NH2-terminal modification of a channel-forming peptide increases capacity for epithelial anion secretion

James R. Broughman1, Kathy E. Mitchell2, Roger L. Sedlacek2, Takeo Iwamoto1, John M. Tomich1, and Bruce D. Schultz2

Departments of 1 Biochemistry and 2 Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

A synthetic, channel-forming peptide, derived from the alpha -subunit of the glycine receptor (M2GlyR), has been synthesized and modified by adding four lysine residues to the NH2 terminus (N-K4-M2GlyR). In Ussing chamber experiments, apical N-K4-M2GlyR (250 µM) increased transepithelial short-circuit current (Isc) by 7.7 ± 1.7 and 10.6 ± 0.9 µA/cm2 in Madin-Darby canine kidney and T84 cell monolayers, respectively; these values are significantly greater than those previously reported for the same peptide modified by adding the lysines at the COOH terminus (Wallace DP, Tomich JM, Iwamoto T, Henderson K, Grantham JJ, and Sullivan LP. Am J Physiol Cell Physiol 272: C1672-C1679, 1997). N-K4-M2GlyR caused a concentration-dependent increase in Isc (k[1/2] = 190 µM) that was potentiated two- to threefold by 1-ethyl-2-benzimidazolinone. N-K4-M2GlyR-mediated increases in Isc were insensitive to changes in apical cation species. Pharmacological inhibitors of endogenous Cl- conductances [glibenclamide, diphenylamine-2-dicarboxylic acid, 5-nitro-2-(3-phenylpropylamino)benzoic acid, 4,4'-dinitrostilben-2,2'-disulfonic acid, indanyloxyacetic acid, and niflumic acid] had little effect on N-K4-M2GlyR-mediated Isc. Whole cell membrane patch voltage-clamp studies revealed an N-K4-M2GlyR-induced anion conductance that exhibited modest outward rectification and modest time- and voltage-dependent activation. Planar lipid bilayer studies yielded results indicating that N-K4-M2GlyR forms a 50-pS anion conductance with a k[1/2] for Cl- of 290 meq. These results indicate that N-K4-M2GlyR forms an anion-selective channel in epithelial monolayers and shows therapeutic potential for the treatment of hyposecretory disorders such as cystic fibrosis.

synthetic peptide; anion conductance; chloride transport; epithelial cells; cystic fibrosis


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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PREVIOUS REPORTS HAVE SHOWN that a 23-residue peptide based in the second transmembrane segment (M2) of the alpha -subunit of the glycine receptor (GlyR) forms a Cl--permeable pathway in lipid bilayers (10). Subsequently, a family of M2GlyR peptides (Kx-M2GlyR) was created by adding lysine residues to the NH2 or COOH termini of the M2GlyR sequence, and channel-forming activity was assessed (15). Addition of the lysine-modified peptides to the apical membrane of epithelial cell monolayers was associated with Cl- secretion and water transport (18). Primarily on the basis of its high aqueous solubility (27.5 mM) (15), C-K4-M2GlyR was characterized extensively and pursued as a model for the development of potential therapeutics for the treatment of anion hyposecretory disorders (17, 18). C-K4-M2GlyR was thought to mimic the orientation of the M2 segment in native GlyR in cell membranes and/or lipid bilayers, with the NH2 terminus positioned intracellularly and the COOH terminus located extracellularly. Additional experiments were conducted to test the hypothesis that reversing the orientation of the M2 segment by lysine modification of the NH2 terminus would significantly alter the effects on epithelial anion transport. Thus we now show that the M2GlyR peptide with four lysines adducted to the NH2 terminus (N-K4-M2GlyR) is more effective in increasing the short-circuit current (Isc) in Madin-Darby canine kidney (MDCK) and T84 cell monolayers and shows substantially more therapeutic potential than any of the previously evaluated Kx-M2GlyR peptides.


