Cryptdin-3 induces novel apical conductance(s) in Clminus secretory, including cystic fibrosis, epithelia

Didier Merlin1, Gang Yue2, Wayne I. Lencer3, Michael E. Selsted4, and James L. Madara1

1 Department of Pathology and 2 Center for Cell and Molecular Signaling, Emory University School of Medicine, Atlanta, Georgia 30322; 3 Combined Program in Pediatric Gastroenterology and Nutrition, Department of Medicine, Children's Hospital and Harvard Medical School, Boston, Massachusetts 02115; and 4 Department of Pathology, University of California College of Medicine, Irvine, California 92717


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Opening of anion-conductive pathways in apical membranes of secretory cells lining mucosal surfaces is a critical step in salt and water secretion and, thus, hydration of sites including airway and intestine. In intestine, Paneth cells are positioned at the base of the secretory gland (crypt) and release defensin peptide, in mice termed cryptdins, into the crypt lumen. Because at least some defensins have been shown to form anion-conductive channels in phospholipid bilayers, we tested whether these endogenous antimicrobial peptides could act as soluble inducers of channel-like activity when applied to apical membranes. To directly evaluate the possibility of cryptdin-3-mediated apical anion conductance (Gap), we have utilized amphotericin B to selectively permeabilize basolateral membranes of electrically tight monolayers of polarized human intestinal secretory epithelia (T84 cells), thus isolating the apical membrane for study. Cryptdin-3 induces Gap that is voltage independent (Delta Gap = 1.90 ± 0.60 mS/cm2) and exhibits ion selectivity contrasting to that elicited by forskolin or thapsigargin (for cryptdin-3, Cl- = gluconate; for forskolin and thapsigargin, Cl- gluconate). We cannot exclude the possibility that the macroscopic current induced by cryptdin could be the sum of cation and Cl- currents. Cryptdin-3 induces a current in basolaterally permeabilized epithelial monolayers derived from airway cells harboring the Delta F508 mutation of cystic fibrosis (CF; Delta Gap = 0.80 ± 0.06 mS/cm2), demonstrating that cryptdin-3 restores anion secretion in CF cells; this occurs independently of the CF transmembrane conductance regulator channel. These results support the idea that cryptdin-3 may associate with apical membranes of Cl--secreting epithelia and self-assemble into conducting channels capable of mediating a physiological response.

intestinal epithelium; conducting channels; amphotericin B


    INTRODUCTION
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INTRODUCTION
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PANETH CELLS RESIDE at the base of small intestinal crypts and release granule contents into the crypt lumen (17). Residency at the crypt base places Paneth cells in a position to affect the biology of adjacent epithelial cells lining the intestinal crypt, including the intestinal stem cells and the cell type making up the bulk of the tubular crypt, i.e., the "undifferentiated" Cl--secreting crypt cell (1).

Along with other soluble proteins, mature human Paneth cells secrete antimicrobial alpha -defensins, termed cryptdins (17, 18, 21, 23). Mouse and human Paneth cell alpha -defensins exhibit selective activity against several microorganisms (21). These antimicrobial activities derive from the ability of alpha -defensins to permeabilize microbial membranes by self-assembly into anion-conductive channels (3, 4, 7, 24). Many known antimicrobial peptides (e.g., cecropin, magainin, and dermaseptin) exhibit cytotoxicity against a diverse array of microorganisms but do not display cytotoxicity against normal eukaryotic cells (3, 6, 12-13, 22).

We recently found that cryptdins-2 and -3 interact with apical membranes of human intestinal T84 cells to induce a physiological Cl- secretory response (11). The secretory response induced by cryptdin-3 appears to correlate with permeabilization of the apical membrane, as judged by use of membrane-impermeant solutes; however, activation of the endogenous cystic fibrosis (CF) transmembrane conductance regulator (CFTR) channel (or an endogenous Ca2+-regulated channel) could not be ruled out. These data support the interpretation that the cryptdin-3 effect was not mediated by endogenous CFTR and included the fact that cryptdin-3 did not induce detectable elevations of intracellular cAMP or cGMP and did not activate apical membrane adenosine receptors (11). On the basis of these results, we hypothesized that cryptdin-3 may act as a novel paracrine regulator of intestinal secretion by forming anion-conductive channels within the apical membrane of neighboring Cl--secreting crypt epithelial cells.

