Cryptdin 3 forms anion selective channels in cytoplasmic
membranes of human embryonic kidney cells
Gang
Yue1,
Didier
Merlin2,
Michael E.
Selsted3,
Wayne I.
Lencer4,
James L.
Madara2, and
Douglas C.
Eaton1
1 Center for Cell and Molecular Signaling and
2 Department of Pathology and Laboratory Medicine, Emory
University School of Medicine, Atlanta, Georgia 30322;
3 Department of Pathology, University of California Irvine,
Irvine, California 92697; and 4 Department of Pediatrics,
Harvard Medical School and Children's Hospital, Boston, Massachusetts
02115
 |
ABSTRACT |
Cryptdins are antimicrobial peptides
secreted by Paneth cells located at the base of intestinal crypts. In
addition to their antimicrobial function, cryptdins may also regulate
salt and water secretion by intestinal epithelial cells. Recent work
with short-circuit current measurements indicated that at least one
cryptdin peptide, cryptdin 3, induces apical conductance(s) in
Cl
secretory, including cystic fibrosis, epithelia. In
the present study, we characterized the cryptdin 3-induced anion
channel activity in human embryonic kidney (HEK) cells with
single-channel patch-clamp techniques. The patch pipette was filled
with solution containing different concentrations of cryptdin 3, and,
after gigaseal formation, the channel activity was recorded with either
cell-attached or inside-out patch modes. We found an anion selective
channel with a conductance of 15 pS and open probability of 0.19, regardless of cryptdin 3 concentration. The mean open and closed times
varied with the cryptdin 3 concentration. For cryptdin 3 concentrations of 10, 4, 1, and 0.5 µg/ml in the pipette, the corresponding mean open times were 1.2, 7.0, 9.0, and 17.4 ms and the corresponding mean
closed times were 1.1, 1.6, 4.2, and 12.5 ms. These results suggest
that cryptdin 3 forms anion-selective channels on the cytoplasmic
membrane of HEK cells and that the kinetics of one such channel are
affected by its interaction with other such channels.
patch clamp; chloride channels; defensins
 |
INTRODUCTION |
INTESTINAL SALT AND
WATER secretion depends on vectorial Cl
transport
across the epithelial cells lining the crypt and secretory glands of
the intestine (49). Cl
ions cross the
basolateral membrane through cotransporters, then they are secreted
into the intestinal lumen through Cl
channels in the
apical membrane. This transepithelial Cl
movement drives
Na+ and water across interepithelial tight junctions to
produce a secretory response. Movement of Cl
through
apical Cl
channels is the rate-limiting step for
Cl
secretion. Therefore, regulation of
Cl
-channel activity from crypt cells plays an important
role in intestinal fluid secretion.
The production of antimicrobial peptides such as defensins is an
important means of host defense against microbial invasion in
multicellular organisms (33). Cryptdins are defensins
produced by Paneth cells located in the small intestine at the crypt
base adjacent to the Cl
-secreting cells
(40). Mouse and human cryptdins are potent antimicrobial
agents especially to Escherichia coli, Listeria, and Salmonella (12). Six mouse defensins,
cryptdins 1-6, have been purified to homogeneity from the small
intestine (32, 40). In addition to their antimicrobial
function, cryptdins may also regulate the salt and water secretion in
intestinal epithelial cells. Recent work based on short-circuit current
measurements indicated that cryptdin 2 and 3 reversibly stimulate
chloride ion secretion when administered apically to T84 cells, a human intestinal epithelial cell line (22). This cryptdin
3-stimulated Cl
transport is not inhibited by
pretreatment with 8-phenyltheophyline or dependent on a concomitant
rise in intracellular cAMP or cGMP, indicating that cellular signaling
pathways that often stimulate Cl
transport are not
involved (22). Interestingly, it has been shown recently
that cryptdin 3 promotes a current in permeabilized cystic fibrosis
(CF) epithelial monolayers, demonstrating that cryptdin 3 induces a
restoration of Cl
secretion in CF cells that is probably
not cAMP or CF transmembrane conductance regulator (CFTR) dependent
(27). In addition, neutrophil defensins induce the
formation of an anion channel when incorporated into artificial
phospholipid bilayers (19). This activity may explain the
bactericidal effect of neutrophil defensins. Because cryptdins are
homologous in structure and function to defensins isolated from
mammalian phagocytic cells (31, 32, 40) and Cl
-secreting crypt cells are exposed to cryptdin released
from Paneth cells in vivo, the cryptdins may induce Cl
secretion by formation of anion channels in the cytoplasmic membrane of
secretory gland cells. In the present study, we characterize the
cryptdin 3-induced single-channel activity in HEK cells with cell-attached and inside-out patch-clamp techniques. We find that extracellular application of mouse cryptdin 3 induces anion-selective channels with a conductance of 15 pS and an open probability of 0.19 regardless of cryptdin concentration.