    MATERIALS AND METHODS
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MATERIALS AND METHODS
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Peptide synthesis. All peptides were synthesized by solid-phase synthesis using fluoren-9-yl methoxycarbonyl chemistry on a peptide synthesizer (model ABI 431A; Perkin-Elmer Biosystems, Norwalk, CT). rho -Hydroxymethylphenoxymethyl resin preloaded with the COOH-terminal amino acid was purchased from Perkin-Elmer, and Nalpha -fluoren-9-yl methoxycarbonyl amino acids were purchased from Perkin-Elmer, Bachem (Torrence, CA), Peninsula Laboratories (Belmont, CA), and Peptides International (Louisville, KY). All peptides were characterized by reverse-phase HPLC and matrix-assisted laser desorption time-of-flight mass spectroscopy. N-K4-M2GlyR was synthesized as the primary focus of this research, and C-K4-M2GlyR was used as a benchmark for comparison. The results include peptides from at least three separate syntheses. Scrambled sequences (Sc-N-K4-M2GlyR and Sc-C-K4-M2GlyR) were used as controls, inasmuch as the scrambled forms of these peptides are not expected to mediate a change in the transepithelial Isc. The scrambled analogs of these peptides were designed to minimize the amphipathic structure while attempting to maintain the helical character of the peptides. The sequences of these peptides are as follows
N-K<SUB>4</SUB>-M2GlyR: KKKKPARVGLGITTVLTMTTQSSGSRA

C-K<SUB>4</SUB>-M2GlyR: PARVGLGITTVLTMTTQSSGSRAKKKK

Sc-C-K<SUB>4</SUB>-M2GlyR: ILASTRSQTGRMALSGTTTPGVVKKKK

Sc-N-K<SUB>4</SUB>-M2GlyR: KKKKILASTRSQTGRMALSGTTTPGVV

Cell culture. MDCK cells were a generous gift of Dr. Lawrence Sullivan (Kansas University Medical Center, Kansas City, KS). T84 cells were obtained from Dr. Daniel Devor (University of Pittsburgh, Pittsburgh, PA). MDCK and T84 cells were maintained with similar culture procedures. The culture medium was a 1:1 mixture of DMEM-Ham's F-12 (GIBCO BRL, Grand Island, NY) supplemented with 5% heat-inactivated fetal bovine serum (BioWhittaker, Walkersville, MD) and 1% penicillin and streptomycin (GIBCO BRL). Cells were grown in plastic culture flasks in a humidified environment with 5% CO2 at 37°C and passaged every 5-7 days. For Ussing chamber experiments, cells were plated on 1.13-cm2 permeable supports (Snapwell; Costar, Cambridge, MA) at a density of ~1 × 106 cells/well and incubated in DMEM-Ham's F-12 supplemented with fetal bovine serum and antibiotics (changed every other day) for 2-3 wk before they were mounted in modified Ussing flux chambers.

Epithelial electrical measurements. Transepithelial ion transport was evaluated in a modified Ussing chamber (model DCV9; Navicyte, San Diego, CA). The Ussing chamber's fluid resistance compensation was completed in Ringer solution (see below). For electrical measurements, cell monolayers were bathed in Ringer solution maintained at 37°C and continuously bubbled with 5% CO2-95% O2. The transepithelial membrane potential was clamped to zero, and the transepithelial Isc, an indicator of net ion transport, was measured continuously with a voltage-clamp apparatus (model 558C; Dept. of Bioengineering, University of Iowa, Iowa City, IA). Data were digitally acquired at 1 Hz with a Macintosh computer (Apple Computer, Cupertino, CA) using Aqknowledge software (version 3.2.6; BIOPAC Systems, Santa Barbara, CA) with an MP100A-CE interface.

Solutions. Ringer solution (290 ± 2 mosmol) composed of (in mM) 120 NaCl, 25 NaHCO3 3.3 KH2PO4, 0.8 K2HPO4, 1.2 MgCl2, and 1.2 CaCl2 was made fresh daily. All components of the Ringer solution were purchased from Sigma Chemical (St. Louis, MO).

Chemicals. Stock solutions of chemicals were prepared as follows: forskolin (Coleus forskohlii; Calbiochem, La Jolla, CA), 10 mM in ethanol; 1-ethyl-2-benzimidazolinone (1-EBIO; Acros Organics), 1 M in DMSO; bumetanide (Sigma Chemical), 20 mM in ethanol; diphenylamine-2-dicarboxylic acid (DPC; Sigma Chemical), 1 M in DMSO; and 4,4'-dinitrostilben-2,2'-disulfonic acid (DNDS; Acros Organics), 10 mM in Ringer solution. The following stock solutions were prepared at 100 mM in DMSO: glibenclamide, indanyloxyacetic acid, 2-[3-(trifluoromethyl)-anilino]nicotinic acid (niflumic acid; Sigma Chemical), and 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB; RBI, Natick, MA). All other chemicals were purchased from Sigma Chemical and were of reagent grade unless otherwise noted.