We now test this idea by defining the biophysical characteristics of cryptdin-3-induced channels. To do so, we electrically isolate the apical membrane of intact T84 cell monolayers by selectively permeabilizing basolateral membranes with amphotericin B. Our data show that cryptdin-3 induces apical membrane anion channels that display biophysical features fundamentally different from those displayed by endogenous apical membrane cAMP- and Ca2+-dependent Cl- channels. We also find that cryptdin-3 induces Cl- secretion in CF (JME/CF15) cells. JME/CF15 cells do not contain the functional cAMP-dependent Cl- channel CFTR (5, 19). Thus cryptdin-3 induces a novel apical conductance(s) (Gap) in human intestinal and tracheal epithelial cell lines. These data are consistent with the idea that cryptdin-3 associates with apical membranes of Cl--secreting epithelia and self-assembles into conducting channels capable of mediating a physiological secretory response.


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Cell culture. T84 cells (American Type Culture Collection), a human colonic carcinoma cell line that functionally and morphologically resembles crypt intestinal epithelia, were grown as confluent monolayers in a 1:1 mixture of Dulbecco's Vogt modified Eagle's medium and Ham's F-12 medium supplemented with 15 mM HEPES buffer (pH 7.5), 14 mM NaHCO3, 40 µg/ml penicillin, 90 µg/ml streptomycin, and 5% newborn calf serum. Monolayers were subcultured every 7 days by addition of 0.1% trypsin and 0.9 mM EDTA in Ca2+/Mg2+-free PBS and grown on collagen-coated permeable supports (area 0.3 cm2, pore size 0.4 µm). All experiments were performed using cells between passages 65 and 92.

Immortalized JME/CF15 cells from CF patients (Delta F508-CFTR; homozygous for the presence of the Phe508 deletion), a human airway epithelial cell line derived from the nasal airway epithelium of a CF patient, were cultured as previously described (5) with some modifications. CF15 cells were cultured as monolayers to 80-100% confluency (~7 days) in 75-cm2 flasks in hormone-defined medium (3:1 DMEM-Ham's F-12 medium containing 0.18 mM adenine, 10% fetal bovine serum, 100 U/ml penicillin, 100 U/ml streptomycin, 1.1 mM hydrocortisone, 5 µg/ml insulin, 5 µg/ml transferrin, 2 nM triiodothyronine, 1.64 mM epidermal growth factor, and 5.5 mM epinephrine) and treated with trypsin as described above for passage (1-2 × 106 cells/75 cm2) or culture on permeable supports. Cells were on permeable supports (5 × 105 cells/cm2) that were pretreated with 50 µl of a 0.5 mg/ml solution of human placental collagen in 0.2% acetic acid. For monolayers on permeable supports, on the day after plating the tissue culture medium was removed from the apical (luminal) surface and maintained as an apical-air interface until use 3 days later.

Cryptdin-3 purification and synthesis. Synthetic, folded, and oxidized cryptdin-3 was prepared as described previously (21). Synthetic and natural cryptdin-3 peptides were shown to have identical physiochemical and antimicrobial characteristics (21).

