Cation-halide transport through peptide pores containing aminopicolinic acid

Article abstract of DOI:10.1039/c6ob00592f

Synthetic pores that selectively transport ions of biological significance through membranes could be potentially used in medical diagnostics or therapeutics. Herein, we report cation-selective octapeptide pores derived from alanine and aminopicolinic acid. The ion transport mechanism through the pores has been established to be a cation-chloride symport. The cation-chloride co-transport is biologically essential for the efficient functioning of the central nervous system and has been implicated in diseases such as epilepsy. The pores formed in synthetic lipid bilayers do not exhibit any closing events. The ease of synthesis as well as infinite lifetimes of these pores provides scope for modifying their transport behaviour to develop sensors.

Full text of DOI:10.1039/c6ob00592f

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Cation-Halide transport through Peptide Pores containing
Aminopicolinic Acid
Debajyoti Basaka, Sucheta Sridharb , Amal. K. Berab and Nandita Madhavana
Received 00th January 20xx,
Accepted 00th January 20xx
Synthetic pores that selectively transport ions of biological significance through membranes could be potentially used in
medical diagnostics or therapeutics. Herein, we report cation-selective octapeptide pores derived from alanine and
aminopicolinic acid. The ion transport mechanism through the pores has been established to be cation-chloride symport.
Cation-chloride co-transport is biologically essential for the efficient functioning of the central nervous system and has
been implicated in diseases such as epilepsy. The pores formed in synthetic lipid bilayers do not exhibit any closing events.
The ease of synthesis as well as infinite lifetimes of these pores, provides scope for modifying their transport behaviour to
develop sensors.
DOI: 10.1039/x0xx00000x
www.rsc.org/
symporters have been previously reported from macrocycles
such as crownethers, azacrownethers, calix[4]pyrrole or
oxacalix[2]arene[2]triazine.30-34 Given the fact that our cation-
selective pores show infinite lifetimes, provides an additional
scope for using them as scaffolds for developing sensors.
Introduction
Synthetic peptides capable of biomimetic ion transport are
emerging as useful materials or therapeutics.1-6 In particular,
molecules designed to mimic the ion-selectivity and/or gating
ability of natural ion channel proteins or carriers are extremely
useful.7-15 Pores such as alpha hemolysin that do not show a
transition between open and close states, have been utilized
as biosensors by genetic engineering or incorporation of
adapters.16-23 There are few examples of synthetic pores that
show open states with infinite lifetimes.24,25 Such pores, if
readily accessible are highly attractive for use as materials.
Furthermore, if these robust pores are able to selectively
transport ions, one can envision using them in therapeutics,
diagnostics or as model systems to standardize ion transport
assays.
Chart 1. Pore forming peptides
Previously reported peptides 3-5 from our research group
Reported herein, are pore forming acyclic octapeptides
1
selectively transported cations and were impervious to halides
and
2 derived from alanine and aminopicolinic acid. The
(Figure 1).35-37 Replacement of aromatic units in peptide
peptides transport cations via an M+/Cl⁻ symport mechanism
and do not show any closing events. Cation-chloride
symporters play a crucial role in maintaining homeostasis and
transmembrane chloride potential in neurons in the body.
scaffold
significantly enhance its cation transport activity.37 Inspired by
these observations, we designed peptide that incorporates
aminopicolinic acid units into our most active peptide scaffold
that we believe forms tetrameric pores.35,36 We envisioned
that the pyridyl rings would significantly enhance the cation
transport activity of peptide . Peptide containing an
aromatic unit at the C-terminus was designed based on our
4 to amino methyl pyridyl units was shown to
2
These symporters are associated with diseases such as
epilepsy and renal acidosis.