Whole cell patch-clamp recording. MDCK cells were maintained as described above. Cells were detached from culture dishes 48 h after they were plated (60-80% confluency) using a trypsin-containing (0.5 mg/ml) physiological salt solution (in mM: 137 NaCl, 2.7 KCl, 2.8 D-glucose, 3.4 NaHCO3, and 0.27 EDTA, sodium salt), pelleted by centrifugation, and resuspended in the external bath solution. Internal (pipette) and external (bath) solutions were selected to measure changes in whole cell Cl- permeability and to minimize cell swelling (13, 14). Recording pipettes were pulled to resistances of 2.5-6 MOmega when filled with intracellular solution containing (in mM) 145 Tris-Cl, 5 MgATP, and 5 HEPES, pH 7.45. The external bath solution consisted of (in mM) 145 Tris-Cl, 1 MgCl2, 1 CaCl2, 60 sucrose, 5 glucose, and 5 HEPES, pH 7.45. Cl- is the primary permeant ion in these solutions. Current-voltage relationships obtained using the standard recording solutions were not corrected for liquid junction potentials, which are <1 mV in these conditions. All chemicals used in the patch-clamp analyses were obtained from Sigma Chemical. Recordings were performed at room temperature (22-24°C) from a Plexiglas chamber mounted on an inverted microscope (Diaphot 300; Nikon). Data acquisition and analysis were accomplished using an IBM-compatible computer interfaced to an Axopatch 200-A amplifier driven by pCLAMP software (Axon Instruments, Foster City, CA).

Planar lipid bilayer experiments. Planar lipid bilayer experiments were conducted as described previously in detail (9). Lipid bilayers were formed across a 100-µm aperture in the wall of a Delrin cup. The bilayer was formed with a solution containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine [22.5% by mass (wt%)], 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (10 wt%), and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (67.5 wt%) in decane at a total lipid concentration of 50 mg/ml. Currents were measured in KCl, with concentrations as indicated in 40 mM HEPES, pH 7.4. The grounded side (equivalent to extracellular) of the bilayer chamber is designated trans; the side to which voltage is applied (intracellular) is designated cis. Peptide solutions were introduced into the trans side of the bilayer chamber. Bilayer recordings were made with a Gene-Clamp 500 and a CV-5B-100GU headstage (Axon Instruments). Data were acquired at 1 kHz after passage through an eight-pole low-pass Bessel filter (cutoff 200 Hz). Microcal Origin (Northhampton, MA) and pCLAMP 6.0 or pCLAMP 7.0 (Axon Instruments) were used for data analysis. Lipids were obtained from Avanti Polar Lipids (Alabaster, AL). All other reagents for the planar bilayer studies were purchased from Sigma Chemical and were of the highest purity available.

Data analysis. All summary results are presented as the arithmetic mean ± SE. The differences between control and treatment data were analyzed using ANOVA, Tukey's test (SAS Institute, Cary, NC), and Student's t-test (Excel; Microsoft, Redman, WA). The probability of making a type I error <0.05 was considered statistically significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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N-K4-M2GlyR mediates anion secretion. Experiments were performed to evaluate the effects of N-K4-M2GlyR on transepithelial ion transport using C-K4-M2GlyR as the basis for comparison. Previously, C-K4-M2GlyR was shown to increase Isc in MDCK and T84 cells (15, 17, 18), with the effect being magnified in the presence of 1-EBIO, an activator of basolateral K+ channels. Data presented in Fig. 1 are from a representative experiment demonstrating the response of paired MDCK cell monolayers to increasing concentrations of N-K4-M2GlyR or C-K4-M2GlyR in the absence or presence of 1-EBIO. The modified M2GlyR peptides were added only to the apical (luminal) compartment. All monolayers exhibited basal resistance that was similar to that typically observed throughout the reported studies (1,260 ± 80 Omega  · cm2, n = 52). In the absence of 1-EBIO, N-K4-M2GlyR was associated with an increase in Isc from a basal level of 0.5 µA/cm2 to a maximal Isc of 8.7 µA/cm2. Results from a paired experiment presented in Fig. 1B demonstrate that C-K4-M2GlyR increased Isc to a lesser extent, yet in accordance with previously reported observations (18). N-K4-M2GlyR and C-K4-M2GlyR stimulated an increase in transepithelial Isc over a concentration range of 30-500 µM. Although starting from a similar basal value, the C-K4-M2GlyR-induced increase in Isc was only half that seen with N-K4-M2GlyR, reaching a maximum value of 4.8 µA/cm2.