Transepithelial current measurement. Studies were carried out at 37°C with the use of confluent monolayers plated on collagen-coated permeable supports and examined 7-16 days later, as previously described (16). Before all studies, inserts were washed with HCO3--free medium warmed to 37°C and transferred to new 24-well tissue culture plates containing the experimental medium. To determine currents, transepithelial potentials, and conductances, a commercial voltage clamp (Bioengineering Department, University of Iowa, Iowa City, IA) was interfaced with equilibrated pairs of calomel electrodes submerged in saturated KCl and with paired Ag-AgCl electrodes submerged in the experimental medium maintained at constant temperature (37°C). Apical and basolateral experimental medium volume remained constant during the course of the experiment, and the electrodes were stably placed on either side of the monolayers. Positive currents correspond to anion secretion/cation absorption, i.e., a lumen-negative potential under open-circuit conditions. Before each experiment, a blank filter was used to compensate for the fluid resistance and the resistance of the filter. In some experiments, transepithelial voltage, short-circuit current (Isc), and conductance were continuously recorded with the aid of an analog-to-digital converter (MacLab, Word Precision Instruments) and a microcomputer.

Measurement of conductance(s) of the apical plasma membrane. To evaluate the ion conductance of the isolated apical plasma membrane, the polyene ionophore amphotericin B (8) was added to the basolateral solution at 100 µM, the lowest concentration that gave a maximal change in steady-state conductance (16). Under these conditions, because of its requirement for cholesterol, amphotericin B permeabilizes only the plasma membrane domain in direct contact with the amphotericin B solution, as previously demonstrated (9, 10, 15, 16). As a result, the other plasma membrane becomes rate limiting for overall, transepithelial ion transport, and "stimulus"-dependent changes of its ion conductances can be assessed as Isc due to ion gradients or as transepithelial conductances changes.

Basolateral membrane in our case incorporates the ionophore, thus electrically isolating the opposing (apical) plasma membrane. After addition of amphotericin B (100 µM, submucosal solution), tissue conductance increased from 0.6 ± 0.08 to 2.8 ± 0.50 mS/cm2 (n = 50) over the course of the experiment. To study the Cl- conductance of the apical plasma membrane [GCl(ap)], the voltage across the monolayer was sequentially stepped from a holding voltage of 0 mV to between -40 and +40 mV over a period of ~10 s. The protocol was performed before and 12 min after addition of cryptdin-3 or agonists. Positive currents correspond to anion secretion/cation absorption, i.e., a lumen-negative potential under open-circuit conditions.


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Cryptdin-3 increases Gap in basolaterally permeabilized T84 cell monolayers. To test the idea that cryptdin-3 may induce Cl- secretion in secretory epithelia by self-assembling into novel anion-conducting channels, we studied confluent monolayers of T84 cells in which the basolateral membranes had been selectively permeabilized with amphotericin B. Initial experiments were performed in the presence of an apically directed 137 mM Cl- gradient, with Cl- representing the major membrane-permeant ion (Table 1; medium A, basolateral; medium B, apical). Under these conditions with the transepithelial voltage clamped to 0 mV, baseline GCl(ap) was 3.10 ± 0.40 mS/cm2 (n = 3). Figure 1 shows that addition of 50 µg/ml cryptdin-3 stimulated GCl(ap) by 1.40 ± 0.6 mS/cm2 (n = 3). Peak conductances were observed after ~10-min incubations and returned to baseline within 30 min. These data demonstrate that cryptdin-3 increases Gap of T84 cell monolayers in the absence of an applied membrane potential and in a nonsustained manner. The characteristics of the cryptdin-3-induced GCl(ap) contrast with those of endogenous GCl(ap) induced using forskolin or thapsigargin as secretory agonists (16).

                              
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Table 1.   Composition of media



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Fig. 1.   At 0 mV transmembrane potential, addition of 50 µg/ml cryptdin-3 stimulates conductance (G) across apical plasma membrane of basolaterally permeabilized T84 cell monolayers. A defined basolateral-to-luminal Cl- gradient was established across the monolayer by substitution of sodium gluconate for NaCl in the solution bathing the luminal side (Table 1, medium B: low-Cl- medium) and addition of 100 µM amphotericin B to the basolateral solution (Table 1, medium A: normal-Cl- medium). Trace is representative of results from 3 different filters.

