Departments of 1 Biochemistry and 2 Anatomy and Physiology, Kansas State University, Manhattan, Kansas 66506
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ABSTRACT |
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A synthetic,
channel-forming peptide, derived from the -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
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INTRODUCTION |
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PREVIOUS REPORTS HAVE
SHOWN that a 23-residue peptide based in the second transmembrane
segment (M2) of the -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.
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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).
-Hydroxymethylphenoxymethyl resin preloaded with the COOH-terminal
amino acid was purchased from Perkin-Elmer, and
N
-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
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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 M
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.
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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 · 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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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
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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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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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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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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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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DISCUSSION |
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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
, that
might substitute for Cl
. Physiological anion channels
have been widely reported to conduct HCO
, and it has
been reported that the ratio of Cl
to
HCO
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.
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ACKNOWLEDGEMENTS |
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The authors are grateful to Ryan Carlin for technical assistance and to Dr. Janice Sargeant for statistical consultation.
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FOOTNOTES |
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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.
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