 |
MATERIALS AND METHODS |
Cryptdin synthesis.
Studies were carried out using synthetic, folded, and oxidized cryptdin
3 prepared using a protocol identical to that previously described for
the synthesis of cryptdin 4 (30). Synthetic and natural
cryptdin 3 peptides have been shown to have identical physicochemical
and antimicrobial characteristics (Ref. 39 and D. Tran and
M. E. Selsted, unpublished data).
Human embryonic kidney cells preparation, patch recording, and
data analysis.
Human embryonic kidney (HEK) cells were purchased from American Type
Culture Collection (Bethesda, MD). The HEK cells were grown in 35-mm
culture dishes in a humidified incubator gassed with 5%
CO2-95% air. The culture media contain 95% DMEM and 5% fetal bovine serum supplemented with 2 mM glutamine.
After 2-4 days of culture, a dish containing HEK cells was placed
on an inverted microscope for patch-clamp experiments. We used the
cell-attached and inside-out patch-clamp methods following standard
procedures. Patch pipettes were fabricated from TW 150 glass (World
Precision Instruments, New Haven, CT) and fire-polished to produce tip
resistances of 5-10 M
when filled with pipette solution.
Experiments were performed at room temperature (22-23°C). Patch
pipette solution contained (in mM) 150 NaCl, 2.8 KCl, 2 MgCl2, 1 CaCl2, and 10 HEPES (pH 7.4). For
cell-attached patch experiments, the bath solution had the same ionic
composition as that of the pipette. For inside-out patch experiments,
the bath was changed to a solution containing (in mM) 3 NaCl, 150 KCl,
1 MgCl2, 5 EGTA, and 10 HEPES. Cryptin 3 appeared to
interfere with seal formation. Therefore, to investigate the effects of cryptdin 3 on single-channel activity, the patch pipettes were filled
with regular pipette solution in the tip portion, then they were
backfilled with solutions containing various concentrations of cryptdin
3. In other experiments using similar electrodes backfilled with
blocking agents for channels, which we observed in other cells, we have
shown that it takes ~5 min for the tip to reach equilibrium. We
always waited 10 min before beginning recording. Single-channel events
were measured with an Axopatch 200 amplifier, low-pass filtered at 5 kHz, recorded on a digital video recorder with a bandwidth of 44 kHz
(Sony, Tokyo, Japan), and then filtered at 500 Hz and digitized at 2 kHz using a Scientific Solutions analog-to-digital converter and
Pentium computer equipped with Axotape software (Axon, Redwood City,
CA). The convention for applied voltage to the membrane patch
represents the voltage deflection from the patch potential (i.e., the
resting membrane potential for cell-attached patches). Outward anionic
current (cell to pipette) is represented as downward transitions in the
single-channel records. Small offset potentials associated with
reduction of chloride in the bath are subtracted from current-voltage records.
Data analysis.
Ten-minute data records were low-pass filtered at 500 Hz using a
software Gaussian filter and analyzed using pCLAMP 6.03 (Axon). Amplitude histograms were generated to obtain unit current values. Although patches containing only a single channel are ideal for performing kinetic analysis, many patches contain multiple opening channels but can be analyzed using previously reported methods (26, 34). In this case, activity of a single channel
(NPo) and open probability
(Po) of a single channel is given by
|
(1)
|
where T is the total recording time,
NA is the apparent number of channels within the
patch, determined as the highest observable current level, i
is the number of channels open, and ti is the time during which i channels are open. We
used this method for calculating NPo
because the calculations were facilitated by having the event
tables generated by pCLAMP. If channels
are uniformly distributed in the membrane and the exact number of
channels in a patch is known, then the mean Po
of one single channel can be calculated by dividing
NPo by the number of channels in a patch. The
total number of functional channels (N) in the patch was
determined by observing the number of peaks detected in all-points
amplitude histograms. The histograms were constructed from event
records of long enough duration to provide 95% confidence of
determining the correct N according to methods we have
previously described (20, 26). The mean open time
(to) of one of the N channels can be
calculated as
|
(2)
|
where n is the total number of transitions between
states during T and the other parameters are the same as in
Eq. 1. This value represents the average time the channel
spends open (in any open state) and should not be confused with the
mean residency time of the channel in a specific state [sometimes
called the mean open time (to) for the state].