Biophysical characteristics of cryptdin-3-induced Gap. To distinguish between anion transport mediated by exogenous cryptdin-3-based channels and that mediated by endogenous apical membrane cAMP- and Ca2+-dependent Cl- channels, the voltage dependency and size selectivity of cryptdin-induced anion conductances were defined. In these experiments, basolaterally permeabilized T84 cell monolayers were studied in symmetrical buffers containing Cl- as the major membrane-permeant ion.

Figure 2A shows that the current-voltage relationships of cryptdin-3-induced apical Cl- current [ICl(ap)] were linear and thus not dependent on membrane potential. To investigate size and charge selectivity, Cl- was replaced with the membrane-impermeant anion gluconate (Table 1, medium D). Under these conditions, the conductance normally stimulated by cryptdin-3 was minimally affected (Fig. 2A): change in Gap (Delta Gap) = 2.20 ± 0.48 and 1.81 ± 0.50 mS/cm2 in the presence and absence of Cl- (n = 3), respectively, at +40 mV. As we have previously shown, the activation of Gap in basolaterally permeabilized T84 cell monolayers by forskolin and thapsigargin [Delta Gap = 3.20 ± 0.4 and 1.3 ± 0.2 mS/cm2 for forskolin and thapsigargin, respectively (n = 30) at +40 mV] is ablated when Cl- is replaced by gluconate: for forskolin, Delta Gap = 3.20 ± 0.4 and 0.04 ± 0.02 mS/cm2 in the presence and absence of Cl-, respectively, at +40 mV (n = 30); for thapsigargin, Delta Gap = 1.30 ± 0.20 and 0.08 ± 0.03 mS/cm2 in the presence and absence of Cl-, respectively, at +40 mV (n = 30). When forskolin and cryptdin-3 were added together (Fig. 2B), the conductance generated was greater than that induced by forskolin alone: Delta Gap = 3.20 ± 0.40 (n = 30) and 5.70 ± 1.10 mS/cm2 (n = 3) for forskolin and forskolin + cryptdin-3, respectively, at +30 mV. Furthermore, when Cl- was replaced by gluconate (monovalent cations were in the millimolar range; Table 1, media C and D), the conductance induced by forskolin and cryptdin-3 was not completely abolished (Fig. 2B): Delta Gap = 5.70 ± 1.10 and 2.60 ± 0.55 mS/cm2 in the presence and absence of Cl-, respectively, at +30 mV (n = 3). In contrast, the conductance induced by forskolin and thapsigargin was completely abolished in the absence of Cl- (Fig. 2C): Delta Gap = 5.20 ± 0.70 and 0.73 ± 0.20 mS/cm2 in the presence and absence of Cl-, respectively, at +30 mV (n = 3). These data provide evidence that cryptdin-3-induced channels display different biophysical characteristics from those induced by forskolin (the cAMP-dependent apical Cl- channel, CFTR) or thapsigargin (the Ca2+-dependent apical Cl- channel). When the membrane was shifted from symmetrical to asymmetrical Cl- solutions, a Cl- gradient was established across the electrically isolated apical membrane by placing "normal Cl-" (buffer A) in the apical reservoir and "low Cl- and high HPO42-" (buffer E) in the basolateral reservoir (Na+ was present in both buffers at 137 mM; Table 1). When HPO42- was substituted for Cl-, the inward (apical reservoir-to-cytosol basolateral reservoir) currents were increased, which is to be expected because of the increased driving force for Cl- but not for HPO42- (the HPO42- gradient is in the opposite direction) through the apical plasma membrane. Under these conditions, apparent reversal potentials (Vrev) for cryptdin-3-induced currents shifted positively, as predicted, by 18.6 ± 7.10 mV (n = 3; Fig. 2A). The observed Vrev do not approximate theoretical values in this model system, suggesting that GCl(ap) is shunted by one or more pathways (tight junction and/or apical plasma membrane with nonspecific permeability). At this point, we still cannot exclude the possibility that the macroscopic current induced by cryptdin could be the sum of the cation and Cl- currents, explaining our observed Vrev. Nonetheless, it can be deduced from the directions of the shift in Vrev and ion selectivities that data obtained from permeabilized monolayers behave as expected for an imperfect Cl- electrode. This agrees with preliminary data obtained from single-channel studies (25).


