inhibition of NaCl cotransport. Several observations indicate that hyperpolarization of psi a and psi b (delta psi a,b) results from inhibition of NaCl entry across the apical membrane: (a) the response was elicited by replacement of mucosal solution Cl or Na by nontransported ions, and (b) mucosal bumetanide or serosal cGMP, inhibitors of NaCl influx, elicited delta psi a,b and decreased the transepithelial potential (psi t) in parallel. Regardless of initial values, psi a and psi b approached the equilibrium potential for K (EK) so that in the steady state following inhibition of NaCl entry, psi a approximately equal to psi b approximately equal to ECl approximately equal to EK. Bumetanide decreased cell Cl activity (aClc) toward equilibrium levels. Bumetanide and cGMP decreased the fractional apical membrane resistance (fRa), increased the slope of the relation of psi a to [K]m, and decreased cellular conductance (Gc) by approximately 85%, which indicates a marked increase in basolateral membrane conductance (Gb). Since the basolateral membrane normally shows a high conductance to Cl, a direct relation between apical salt entry and GClb is suggested by these findings. As judged by the response to bumetanide or ion replacement in the presence of mucosal Ba, inhibition of Na/K/Cl co-transport alone is not sufficient to elicit delta psi a,b. This suggests the presence of a parallel NaCl co-transport mechanism that may be activated when Na/K/Cl co-transport is compromised. The delta psi a,b response to reduced apical NaCl entry would assist in maintaining the driving force for Na-coupled amino acid uptake across the apical membrane as luminal [NaCl] falls during absorption.

Electrophysiology of flounder intestinal mucosa. I. Conductance properties of the cellular and paracellular pathways.

We evaluated the conductances for ion flow across the cellular and paracellular pathways of flounder intestine using microelectrode techniques and ion-replacement studies. Apical membrane conductance properties are dominated by the presence of Ba-sensitive K channels. An elevated mucosal solution K concentration, [K]m, depolarized the apical membrane potential (psi a) and, at [K]m less than 40 mM, the K dependence of psi a was abolished by 1-2 mM mucosal Ba. The basolateral membrane displayed Cl conductance behavior, as evidenced by depolarization of the basolateral membrane potential (psi b) with reduced serosal Cl concentrations, [Cl]s. psi b was unaffected by changes in [K]s or [Na]s. From the effect of mucosal Ba on transepithelial K selectivity, we estimated that paracellular conductance (Gp) normally accounts for 96% of transepithelial conductance (Gt). The high Gp attenuates the contribution of the cellular pathway to psi t while permitting the apical K and basolateral Cl conductances to influence the electrical potential differences across both membranes. Thus, psi a and psi b (approximately 60 mV, inside negative) lie between the equilibrium potentials for K (76 mV) and Cl (40 mV), thereby establishing driving forces for K secretion across the apical membrane and Cl absorption across the basolateral membrane. Equivalent circuit analysis suggests that apical conductance (Ga approximately equal to 5 mS/cm2) is sufficient to account for the observed rate of K secretion, but that basolateral conductance (Gb approximately equal to 1.5 mS/cm2) would account for only 50% of net Cl absorption. This, together with our failure to detect a basolateral K conductance, suggests that Cl absorption across this barrier involves KCl co-transport.

Bicarbonate and chloride secretion in Calu-3 human airway epithelial cells.

Serous cells are the predominant site of cystic fibrosis transmembrane conductance regulator expression in the airways, and they make a significant contribution to the volume, composition, and consistency of the submucosal gland secretions. We have employed the human airway serous cell line Calu-3 as a model system to investigate the mechanisms of serous cell anion secretion. Forskolin-stimulated Calu-3 cells secrete HCO-3 by a Cl-offdependent, serosal Na+-dependent, serosal bumetanide-insensitive, and serosal 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive, electrogenic mechanism as judged by transepithelial currents, isotopic fluxes, and the results of ion substitution, pharmacology, and pH studies. Similar studies revealed that stimulation of Calu-3 cells with 1-ethyl-2-benzimidazolinone (1-EBIO), an activator of basolateral membrane Ca2+-activated K+ channels, reduced HCO-3 secretion and caused the secretion of Cl- by a bumetanide-sensitive, electrogenic mechanism. Nystatin permeabilization of Calu-3 monolayers demonstrated 1-EBIO activated a charybdotoxin- and clotrimazole- inhibited basolateral membrane K+ current. Patch-clamp studies confirmed the presence of an intermediate conductance inwardly rectified K+ channel with this pharmacological profile. We propose that hyperpolarization of the basolateral membrane voltage elicits a switch from HCO-3 secretion to Cl- secretion because the uptake of HCO-3 across the basolateral membrane is mediated by a 4,4 '-dinitrostilben-2,2'-disulfonic acid (DNDS)-sensitive Na+:HCO-3 cotransporter. Since the stoichiometry reported for Na+:HCO-3 cotransport is 1:2 or 1:3, hyperpolarization of the basolateral membrane potential by 1-EBIO would inhibit HCO-3 entry and favor the secretion of Cl-. Therefore, differential regulation of the basolateral membrane K+ conductance by secretory agonists could provide a means of stimulating HCO-3 and Cl- secretion. In this context, cystic fibrosis transmembrane conductance regulator could serve as both a HCO-3 and a Cl- channel, mediating the apical membrane exit of either anion depending on basolateral membrane anion entry mechanisms and the driving forces that prevail. If these results with Calu-3 cells accurately reflect the transport properties of native submucosal gland serous cells, then HCO-3 secretion in the human airways warrants greater attention.