26-29
3
Synthetic cation-chloride
2
1
a.Department of Chemistry, Indian institute of Technology, Madras, Chennai-
600036, India.
prior hypothesis that peptide
3 formed tetrameric pores with
b.Department of Biotechnology, Indian institute of Technology, Madras, Chennai-
600036, India.
the peptide C-termini at the lipid-water interface.35 We
believed that the closer proximity of the aminopicolinic acid
c. † Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [Detailed experimental units to the lipid-water interface could enhance its influence
procedures, characterization of compounds, analysis of fluorescence data,
microscopy data, dynamic light scattering data]. See DOI: 10.1039/x0xx00000x
on the ion transport properties of the pore. At the outset, we
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had not expected these peptides to co-transport cations and Vesicles entrapped with pH sensitive HPTS dye38-40
View ArticlewOnelirnee
DOI: 10.1039/C6OB00592F
halides.
prepared in HEPES-NaCl buffer to investigate ion transport
through peptides and . The vesicles were equilibrated with
1
2
a)
b)
the peptide, following which NaOH was added to increase the
external pH by 0.6 units (Figure 2a). Ion transport was gauged
by monitoring an increase in the concentration of the
deprotonated HPTS⁻ due to Na+/OH⁻ symport, Na+/H+ antiport
or OH⁻/Cl antiport. The fluorescence intensity was normalized
based on the concentration of HPTS⁻ obtained after adding the
detergent Triton X. Surprisingly, a negligible increase in the
concentration of HPTS⁻ in the presence of peptides
1 and 2
was observed after the base pulse (Figure 2b). Rate constants
obtained by fitting the curves obtained (with the peptides) to
first order exponential equations were marginally higher than
the control experiments. Such a drastic decrease in the ion
transport activity due to replacement of aminobenzoic acid36
by aminopicolinic acid units was extremely intriguing.
Figure 1 a) Basic design and comparison of peptide derived pores; b) schematic
representation of tetrameric pore formed by peptide 3.
Results and discussion
Octapeptide
1), which is obtained in three steps from 2-amino-6-picoline.
Amine was coupled with Boc-Alanine using HCTU to form
dipeptide 7 in 75% yield. Boc deprotection and subsequent
1 was synthesized starting from amine 6 (Scheme
a)
b)
6
coupling with Boc-
70% yield. Tetrapeptide
peptides and 10, which were coupled to afford octapeptide
in 77% yield. Similarly, octapeptide
L
-Ala-
D
-Ala-OH afforded tetrapeptide
was selectively deprotected to get
8
in
8
9
1
2
was synthesized in
solution from dipeptide
7
as shown in Scheme 2.
Scheme 1. Synthesis of peptide
1
Figure
2
a) Schematic representation of the HPTS assay. b) Change in HPTS⁻
concentration after NaOH pulse until addition of Triton X (i.e. between Step 2 to step 3
in the schematic represention). 20.4 μM = 5 mol % peptide with respect to lipid.
Average k values reported in the legend. Origin 8.1 was used to fit the curves.
To investigate whether the peptides transport halides across
lipid membranes, vesicles entrapped with lucigenin dye were
prepared. The fluorescence of lucigenin is quenched in the
Scheme 2. Synthesis of peptide
2
presence of halides (Figure 3a).41 In contrast to peptide
3,
which was impervious to halides, peptides
1 and 2 were
transporting Cl⁻, Br⁻ (Figure 3b) and I⁻ (Figure S7). The results
from the lucigenin assay indicated that the presence of the
aminopicolinic acid units increased the affinity of the peptides
for halides.
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DOI: 10.1039/C6OB00592F
a)
b)
c)
Figure 4. A) Current traces of peptides 1 and 2 recorded at -80 mV. The current traces
shown besides ‘0 pA’ is the base line current at 0 mV holding potential. B) I-V Plots of
Peptides 1 and 2. The currents generated in symmetric solution of 1 M KCl are shown
with black line, while the red line represents currents recorded in 0.5M:1M KCl
(cis:trans) gradient. Cis chamber was grounded.
Figure
3
a) Schematic representation of Lucigenin assay. Change in lucigenin
fluorescence after addition of peptides (i.e. between step 2 and step 3 of schematic
representation) in the presence of b) NaCl; c) NaBr. 20 μM = 5 mol % peptide with
respect to lipid.
The lucigenin assay and the conductance measurements
showed that peptides
1 and 2 transported cations as well as
halides, with cation transport being the driving force. This
observation could be explained by cation transport through
the peptides via an M+/Cl⁻ symport mechanism. The symport
mechanism could also explain the minimal change in
intravesicular pH for the HPTS assay with an NaOH pulse
(Figure 1b). The HPTS assay uses change in pH as an indirect
probe to study ion transport. As suggested by the conductance
studies, if the peptides are cation-selective, one can envision
the system maintaining charge neutrality via the mechanisms
shown in Figure 5 (for a HEPES:NaCl buffer). A dominant
Na+/Cl- symport mechanism as hypothesized will lead to
negligible change in the internal pH of the vesicle as observed.