Alternatively, in patches with multiple channels, we determined the
most likely value for N and then calculated values for
to by measuring the duration of the events in
which all N channels were open (tN)
and remembering that to = tN × N. The difficulty with this
method is the assumption that N can be determined accurately
and that there are enough events with all channels open to obtain a
meaningful distribution of intervals. To ensure the accuracy of this
approach, we only used patches for which we had a >95% probability of
having estimated the number of channels correctly [following the
methods of Marunaka and Eaton (26) or in which
to can be determined unambiguously (patches with
no overlapping open events)]. Either measure we used to calculate
to provided an easy way to distinguish whether experimental manipulations (e.g., cryptdin 3) modify
Po by affecting the channel's open or closed states.
Besides the method described above to calculate
Po, in theory, one could also calculate
Po according to
|
(3)
|
However, in general, the variability in the two parameters, mean
open and closed times, produces an estimate for
Po that tends to have a large error (for an
example of the problem, see Ref. 46). For the cryptdin
channels described in this work, the problem is exacerbated by
two properties of the channels. First, at high cryptdin concentrations,
the mean open and closed times are short, making determinations of the
mean values from interval histograms somewhat inaccurate, although we
used methods described elsewhere to improve the accuracy (4,
41). Second, there is a large cell-to-cell variability in mean
open and closed times. However, the variability in mean open and closed
times appears to be on a cell by cell basis (see Fig. 8); that is, if mean to is short, mean closed time is also
short. This means that estimating Po from
NPo is more accurate than calculating
Po from the mean open and closed times.
Statistics.
The data were presented as means ± SD. Student's
t-tests were used as appropriate to compare experimental
groups. For more than one group, one-way ANOVA was used. For comparison
of fits, a
2-test was used. Results were considered
significant when P was <0.05.
 |
RESULTS |
A recent report suggests that cryptdin 3 may induce anion
permeability in T84 cell monolayers (22). However, T84
cells express large numbers of several different types of
Cl
channels. Therefore, it might be difficult to
determine whether cryptdin 3 induced new channels or activated
preexisting ones. On the other hand, HEK cells express few endogenous
channels of any type, and no anion channels have been reported (see
DISCUSSION). Therefore, we used HEK cells to determine
whether cryptdin 3 could form anion channels. As shown in Fig.
1, there was no single-channel activity
when cryptdin 3 was absent from the pipette solution (and none in 142 additional untreated patches). Application of 10 µg/ml cryptdin 3 in
the pipette elicited multiple channel openings with very rapid
transitions between open and closed states. As cryptdin 3 concentration
increased, the number of channel openings increased and open and closed
times decreased (as summarized in Table 1). The relationship between
the cryptdin 3 dose and the number of channels is shown in Fig.
2. At cryptdin 3 concentrations higher
than 10 µg/ml, it was not possible to count the number of channels
and patches were not stable so that it was not possible to determine
whether there was a saturating dose of cryptdin 3. Figure
3 illustrates the current-voltage
relationship (I-V) from inside-out patch
experiments. This result shows that formation of cryptdin 3 channels
does not require any cytoplasmic components and does not involve
changes in cell volume. The Po of the channels was not voltage dependent, and the channels did not rectify. A conductance of 15 pS was obtained from the slope of the I-V
curves. In five experiments, when part of the Cl
in the
pipette was replaced with 120 mM gluconate, a nonpermeable anion, the
reversal potential shifted from 0.2 ± 1.6 to 39.6 ± 2.9 mV,
close to the calculated equilibrium potential for Cl
(+42 mV). This indicated that cryptdin 3 induced channels that were
strongly selective for anions over cations in HEK cells.