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Fig. 1.   M2GlyR peptides facilitate anion secretion by Madin-Darby canine kidney (MDCK) epithelial cell monolayers. Representative responses of paired monolayers are displayed. N-K4-M2GlyR (A and C) and C-K4-M2GlyR (B and D) were added to solutions bathing the apical membrane of epithelial monolayers at increasing concentrations from 10 to 500 µM (total solute concentration). Monolayers in C and D were exposed to 300 µM 1-ethyl-2-benzimidazolinone (1-EBIO) before the addition of peptide.

Stimulatory effects of channel-forming peptides were enhanced in the presence of 1-EBIO. As was typical of all experiments employing MDCK cells, 1-EBIO had no effect on Isc (Fig. 1, C and D, see also Fig. 3), which suggests that apical conductance is the rate-limiting component of ion transport in basal conditions. Subsequent exposure of the apical membrane to channel-forming peptides resulted in magnified secretory responses. Effects of N-K4-M2GlyR and C-K4-M2GlyR were approximately threefold greater in the presence of 1-EBIO, reaching 29 and 11 µA/cm2, respectively. N-K4-M2GlyR consistently showed an ability to elicit a greater Isc across the MDCK monolayers in the presence and absence of 1-EBIO than the C-K4-M2GlyR peptide and was able to elicit a greater response than forskolin (see Fig. 3).

Neither of the scrambled peptides, Sc-C-K4-M2GlyR nor Sc-N-K4-M2GlyR, induced a change in Isc when applied to the apical membrane of MDCK cells in the absence or presence of 1-EBIO (data not shown).

Concentration dependence of N-K4-M2GlyR-mediated anion secretion. Data from 52 MDCK cell monolayers treated with various concentrations of N-K4-M2GlyR, in the presence or absence of 1-EBIO, are summarized in Fig. 2. The response to N-K4-M2GlyR was concentration dependent over the range tested (10-500 µM), and the response in the presence of 1-EBIO was always greater than in its absence. Visual inspection of the data suggested that the response increased as a saturating function of peptide concentration, as was previously observed for C-K4-M2GlyR (18). Thus a nonlinear least-squares fit to the data was obtained using a modified Hill equation, Isc Imax [xn/(kn + xn)], where Isc is the observed Isc, Imax is the predicted maximal Isc, k represents the peptide concentration at 0.5 Imax [or the apparent dissociation constant (KD)], x represents the peptide concentration, and n is the Hill coefficient. All variables were allowed to vary until chi 2 (a measure of goodness of fit) was minimized. In cell monolayers exposed to N-K4-M2GlyR, Imax is predicted to be 8.5 ± 0.6 µA/cm2, with half-maximal Isc attained at 190 ± 15 µM peptide and a Hill coefficient of 3.1 ± 0.7. When experiments were conducted in the presence of 1-EBIO, the fitted parameters for k (208 ± 6 µM) and n (2.6 ± 0.1) were similar to values derived in the absence of 1-EBIO, although Imax is predicted to be three times greater (24.3 ± 0.5 µA/cm2).


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Fig. 2.   Concentration dependence of N-K4-M2GlyR (N-K4) on MDCK epithelial monolayer short-circuit current. Values are means ± SE of 3-10 observations from a total of 52 epithelial monolayers. Solid lines, best fit of a modified Hill equation to the data. Parameters of the fitted lines are indicated in RESULTS.