To gain further insights into the ion transport mechanism of
peptides
1 and 2, the two peptides were incorporated in lipid
bilayers (composed of DPhPC – 20 µL, 23.6 mM). Incubation of
40 µL of peptide (0.125 mM) leads to channel formation.
Channel incorporation was observed every time the
experiment was conducted. The membrane currents increased
linearly, proportional to the applied voltages. The current
traces recorded at -80 mV holding potential are shown in
Figure 4a. No single channel opening and closing events or
gating was observed, indicating that the peptides possibly
formed pores. The reversal potential was estimated
experimentally by creating an ionic gradient (0.5M:1M KCl in
cis:trans side respectively with the cis side being held in virtual
ground) across the bilayers. Currents were measured at
different voltages using step protocol, and current-voltage (I-
V) plots were generated. The reversal potential was found to
be 17.9 mV for Peptide 1 and 21.6 mV for Peptide 2 (Figure 4b)
both of which were close to the theoretical reversal potential
of K+ ions (17.8 mV). This suggests that the peptides
preferentially transport cations over anions.
Figure 5 Schematic representation explaining minimal pH change in HPTS assay.
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To confirm the M+/Cl⁻ symport mechanism, counter cations ion transporting pore, it indicates that these peptides have a
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(M+ = Na+, K+, Ni2+, Co2+) to chloride were varied in the propensity to form stable nano-assembDliOesI:.1T0.h10e39c/oCn6dOuBc0t0a5n9c2eF
lucigenin assay. In this assay where the vesicles are prepared studies corroborate the hypothesis that the peptides form
in a solution of NaNO3, likely ion transport mechanisms are stable pores as they show infinitely open lifetimes. Low ion
M+/Cl⁻ symport and Cl⁻/NO3⁻ antiport (Figure 6a). Chloride transport for the HPTS assay rules out non-specific ion
transport was only observed with alkali metal ions and not transport due to detergent like behaviour of peptides
1 and 2.
with Ni2+ and Co2+ that exist as large pore impermeable Furthermore, dynamic light scattering studies before and after
[M(H2O)6]2+ complexes (Figure 6b and S9). Had the antiport peptide addition to vesicles indicated that the vesicle integrity
mechanism been operative, quenching of lucigenin dye would was maintained after addition of peptide (Figure S30 and S31).
be observed in the presence of all cations. Peptide
1 also
showed a marginal preference for K+ ion over Na+ ion,
indicating that M+ transport could be the driving force for the
M+/Cl⁻ symport. To rule out the possibility of Ni2+/Co2+ binding
with the peptide prior to membrane insertion, the assay was
carried out by premixing peptide and vesicles before addition
of MX (Figure S19 and S20). Similar results were obtained with
the modified lucigenin assay confirming that the lack of
peptide activity in the presence of Ni2+/Co2+ was not due to
binding of these ions to the peptides. In addition, the
absorbance spectra of each peptide, metal salt and peptide-
salt mixture were measured in the absence of vesicles (Figures
S32 & S33). The peptide to metal salt ratio was same as that
used in the Lucigenin assay. The absorbance spectra of the
peptide-salt mixtures were similar to the free peptide, which
further ruled out metal-binding to peptide.
a)
b)
Figure 7 a) TEM image of peptide 1. b)TEM image of peptide 2.
As with the case of our earlier peptides, the ability of these
pores to conduct cations could be attributed to the affinity of
cations to the carbonyl groups from the peptide backbone. In
contrast to our earlier peptides, halide transport is observed
with peptides
1 and 2 containing aminopicolinic acid units.
Aminopicolinic acid units are known to facilitate halide binding
in macrocyclic peptide based receptors.43, 44 The presence of
these units in our peptide scaffolds appears to significantly
improve the affinity of the pores for halides.
a)
b)
Conclusions
In conclusion, peptides
1 and 2 derived from aminopicolinic
acid and alanine have been developed as ion transporting
pores. The ion transport mechanism has been established
using vesicles entrapped with pH sensitive HPTS or halide
sensitive lucigenin dyes. The assays show that the peptides
transport cations through lipid membranes via an M+/Cl⁻
symport mechanism. The activity of peptide
picolinic units at the C-terminus has been found to be
marginally higher than peptide . Both peptides form cation
selective pores in synthetic lipid bilayers which do not show
any transition to the closed state. Such readily accessible
cation–selective pores with prolonged open states are highly
attractive for the development of sensors and materials.