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Fig. 1.
Records in which transitions of single cryptdin channels are
visible from cell-attached patches with different concentrations of
cryptdin 3 in pipette solution. Bath and pipette solutions contained
patch pipette solution, which contained (in mM) 150 NaCl, 2.8 KCl, 2 MgCl2, 1 CaCl2, and 10 HEPES (pH 7.4).
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Fig. 2.
The relationship between the cryptdin 3 dose and the
number of channels. At cryptdin 3 concentrations >10 µg/ml, it was
not possible to count the number of channels, and patches were not
stable so that it was not possible to determine whether there was a
saturating dose of cryptdin 3.
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Fig. 3.
Current-voltage relation of cryptdin 3-induced anion channels from
inside-out patch recordings. For recording conditions with almost equal
chloride in the bath and pipette, the reversal potential in 5 experiments was 0.2 ± 1.6 mV [ ; bath contained
(in mM) 3 NaCl, 150 KCl, 1 MgCl2, 5 EGTA, and 10 HEPES; and
pipette contained (in mM) 150 NaCl, 2.8 KCl, 2 MgCl2, 1 CaCl2, and 10 HEPES, with 0.4 µg cryptdin]. When the
chloride concentration in the pipette was reduced, the reversal
potential shifted to 39.6 ± 2.9 mV in a manner expected for a
chloride permeable channel [ : bath contained (in mM) 3 NaCl, 150 KCl, 1 MgCl2, 5 EGTA, and 10 HEPES; and pipette
contained (in mM) 30 NaCl, 120 Na+ gluconate, 2.8 KCl, 2 MgCl2, 1 CaCl2, and 10 HEPES, with 0.4 µg
cryptdin 3]. Lines are the best-fit linear least squares regression
line through the data points.
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The mean open and closed times of these channels increased with
decreasing concentration of cryptdin 3 in the pipette (Fig. 4). Examination of the open and closed
interval histograms revealed only a single exponential component of
each histogram. However, despite the changes in open and closed times,
there is no significant variation of the Po at
different concentrations of cryptdin 3 in pipettes (Fig.
5). These results suggest that the
kinetics of one channel in the membrane is modified by its interaction with other cryptdin 3 molecules.

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Fig. 4.
Mean open and closed times of cryptdin 3-induced single
anion channel activity vary with cryptdin 3 concentration. Mean open
and closed times were dependent on the cryptdin 3 concentrations in the
pipette solution. Both open time and closed times decreased with
increasing cryptdin 3 concentrations from 0.5 to 10 µg/ml.
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Fig. 5.
Single channel open probability
(Po) was not affected by the cryptdin 3 concentrations in the pipette solution. Despite changes in mean open
and closed times, Po remained near 0.2 when
cryptdin 3 concentration was varied from 10 to 0.5 µg/ml.
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 |
DISCUSSION |
In this study, we report that cryptdin 3 induces an
anion-selective channel in the cytoplasmic membrane of HEK cells.
HEK293 cells were originally described in 1990 (42, 43).
We used HEK293 cells because they have few endogenous ion channels and are often used for heterologous expression of cloned ion channels. By
using these cells, we hoped to eliminate the possibility that cryptdin
3 was activating an endogenous chloride channel. In 823 PubMed
references to HEK293 cells, only four describe any endogenous channels,
and these are all cation channels. However, more information about
endogenous channels is available from expression studies in which
investigators often establish that there are no endogenous channels of
a specific type before transfecting and expressing cloned channels. In
this context, several chloride channels (CLC; CLC-0, CLC-1, CLC-2,
CLC-4, CLC-5, CLC-6, and CLC-7) cannot be detected in HEK293 as protein
(by Western blotting), as mRNA (by RT-PCR), or functionally (by patch
clamp) (3, 8, 9, 14-17, 24, 35, 37, 44, 45,
48). In addition, unit conductance, single-channel
kinetics, and voltage dependence of these channels as well as CLC-3 are
all different from the cryptdin channels we describe in this work
(3, 16, 17, 24, 36, 37, 45). Thus all known members of the
CLC family are unlikely candidates for activation by cryptdin 3 unless
the peptide is capable of changing virtually every biophysical
characteristic of the channel.