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Fig. 2.   A: current-voltage relationships in basolaterally permeabilized monolayers of T84 cells after addition of 50 µg/ml cryptdin-3 to the apical reservoir. Reservoir concentrations were as follows: 144/144 mM basolateral/apical Cl- with 0.5 mM Na+ on both sides (; Table 1, medium C) and 137/137 mM basolateral/apical with substitution of Cl- by gluconate (; Table 1, medium D). A defined luminal-to-basolateral Cl- gradient (144/7.90 mM) was established across the monolayer by substitution of NaHPO42- for NaCl in the solution bathing the basolateral side (×; Table 1, medium E: low Cl- and high HPO42-). Ordinates, difference current (ICl) between stimulated and unstimulated cells. B: current-voltage relationships in basolaterally permeabilized monolayers of T84 cells after addition of 10 µM forskolin (to basolateral reservoir) + 50 µg/ml cryptdin-3 (to apical reservoir). Reservoir concentrations were as follows: 144/144 mM basolateral/apical Cl- (forskolin + cryptdin, ; Table 1, medium C) and 137/137 mM basolateral/apical with substitution of Cl- by gluconate (forskolin + cryptdin-3, open circle ; Table 1, medium D). C: current-voltage relationships in basolaterally permeabilized monolayers of T84 cells after addition of 10 µM forskolin (to basolateral reservoir) + 10 µM thapsigargin (to basolateral reservoir). Reservoir concentrations were as follows: 144/144 mM basolateral/apical Cl- (forskolin + thapsigargin, black-triangle; Table 1, medium C) and 137/137 mM basolateral/apical with substitution of Cl- by gluconate (forskolin + thapsigargin; triangle ; Table 1, medium D). Ordinates, difference current between stimulated and unstimulated cells. Traces are representative of results from 3 different filters for each condition.

Cryptdin-3 activates Gap in epithelial cells deficient in functional CFTR. To further distinguish between Gap mediated by cryptdin-3-based channels and Gap mediated by the endogenous cAMP-dependent Cl- channel CFTR, we used the airway cell line JME/CF15 containing CFTR with the inactivating Delta F508 mutation. These epithelial cells, derived from a patient with CF, grow in monolayer culture with high transepithelial resistance but do not exhibit cAMP-dependent Cl- secretion because of the inactivating mutation in CFTR (5).

Transepithelial conductances of JME/CF15 cell monolayers at baseline were ~0.8 mS/cm2. Preliminary studies confirmed that transepithelial currents were not induced by 10 µM forskolin, consistent with the absence of functional CFTR (19, 20). In contrast, elevation of intracellular Ca2+ by 10 µM thapsigargin induced a small increase in Isc of ~2 µA/cm2.

These data (not shown) confirmed the Cl- secretory phenotype of this cell line and the absence of functional CFTR.

To study cryptdin-3-induced channels, JME/CF15 cell monolayers were basolaterally permeabilized with amphotericin B, and an apically directed 137 mM Cl- gradient was established using buffers A and B (Table 1). Under these conditions, forskolin did not increase GCl(ap) (Fig. 3A). In contrast, 50 µg/ml cryptdin-3 stimulated a 0.72 ± 0.25 mS/cm2 increase in GCl(ap) (Fig. 3A). These data indicate that cryptdin-3-induced ICl(ap) is not due to activation of CFTR. This interpretation was confirmed by examining the voltage dependency of cryptdin-3-induced ICl(ap) in symmetrical buffers containing Cl- as the major permeant ion (Table 1, buffer C, and Fig. 3B). Cl- currents were not observed under any applied membrane potential after stimulation with forskolin. In contrast, Cl- currents induced by 50 µg/ml cryptdin-3 were clearly present and voltage independent: Delta Gap = 0.80 ± 0.06 mS/cm2 at +40 mV (n = 3). These results are consistent with those obtained from T84 cells and demonstrate that the cryptdin-3-induced Gap is unrelated because of activation of endogenous CFTR.