N-K4-M2GlyR-mediated current is independent of cAMP stimulation. There was the possibility that the N-K4-M2GlyR peptide elicited an increase in Isc by stimulation of adenylyl cyclase and an endogenous conductance [e.g., cystic fibrosis transmembrane conductance regulator (CFTR)]. Therefore, the effects of M2GlyR peptides were compared with the effects of a known stimulant of adenylyl cyclase, forskolin, in the absence and presence of 1-EBIO. Data presented in Fig. 3 show a comparison of the activity of channel-forming peptides and forskolin in MDCK (Fig. 3A) and T84 (Fig. 3B) cell monolayers. These cell types were employed for these studies, because it is known that they differ substantially in their ability to secrete anions, with T84 cells being more responsive at least in part because of greater expression of the apical membrane channel CFTR. Basal ion transport across the monolayers was minimal in both cell lines (0.1 ± 0.1 and 1.0 ± 0.1 µA/cm2 in MDCK and T84 cells, respectively). Exposure of MDCK cell monolayers to N-K4-M2GlyR (300 µM) resulted in an increase in Isc of 7.4 ± 0.8 and 17.8 ± 1.8 µA/cm2 in the absence and presence of 1-EBIO, respectively; these values are similar to those reported for other experiments (Figs. 1 and 2). These responses are two times greater than the paired responses to forskolin (10 µM) or C-K4-M2GlyR (300 µM). C-K4-M2GlyR and forskolin caused increases in Isc that were of similar magnitude. The responses to N-K4-M2GlyR, C-K4-M2GlyR, and forskolin were greater in T84 than in MDCK cell monolayers (note scale bars). More importantly, the rank order of potency is unique to each epithelial cell type: N-K4-M2GlyR > C-K4-M2GlyR = forskolin for MDCK cells and forskolin > N-K4-M2GlyR > C-K4-M2GlyR for T84 cells. The relative magnitude of the cAMP-stimulated response differed between cell types, reflecting their differences in endogenous secretion pathways. Again, neither of the scrambled peptides induced an increase in the transepithelial Isc in either cell type (data not shown). Thus it is unlikely that N-K4-M2GlyR is acting solely or predominantly via an endogenous pathway, since closely related peptides are either less effective or have no effect on either cell type.


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Fig. 3.   M2GlyR peptides [C-K4-M2GlyR (C) and N-K4-M2GlyR (N)] and forskolin (F) exhibit different rank orders of potency for stimulation of short-circuit current in MDCK (A) and T84 (B) epithelial cell monolayers. Values are means ± SE for 3-15 observations in each condition. M2GlyR peptides (300 µM) were added to the apical compartment of modified Ussing chambers. Concentrations of forskolin and 1-EBIO (E) were 10 and 300 µM, respectively.

N-K4-M2GlyR forms channels in planar lipid bilayers. To further verify that N-K4-M2GlyR was introducing a novel anion conductance rather than stimulating an endogenous conductance, experiments were performed in which N-K4-M2GlyR was introduced into planar lipid bilayers to examine the biophysical characteristics of the conductance. Channel activity was observed at N-K4-M2GlyR concentrations as low as 100 nM. An example of N-K4-M2GlyR channel activity in a planar lipid bilayer recorded at -50 mV in a 500/100 mM KCl gradient is shown in Fig. 4A. Two channels were present in the bilayer with open levels as indicated. Single-channel amplitude was 3.2 pA. In another bilayer with a single active channel, membrane voltage was repeatedly ramped from -100 to +100 mV (1 mV/ms), with the average current (corrected for capacitive current before peptide addition) from 32 episodes in each condition presented in Fig. 4B. Increasing the concentration of KCl in the trans solution from 100 to 500 mM caused a leftward shift in the reversal potential of 30.2 mV, which indicates a Cl--to-K+ selectivity ratio of ~16:1 (2), similar to that of the native glycine receptor (1). The average single-channel conductance of N-K4-M2GlyR determined from the slope of current-voltage relationships was 50 pS when measured in symmetrical 0.1 M KCl. This channel conductance is similar to that reported for the native M2GlyR peptide (10). The effects of incrementally increasing Cl- concentration on N-K4-M2GlyR conductance were also assessed (Fig. 4C). The resulting maximum conductance was 192 pS, and the half-maximal saturating concentration (k1/2 or apparent KD) of Cl- was 290 meq.