Rabbit neutrophil defensin NP3A stimulates a calcium-dependent
volume reduction in villus enterocytes (25). This cell
shrinkage is prevented by 9-anthracenecarboxylic acid, a
Cl
channel blocker. The results might suggest that NP3A
activates Ca2+-dependent Cl
channels in
enterocytes, but this activation could easily be due to an
NP3A-mediated increase in the membrane permeability to calcium that
indirectly activates calcium-dependent chloride channels. However, it
is unlikely that cryptdin 3 activates Ca2+-dependent
Cl
channels in HEK cells, because such channels have
never been reported in HEK cells (in the 893 references mentioned
above). Moreover, cryptdin 3-induced channels have very different
biophysical properties (e.g., no voltage dependence and a very
different conductance) from those of Ca2+-dependent
Cl
channels (2, 11). Also, cryptdin can
induce channels in excised patches when the calcium concentration on
the cytosolic surface is well below the level necessary for
calcium-dependent chloride channel activation.
CFTR, a cAMP-activated anion channel with a conductance of 7-10
pS, also does not appear to be a candidate for a cryptdin 3-induced
channel, because the biophysical properties are different (5,
15). Moreover, cryptdin 3 still induces a current in basolaterally permeabilized epithelial monolayers derived from airway
cells with a nonfunctional F508 mutation of CFTR (27), and
cryptdin 3 does not increase intracellular cAMP (22),
which is necessary for CFTR activation.
Volume-sensitive anion channels, which are stimulated by extracellular
hyposmolality, are broadly expressed in animal cells (47).
They are also poor candidates for cryptdin 3-induced channels, because
they are all voltage dependent, outwardly rectifying channels (6,
10, 50) with unit conductances in excess of 60 pS
(29). Moreover, there was no alteration of osmolality
during our patch-clamp recording in HEK cells, making activation of
such channels unlikely. Also, cryptdin channels appear in excised
patches, a condition under which volume-sensitive channels are not active.
Therefore, cryptdin 3 channels in HEK cells do not appear to be
associated with any of the major categories of anion channels often
present in animal cell membranes.
The remaining possibility is that cryptdin 3 forms new anion channels
in the cytoplasmic membrane. Previous studies show that a rabbit
neutrophil defensin (NP-1) forms ion channels in a planar lipid bilayer
membrane (19). This channel is voltage dependent and
weakly anion selective with heterogenous single-channel conductance ranging from 10 to 1,000 pS. Because the structure of NP-1 is similar
to cryptdin 3, it is possible that cryptdin 3 might also form new
membrane channels. However, the biophysical properties of NP-1 channels
are very different from those we observed for cryptdin 3-induced anion
channels: cryptdin 3 channels are voltage-independent, highly
anion-selective channels with a constant conductance of 15 pS. The
differences could be due to differences between incorporation in native
cell membranes rather than artificial bilayer membranes or from
intrinsic differences in amino acid structure between the two
defensins. The second explanation is supported by the recent study
showing that cryptdin 3 induces an apical conductance that is voltage
independent in basolaterally permeabilized epithelial cell monolayers
(T84) (27). Defensins are cationic molecules with
spatially separated hydrophobic and charged residues (51). This arrangement allows their hydrophobic regions to be buried within
interior apolar regions of membrane lipid and their cationic regions to
interact with the anionic polar head group of phospholipid and water so
that the defensin molecules can incorporate into cytoplasmic membranes.
Beyond the primary structure, defensins are folded to form antiparallel
-sheets, which are stabilized by three disulfide bonds (13,
40). Despite these general structural similarities among the
defensin peptide family, mouse cryptdin 3 and rabbit NP-1 differ in
amino acid sequence. Figure 6 shows the
sequences of these two peptides. There are a number of structural differences between NP-1 and cryptdin 3. By analogy with the neutrophil defensins HNP-1, HNP-3, NP-2, and NP-5, amino acids at positions 10 and 15 are predicted to be located at conserved
turns on the surface of the molecule (31). Positively
charged Arg-11, Lys-14, and Arg-15 in cryptdin 3 are replaced by
neutral residues Ala, Leu, and Pro in the corresponding positions in
NP-1. Positive charges in the turn regions are critical for defensin
molecules to interact with negatively charged polar head groups of
membrane lipid. Thus such positive charges might logically favor the
anion selectivity of cryptdin 3 channels compared with NP-1 channels. The extra positive charges on cryptdin 3 molecules might also produce a
different peptide stoichiometry for the channel than NP-1, thus leading
to the conductances which differ. Alternatively, the longer
NH2 terminus of cryptdin 3 compared with NP-1 could contribute to differences in stoichiometry and gating.