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Fig. 3.   A: at 0 mV transmembrane potential, addition of 50 µg/ml cryptdin-3 to the apical reservoir stimulates conductance(s) across apical plasma membrane of basolaterally permeabilized JME/CF15 cell monolayers. A defined basolateral-to-luminal Cl- gradient (144/7.9 mM) was established across the monolayer by substitution of sodium gluconate for NaCl in the solution bathing the luminal side (Table 1, medium B) and addition of 100 µM amphotericin B to the basolateral solution (Table 1, medium A). Trace is representative of results from 3 different filters for each condition. B: current-voltage relationships in basolaterally permeabilized monolayers of JME/CF15 cells after addition of 10 µM forskolin (triangle ) to the basolateral cytosol reservoir or 50 µg/ml cryptdin-3 () to the apical reservoir. Basolateral/apical reservoir Cl- concentration (in mM) was 144/144 mM (Table 1, medium C). Traces are representative of results from 3 different filters for each condition. Isc, short-circuit current.


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Members of the cryptdin family display an amphipathic structure constrained by three disulfide bridges. Several studies indicate that these peptides act by altering the permeability of biological membranes. However, the mechanism of the membrane permeabilization is not well understood.

In the present study we found that cryptdin-3, at concentrations (50 µg/ml) comparable to those at which they exert antimicrobial effects (22), increased Gap in basolaterally permeabilized T84 cell monolayers. The reversible effect of cryptdin-3 could be that cryptdin channels could be removed from the apical membrane by partitioning into the aqueous phase or by endocytosis. More specifically, cryptdin-3 induced voltage-independent Gap in apical membranes of T84 cells. In previous studies on artificial bilayers (7), human alpha -defensin human neutrophil peptide-1 (HNP-1) induced voltage-dependent ionic conductances. This discrepancy may reflect differences in the lipid composition of T84 cell membranes vs. phospholipid bilayers, presence or absence of phosphates, divalent cations, other factors present in T84 cells but not in bilayers, or structure-function differences in monomeric cryptdin-3 and dimeric HNP-1. Alternatively, a voltage-dependent conformation in T84 cells could be short lived and progress rapidly to a voltage-independent conductance.

The increase of Gap by cryptdin-3 in the basolaterally permeabilized T84 cells could be the result of 1) formation of novel conductive channels by this peptide or 2) the peptides (cryptdin-3) perturbing lipid organization and, thereby, enhancing endogenous apical membrane channels (CFTR or Ca2+-dependent Cl- channel). The present data show that 1) the combination of forskolin and cryptdin-3 induces an increase in conductance greater than that stimulated by forskolin alone; 2) cryptdin-3, but not thapsigargin, stimulates Gap at 0 mV; and 3) cryptdin-3 does not increase cAMP, cGMP (11), or Ca2+ (data not shown) in intact cells. It is unlikely that cryptdin-3 activates volume-sensitive channel(s) (volume-sensitive Cl- channels are Ca2+ and voltage dependent), since channels formed by cryptdin-3 are Ca2+ and voltage independent. These results, in combination with a recent study (25) demonstrating that cryptdin-3 forms anion-selective channels in cytoplasmic membranes of human embryonic kidney cells, suggest that, as reported for human myeloid defensins, it is likely that cryptdin-3 self-assembles into anion-conducting channel(s) by interacting with the apical plasma membrane of T84 cells.