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Fig. 4.   Characteristics of N-K4-M2GlyR channels in a planar lipid bilayer. A: typical recording of N-K4-M2GlyR channel activity made at -50 mV at 0.5 M KCl on the trans (grounded) side and 0.1 M KCl on the cis side. Two channels are present and actively gating in the membrane. Single-channel amplitude is 3.2 pA. B: current-voltage relationship of a bilayer with a single N-K4-M2GlyR channel in symmetrical (100 mM) and nonsymmetrical (500 mM trans/100 mM cis) KCl. Membrane voltage was repeatedly ramped from -100 to +100 mV (1 mV/ms), and records were corrected by subtraction of capacitive current. Averaged results from 32 episodes in each condition are presented. Increasing the concentration of KCl in the trans solution from 100 to 500 mM caused a leftward shift in the reversal potential from 6.3 to -23.4 mV and a shift in the slope at the reversal potential from 44 to 117 pS. C: saturation of N-K4-M2GlyR conductance in planar lipid bilayer. Slope conductances from current-voltage relationships were determined at 0.1-0.6 M KCl in the trans chamber while the cis chamber was held constant at 0.1 M KCl. Solid line, best fit of a hyperbolic function to the data: g = gmax(i)/([Cl-] + KD), where g is the observed conductance, gmax is the predicted maximal conductance, i is the single-channel amplitude, [Cl-] is Cl- concentration, and KD represents the concentration where g = gmax/2. Parameters of the fitted line are indicated.

N-K4-M2GlyR increases whole cell Cl- current in MDCK cells. The whole cell configuration of the patch-clamp technique was used to examine the electrophysiological properties of the current induced by N-K4-M2GlyR in MDCK cells. An example of the whole cell Cl- current of an MDCK cell before and after treatment with 250 µM N-K4-M2GlyR is presented in Fig. 5. In contrast to currents produced by C-K4-M2GlyR (7), N-K4-M2GlyR induced a current that exhibits modest outward rectification with a slight time-dependent activation. In addition, a minor component of N-K4-M2GlyR-induced current at positive potentials was blocked by DIDS (200 µM). These properties, outward rectification, time-dependent activation, and sensitivity to DIDS, are similar to those of the calcium-dependent Cl- conductance (CaCC) reported in secretory epithelial cells (3) and suggest that N-K4-M2GlyR may activate this endogenous conductance. However, >80% of the N-K4-M2GlyR-induced current remained in the presence of DIDS, indicating that the synthetic conductance largely accounts for the observed current and that an endogenous channel mediated only a minor component of the overall response. Sc-C-K4-M2GlyR has previously been shown to have no effect on whole cell conductance (7).


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Fig. 5.   Whole cell recordings of N-K4-M2GlyR-mediated Cl- currents in MDCK cells. Whole cell currents were recorded before and 20 min after treatment with 250 µM N-K4-M2GlyR and after subsequent treatment with 200 µM DIDS. N-K4-M2GlyR-induced current was obtained by subtracting the current measured before peptide treatment from the current recorded 20 min after peptide treatment. DIDS-sensitive current was obtained by subtracting the current measured in the presence of DIDS from the current measured 20 min after peptide treatment. The current-voltage relationships of the N-K4-M2GlyR-induced () and DIDS-sensitive currents (open circle ) are shown at bottom left. Whole cell currents were recorded during 20-mV step changes in voltage from -80 to +80 mV from a holding potential of -40 mV. Results are typical of 3 similar experiments.

Anion conductance inhibitors have minimal effects on N-K4-M2GlyR-mediated currents. Anion conductances have been extensively characterized by their pharmacology (12). Therefore, the pharmacological profile of inhibition by known anion channel blockers was evaluated for forskolin- and N-K4-M2GlyR-treated cell monolayers (Table 1). Statistical analysis was completed to test the hypothesis that the profile of inhibition was different for forskolin-stimulated and N-K4-M2GlyR-mediated secretion. The results indicated a significant difference in inhibitor sensitivity between the N-K4-M2GlyR- and the forskolin-induced current in MDCK cells (ANOVA, P < 0.01), although there was not a significant difference in the effects of the inhibitors on cell monolayers treated with 250 µM N-K4-M2GlyR in the absence and presence of 1-EBIO (Tukey's test, P > 0.5). None of the anion channel blockers that were tested caused a >30% reduction in the N-K4-M2GlyR-stimulated Isc, regardless of the presence of 1-EBIO. In contrast, the Isc stimulated by forskolin in the presence of 1-EBIO was significantly reduced by known inhibitors of CFTR (DPC, NPPB, and glibenclamide) and inhibitors of CaCC (DNDS and niflumic acid). DNDS was virtually without effect on N-K4-M2GlyR-mediated Isc, which supports the observation that CaCC-mediated current is reduced or absent in confluent epithelial monolayers (8). Surprisingly, bumetanide had less of an inhibitory effect on the N-K4-M2GlyR-mediated Isc than on the forskolin-stimulated Isc. The basis of this difference remains to be determined (see DISCUSSION).