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Fig. 6.
Amino acid sequences of mouse cryptdin 3 and rabbit defensin
(NP-1). Primary structures are shown in single-letter amino acid code.
Charged residues are marked + or as positive or
negative charge. Three disulfide bonds are indicated as line
connections.
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|
In our work, we observe cryptdin-induced channels that are remarkably
selective for chloride over sodium or potassium, but we have not
examined the permeability sequence for different anions. Nonetheless,
the anion-to-cation selectivity is maintained at all of the cryptdin
concentrations we have examined. Although the concentrations we
examined tend to be lower than those used by other investigators, our
results are still in marked contrast to reports that, after exposure to
higher cryptdin concentrations, cells become much more permeable and
the cryptdin-induced permeability becomes much less selective. For
example, at high concentrations, cryptdin 3 increases the permeability
of T84 cells to even allow entry of the usually membrane-impermeant
fluorophore
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein acid
(22). This observation appears to imply that larger, less selective pores are formed at high cryptdin 3 concentrations. However,
an alternative explanation, consistent with the actions of other
pore-forming peptides, is that as the cryptdin concentration and the
number of cryptdin channels increase, the selectivity of the channel
remains unchanged. However, when the concentration is high enough,
there is a generalized disruption of the cell membrane and cell
function that leads to a increased nonspecific permeability of the cell
membrane to large molecules even though the cryptdin pore is still only
permeable to anions. This idea is consistent with the actions of a
variety of pore-forming agents such as filipin, nystatin, and
amphotericin (7, 21, 23, 28, 38), which form pore
complexes with fixed properties but at high concentrations disrupt
cellular membranes.
In brief, the present studies show that cryptdin 3 is, at the least,
associated with and is likely capable of forming anion channels in the
cytoplasmic membranes of HEK cells. Thus cryptdin 3 in vivo, after
release from Paneth cells at the base of the crypt, likely forms
channels in the apical membrane of adjacent glandular cells. Such
channel formation would lead to salt and water secretion, causing the
crypt lumen to be flushed after Paneth cell discharge, removing
bacteria as well as diluting the cryptdin 3 concentration to levels
below the effective cytotoxic concentration for epithelial cells in the
crypt lumen. Meanwhile, cryptdin 3 channels could also be removed from
the apical membrane by partioning into the aqueous phase or by
endocytosis. This balance between luminal cryptdin 3 concentrations
sufficient to induce Cl
secretion but below the cytotoxic
range coupled with membrane recycling of cryptdin 3 channels may serve
to protect crypt epithelial cells from possible cytotoxic effects while
delivering these antimicrobial peptides to pathogen cell surfaces at
high enough concentrations in the crypt or within the gut lumen itself
to promote lysis of the bacterial cells.
 |
APPENDIX |
A Potential Mechanism for Cryptdin 3 Channel Formation
Presently, there is not enough information available to propose
a complete structural model of cryptdin channels; however, some
conclusion can be drawn about the mechanism of the interaction of
cryptdin 3 with the membrane. Of course, other small molecules produced
in eukaryotic cells can form pores. For example, a 27-amino-acid fragment of the so-called minK potassium channel forms a cation pore by
forming a homotetramer (1, 18). Nonetheless, it seemed unlikely to us that one cryptdin 3 peptide could form a channel, because the size of the molecule (~4 kDa) would appear to preclude a
single peptide from having the multiple membrane spanning domains usually required for channel formation. In an attempt to gain more
information about this issue, we reexamined the dose-response data of
Fig. 2. If n peptide molecules in the aqueous phase are necessary to form one channel in the membrane according to the following reaction
|
(A1)
|
then at equilibrium
|
(A2)
|
and
|
(A3)
|
Therefore, in a plot of the log of the number of channels vs.
the log of the cryptdin 3 concentration, the slope will be equal to the
number of cryptdin 3 molecules forming a channel and the value at a
cryptdin 3 concentration of 1 {i.e., log [cryptdin concentration] = 0} will be the log of the equilibrium distribution (log
Keq) of cryptdin 3 molecules between the aqueous
and membrane phases. An examination of such a plot (Fig.