The ability of other anions to substitute for Cl- in transepithelial anion secretion has been determined in intact monolayers of T84 cells. For example, in a previously reported study, when Cl- is replaced by gluconate, the cryptdin-3-dependent Isc was nearly abolished (11). Net secretion, however, reflects the selectivities of two serial steps in transepithelial movement: uptake at the basolateral membrane and exit across the apical membrane, with the overall selectivity determined by the most restrictive step. The basolateral cotransport process responsible for anion uptake from the serosal solution restricts influx of halides other than Cl- and determines the anion selectivity for secretion. In contrast, in basolaterally permeabilized monolayers, the only restrictive step for anion secretion is represented by the apical plasma membrane. In the present study we find that when Cl- is replaced by gluconate, the increase of Gap by forskolin and thapsigargin is completely ablated. In contrast, the Gap stimulated by cryptdin-3 was only slightly reduced in the presence of gluconate. Furthermore, as previously reported and confirmed here, forskolin and thapsigargin have no effect on gluconate current (16). In addition, the finding that, in the presence of gluconate as the major charge carrier, the increase of Gap stimulated by the combination of forskolin and cryptdin-3 is only partially reduced is consistent with the residual current being a gluconate current that is cryptdin-3 dependent. Interestingly, the cryptdin-3 channels are more selective to Cl- than HPO42- and Na+, showing its preference for Cl-. We cannot exclude the possibility that the macroscopic current induced by cryptdin could be the sum of the cation and Cl- currents. The question of anion selectivity induced by cryptdin-3 could be solved using methods such as patch clamp and lipid bilayer. With the use of single-channel analysis, it has also been recently reported that cryptdin-3 at low concentration (1 µg/ml) may activate an anion-selective channel with unique features, including a conductance of 15 pS (25). As for other defensins, the selectivity of the channel(s) formed by cryptdin-3 could be dose dependent (4, 25).

In the most common form of CF, the gene that encodes for CFTR anion channel is mutated, resulting in a protein that is capable of conducting Cl- (19) but is absent from the plasma membrane because of aberrant intracellular processing (2, 20). For example, in CF airway epithelia, Ca2+ stimulated Ca2+-sensitive (i.e., non-CFTR) Cl- channels, but the response to cAMP (via CFTR) is defective. Recently, many studies have been focused on methods to promote functional correction of cAMP-mediated Cl- transport in CF epithelial cells. These studies have included pharmacological approaches aimed at restoration of the amount of Delta F508-CFTR on the surface epithelium of patients with CF as well as gene-targeting strategies to promote normal Cl- secretion in epithelia (2). One logical extension of our present study was to investigate the effect of cryptdin-3 on CF cells. Using an airway CF cell line (JME/CF15), we show that cryptdin-3 promotes a current in permeabilized CF monolayers. These results show that cryptdin-3 induces a restoration of Cl- secretion in CF cells that is probably not cAMP or CFTR dependent.

In summary, our results demonstrate that cryptdin-3 induces Gap with ion selectivity different from that elicited by forskolin or thapsigargin. Cryptdin-3 is positively charged at physiological pH and might bind to negatively charged phospholipid membranes with the aid of electrostatic interactions, forming channel(s) and permeabilizing the apical plasma membrane of normal and CF phenotypes of epithelial cells, even in the absence of the transmembrane potential. Such cryptdin-dependent channel formation is capable of supporting electrogenic Cl- secretion in cells electrochemically poised for this key physiological event.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants DK-35932 (J. L. Madara and W. I. Lencer) and AI-22931 (M. E. Selsted) and Large Scale Biology (M. E. Selsted). This work was initiated with National Institute of Diabetes and Digestive and Kidney Diseases National Research Service Award DK-09800 (D. Merlin). D. Merlin is a recipient of a Career Development Award from the Crohn's and Colitis Foundation of America.


    FOOTNOTES

Address for reprint requests and other correspondence: D. Merlin, Dept. of Pathology and Laboratory Medicine, Emory University, WMRB-2, Rm. 2329, 1639 Pierce Dr., Atlanta, GA 30322 (E-mail: dmerlin{at}emory.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 27 April 2000; accepted in final form 8 September 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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