                              
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Table 1.   Percent inhibition of N-K4-M2GlyR- and forskolin-stimulated Isc by anion transport inhibitors


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

N-K4-M2GlyR forms an anion-selective conductive pathway across epithelial cell membranes that exhibits unique biophysical and pharmacological characteristics compared with other M2GlyR peptides. N-K4-M2GlyR is similar to the previously characterized analog, C-K4-M2GlyR, in that both adducts elicit an increase in the anion-selective current in lipid bilayers, whole cell membrane patches, and epithelial monolayers. Furthermore, similar concentration dependence was observed for the two adducts; effects were observed with as little as 25-30 µM, and maximal effects were observed with 500 µM. The observed results allow us to estimate a half-maximal effective concentration (190 µM) and a Hill coefficient of ~3. This high degree of cooperativity suggests that that there is a minimal density of peptide required to form functional channel assemblies. Whether the cooperative step involves membrane partitioning, assembly of the helical bundles into structures, or a combination of these necessary steps for channel formation is not known.

When employed at similar concentrations, N-K4-M2GlyR provides for greater anion secretion across epithelial cell monolayers than any related channel-forming peptide tested so far (Figs. 1 and 3). Numerous possibilities exist that could account for the enhanced level of Isc. In solution, the M2GlyR peptides self-associate to form di-, tri-, or higher-order aggregates (15). If only monomers or perhaps dimers easily partition into cell membranes, then modified peptides that are most likely to be in the monomeric form will demonstrate the most rapid and most complete partitioning into membranes. N-K4-M2GlyR exhibits a more rapid onset of activity than has been reported for its C-K4-M2GlyR counterpart (17, 18), although we did not observe such a dramatic difference (Fig. 1). Once in the membrane, there are likely differences in the stability of the multimeric form(s) that produces channel activity. A decreased dissociation rate (i.e., longer channel lifetime) could account for the higher current observed with N-K4-M2GlyR than with C-K4-M2GlyR. Uniquely modified peptides might also exhibit an elevated open probability or conductance relative to the benchmark compound, although our observations indicate that N-K4-M2GlyR exhibits a conductance that is similar to that reported for C-K4-M2GlyR (7) or M2GlyR (10). M2GlyR peptides might also activate endogenous secretory pathways in epithelial cells. Whole cell recordings show that a component of the N-K4-M2GlyR-mediated current is sensitive to DIDS, which might suggest the activation of CaCC. However, DNDS had little effect on N-K4-M2GlyR-mediated secretion across epithelial monolayers. These apparently conflicting observations can be reconciled in two ways: 1) little DIDS-sensitive current was observed in whole cell membrane patches at membrane potentials expected for epithelial cells, and 2) the relative amount of CaCC in epithelial cells has been reported to decrease when epithelial cells reach confluency (8). Nonetheless, differences in maximal current levels between modified M2GlyR peptides likely reflect some combination of these possibilities.