7) shows that, surprisingly, the slope is
1.07 ± 0.094 and the value at a cryptdin concentration of 1 (the
log of equilibrium constant) is 0.734 ± 0.133, implying that only
one cryptdin 3 molecule is required to form a channel and that cryptdin
3 molecules are about fivefold more likely to reside in the membrane as
a channel as in the aqueous phase.

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Fig. 7.
A plot of the log of the number of channels vs. the log
of the cryptdin 3 concentration. In this plot, the slope will be equal
to the number of cryptdin molecules forming a channel, and the value at
a cryptdin 3 concentration of 1 (at which log concentration is 0) will
be the log of the equilibrium distribution of cryptdin 3 molecules
between the aqueous and membrane phases. An examination of the plot
shows that the slope is 1.07 ± 0.094 and the intercept is
0.734 ± 0.133, implying that only one cryptdin 3 molecule or only
one cryptdin 3 molecular complex in solution is required to form a
channel and that cryptdin 3 molecules are ~5-fold more likely to
reside in the membrane as a channel as in the aqueous phase. The means
include patches with 0 channels for lower cryptdin concentrations,
because the patches with no channels also reflect the probability that
cryptdin at low concentration will enter the membrane.
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|
An alternative method for approaching the issue of the distribution of
cryptdin molecules depends on the observation that the open- and
closed-interval histograms for cryptdin channels indicate that there is
only one open and one closed state of the channel. Also, once we
observed activity, we never subsequently observed a loss of activity
before seal breakdown (usually ~30-40 min). If the channel in
the membrane was changing from a closed to an open conformation and, in
addition, was partioning and departioning between membrane and aqueous
phases, we would expect two components to the closed-interval histogram
or we would expect occasional loss of activity. Therefore, the observed
pattern is only consistent with two possibilities. The first is that
the channel partitions into the membrane and never partitions out. This
seems a thermodynamically unlikely possibility. The second is that the
transitions actually represent partitioning and departitioning of
channels, in which case we would expect only one component of the
closed- and open-interval histograms (as we see) and the reciprocal of
the closed time is the partitioning rate and the reciprocal of the open
time is the departioning rate. A calculation of the equilibrium
partition constant from the mean open and closed time gives a value of
2.78 ± 1.56, which is not significantly different from the value
of 5.42 ± 2.06 obtained from Fig. 7.
However, these results do not necessarily imply that only one peptide
forms a channel. The alternative explanation is that the peptides can
only insert into the membrane as a multimer of fixed stoichiometry. On
the basis of a variety of approaches including the crystal structure
and nuclear magnetic resonance measurements, investigators have
reported that some defensins appear to form a dimer in aqueous phase
(13, 51). If cryptdin forms a dimer, the dimeric
channel would appear to have a structure that might be consistent with
the formation of an aqueous pore (13). If cryptdin 3 were
either present in aqueous solution or at least inserted into the
membrane as a dimer, then it would be possible to explain the
first-order power dependence of the number of channels on cryptdin 3 concentration and still invoke a multimeric channel.
It is not surprising that increasing cryptdin 3 concentrations increase
the channel density (number of channels per patch). Some investigators
have suggested that as the concentrations of some defensins increase,
the number of peptides that form a channel also increases
(51). In these cases, the conductance increases with
increasing cryptdin concentration, and the selectivity decreases (implying a larger pore). However, for cryptdin 3, the fact that the
unit conductance and even the Po do not change
with increasing cryptdin 3 concentration implies that the stoichiometry
of a cryptdin 3 channel, once formed, is constant. Nonetheless, when
there are a large number of channels in the membrane, the properties of channels do change: both mean open and closed times decrease with increasing cryptdin 3 concentration. Despite a large variability in the
mean open and closed times from cell to cell, in any given cell, the
mean open and closed times are correlated; i.e., if the mean open time
is short, the mean closed time is also (Fig. 8). Thus the ratio of the mean open to
closed time remains close to the same so that Po
does not appreciably change. This result also implies that the ratio of
partitioning to departioning of cryptdin into and out of the membrane
does not change. The nature of this interaction can best be understood
by examining the properties of a simple kinetic model that describes
the properties of cryptdin 3 channels once a cryptdin molecule in
aqueous solution has entered the membrane to form a channel.
|
(A4)
|
The transition from a closed to an open state represents a
conformational change of the cryptdin 3 molecule. Such a change requires a certain amount of energy, and the mean duration in the open
or closed state is proportional to the amount of energy required to
cause a transition. Likewise, the rate of transition from one state to
another is inversely proportional to the mean duration and inversely
proportional to the transition energy. A decrease in mean duration can
only mean that the energy barrier between the two states is uniformly
decreased. Therefore, addition of extra cryptdin 3 molecules does not
alter the fact that functional conducting pores are present at both low
and high cryptdin 3 concentrations and that they have the same
selectivity, conductance, and Po. It does mean,
however, that at high concentrations, functional cryptdin molecules in
the membrane must interact and that this interaction reduces the
transition energy (and mean durations for closed and open states) for
the interacting cryptdin molecules. The power relationship
(t = aNb; where t is the open or
closed time, N is the number of channels, and a
is the value when N = 1) between the number of channels and the mean open and closed times varies as approximately the first
power of the number of channels (Fig. 9,
slope =
1.00 ± 0.376 with r = 0.72 for
open times and
0.958 ± 0.193 with r = 0.69 for
closed times) and close to the first power of the cryptdin 3 concentration (slope =
0.796 ± 0.0931 for open times and
0.708 ± 0.122 for closed times). Adding additional power
components does not improve the goodness of fit (based on
2 analysis). Cryptdin 3 (10 µg/ml) produces a
flickering channel with shorter open and closed durations. Five
micrograms per milliliter or less are required to observe clear
single-channel records. This result is consistent with the idea that a
single cryptdin molecule (regardless of its stoichiometry) can form a
pore but that two molecules in the membrane can associate, and when
they do associate, the rate of transitions between the open and closed states is increased. This result is also consistent with the finding that low (5 µg/ml) but not high concentrations (50 µg/ml) of NP-1 are necessary to observe a stable unitary conductance in artificial lipid bilayers (19). The formation of a multimeric channel
depends on the interaction among cryptdin 3 peptides, which is related to their concentration. Thus the kinetics of one channel are affected by interaction with other cryptdin 3 molecules.

View larger version (12K):
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|
Fig. 8.
Mean open and closed times are correlated. Plotting all
mean open times as a function of closed times yields a linear
relationship with an r value of 0.804. Despite a large
variability in the mean open and closed times from cell to cell, in any
given cell, the mean open and closed times are correlated; i.e., if the
mean open time is short, the mean closed time is, also. Thus the ratio
of the mean open to closed time remains close to the same so that
Po does not appreciably change as cryptdin
concentration increases.
|
|

View larger version (15K):
[in this window]
[in a new window]
|
Fig. 9.
Plots of the log of the number of channels vs. the log of
the cryptdin 3 channel mean open and closed times. In these
plots, the slope will be equal to the average number of cryptdin
complexes in the membrane that interact to alter mean open and closed
times. An examination of the plot shows that the slopes are 1.10 ± 0.082 for open time and 0.958 ± 0.193 for closed time,
values not significantly different from 1. This result is consistent
with the idea that a single dimer can form a pore but that 2 dimers in
the membrane can associate, and when they are associated, the rate of
transitions between the open and closed states is increased.
|
|
 |
ACKNOWLEDGEMENTS |
We thank B. J. Duke for maintaining the cells in culture.
 |
FOOTNOTES |
This work was supported by R01 AI-22931 and the Large Scale Biology (to
M. E. Selsted), P01 DK-33506 (to J. L. Madara), K01 DK-0283
(to D. Merlin), R01 DK-48106 to (W. I. Lencer), R01 DK-56305, P01
DK-50268, and R01 DK-37963 (to D. C. Eaton), and the Center for
Cell & Molecular Signaling.
Address for reprint requests and other correspondence: D. C. Eaton, Center for Cell and Molecular Signaling, Emory Univ. School of Medicine, 615 Michael St., Atlanta, GA 30322 (E-mail:
deaton{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.
First published February 20, 2002;10.1152/ajpgi.00152.2001
Received 10 April 2001; accepted in final form 10 January 2002.
 |
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