Pharmacological profiles are frequently employed to characterize ion conductances. Thus a panel of known anion channel modulators was employed to determine which might affect N-K4-M2GlyR-mediated ion transport. None of the tested compounds provided consistent evidence of inhibition: N-K4-M2GlyR exhibits a unique response to putative Cl- channel inhibitors differing from forskolin-stimulated secretion (likely CFTR, although the pharmacological profile suggests that another forskolin-stimulated pathway might also be present in these cell monolayers) and C-K4-M2GlyR-mediated ion transport. These results support two conclusions. 1) N-K4-M2GlyR does not merely enhance the function of an existing channel. If such were the case, inhibitors of that channel would consistently reduce the N-K4-M2GlyR-induced secretion. 2) The exposed epitopes of N-K4-M2GlyR are substantially different from those exposed in C-K4-M2GlyR. Previously, Wallace et al. (18) reported that niflumic acid, NPPB, DPC, and glibenclamide significantly inhibited C-K4-M2GlyR-mediated ion transport, which is in stark contrast to the present results. It is possible that inhibitory binding sites are masked by NH2-terminal lysyl modification or that the lysyl adducts compose a portion of the binding site at the COOH, but not the NH2, terminus. Nonetheless, the COOH- or NH2-terminal modifications expose dramatically different interaction sites for pharmacological modulators.

It was somewhat surprising that bumetanide inhibited little of the N-K4-M2GlyR-mediated ion transport. Transepithelial Cl- secretion is thought to require the activity of the bumetanide-sensitive Na+/K+/2Cl- cotransporter in the basolateral membrane as a loading step. Thus one would expect a substantial bumetanide-induced reduction in Isc as cotransporter activity becomes rate limiting. Such results were observed for forskolin-stimulated Isc (Table 1) and were previously reported for C-K4-M2GlyR (18). One possibility to account for the bumetanide-insensitive current is that N-K4-M2GlyR is permeant to another anion, such as HCO<SUB>3</SUB><SUP>−</SUP>, that might substitute for Cl-. Physiological anion channels have been widely reported to conduct HCO<SUB>3</SUB><SUP>−</SUP>, and it has been reported that the ratio of Cl- to HCO<SUB>3</SUB><SUP>−</SUP> that is secreted depends on the identity and activity of basolateral components (5). Clearly, additional experiments are required to identify and characterize the basis of this secretory current.

Although introduction of an apparently unregulated pore into the apical membrane of epithelial cells might be expected to cause severe and detrimental effects to the cell, anion flux through this pathway is modulated by the permeability of the basolateral membrane. Addition of N-K4-M2GlyR to the apical surface of cultured epithelial cells results in a shift from the basal conductance to an increased conductance that is independent of the concentration of endogenous anion channels. Exposure to 1-EBIO increases the electrochemical driving force for Cl- conductance by activating basolateral K+ channels and hyperpolarizing the cell. This results in an increase in anion flow through channels in the apical membrane. This effect is much more pronounced in MDCK cells, which have a much lower level of expression of CFTR than T84 cells, which express high levels of CFTR. Thus potential exists for the modulation of anion secretion in persons with hyposecretory disorders, even in the absence of a functional CFTR. The effects of the anion-conducting peptide can be modulated by agents that alter the permeability of the basal membrane in epithelial cells.

Several lines of evidence indicate that cystic fibrosis (CF) may be ameliorated by the presence of an alternate Cl- channel. The CFTR knockout mouse does not develop lung disease because of the presence of a non-CFTR Cl- conductance (11). Severity of disease in the CF knockout mice varies in different organs and correlates with the presence of a non-CFTR Cl- conductance (4). Congenic CFTR knockout mice lacking a non-CFTR Cl- channel normally found in CFTR knockout mice with mixed genetic background develop severe lung disease (6). Residual Cl- secretion in CF patients correlates with preserved pancreatic function and delayed presentation of the disease (16). Thus introduction of an anion channel such as N-K4-M2GlyR into the apical membrane of airway or other epithelial cells holds therapeutic potential for CF patients.


    ACKNOWLEDGEMENTS

The authors are grateful to Ryan Carlin for technical assistance and to Dr. Janice Sargeant for statistical consultation.


    FOOTNOTES

This work was supported by National Institute of General Medical Sciences Grants GM-43617 (J. M. Tomich) and GM-19332 (K. E. Mitchell) and Cystic Fibrosis Foundation Grant SCHUL960 (B. D. Schultz).

This work is contribution 00-429-J from the Kansas Agricultural Experiment Station (J. M. Tomich and B. D. Schultz).

Address for reprint requests and other correspondence: B. D. Schultz, Dept. of Anatomy and Physiology, Kansas State University, 1600 Denison Ave., VMS 228, Manhattan, KS 66506 (E-mail: bschultz{at}vet.ksu.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 9 June 2000; accepted in final form 25 September 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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