1 with the
Figure 6 a) Schematic representation of ion transport mechanism. b) Change in
lucigenin fluorescence in the presence of MX salts and peptide 1 (20 μM = 5 mol %
peptide with respect to lipid ). Average k values reported in the legend. Origin 8.1 was
used to fit the curves.
2
Peptides
alanine units and two aromatic units similar to our previously
reported octapeptides . In particular, the peptides
reported here are the pyridyl analogs of peptide . Peptide
1 and 2 contain a scaffold of alternating L and D
3
- 5
3
3
was shown to have an S-shaped structure with β-sheet like
dihedral angles and formed thermodynamically stable pores in
the lipid bilayer.35 The replacement of phenyl groups with
pyridyl units should not drastically change peptide
Experimental Section
Preparation of vesicles for HPTS assay: Cholesterol (1.6 mg,
4.1 µmol, 1 equiv) was added to a solution of EYPC lipids (28.4
mg, 36.9 µmol, 9 equiv) in chloroform (0.284 mL). Chloroform
was removed under a stream of nitrogen and further in vacuo
for 5 h at 0 ºC to give a lipid film. The lipid film was hydrated
with 1 mL of a buffer solution containing HPTS dye (0.1 mM
HPTS, 10 mM NaCl, 100 mM, HEPES) at pH 7.2. The resulting
suspension was allowed to stir at room temperature for 1 h.
conformation. Therefore, we believe that peptides
self-assemble to form thermodynamically stable pores similar
to peptide . The TEM images of peptides and indicate that
1 and 2
3
1
2
they form nanotubes or nanofibres (Figure 7a & b). While the
TEM image does not directly correlate to the structure of the
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and then subjected to five freeze-thaw cycles. The vesicle (PLB) recording. Synthetic lipid 1,2-diphytanoyl-sn-glycero-3-
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solution was placed in an ice bath and subsequently sonicated phosphocholine (Avanti Polar Lipids, DOAIl:a1b0a.1s0t3e9r/,C6AOLB)005w9a2Fs
in a bath sonicator at 0-4 °C for a total time of 2 minutes (30 s dissolved in n-decane at a concentration of 20 mg/mL and
on & 15 s off in degas mode). The vesicles were allowed to used to make the lipid bilayer. Briefly, the lipid was painted on
anneal for one hour in the refrigerator, following which they to the aperture (pore diameter of 150 m) of a polystyrene
were extruded 20 times through 100 nm polycarbonate bilayer cuvette (Warner Instruments, USA). Both chambers of
membranes using a mini-extruder from Avanti Polar Lipids. The the cuvette contained symmetrical solution (1 M KCl). The cis
extra-vesicular dye was removed by size exclusion chamber was held at virtual ground and the trans chamber
chromatography using Sephadex G-50 (eluent: HEPES buffer at connected to a recording amplifier via a PC501A head-stage
pH 7.2 (10 mM NaCl, 100 mM HEPES). The milky white solution (Warner instrument). Ag-AgCl electrodes were used. Currents
containing vesicles were collected and used for further study.
were low pass filtered at 1 kHz and digitized at 5 kHz using the
Digidata 1440A (Axon Instruments). Once a stable bilayer was
HPTS Assay: Vesicle solution (100 µL), HEPES, NaCl buffer (2.9 obtained, the synthetic peptide was added to the cis chamber
mL) and an appropriate amount of peptide in methanol (0.5% and mixed with a magnetic stirrer. Membrane currents were
DMSO) were added to a cuvette equipped with a magnetic stir frequently checked to monitor channel incorporation. After
bar. The solution in the cuvette was stirred for 2 minutes stable insertion of the peptides in bilayer, membrane current
before the fluorescence experiment was started. After 50 s, a was recorded at different positive and negative voltages. For
solution of NaOH (20 µL, 0.5 N) was added to the vesicles. 5% measuring reversal potential, the solution of the cis chamber
Triton X (50 µL) solution was added at 250 s to achieve final was replaced with 0.5 M KCl while the trans chamber had 1 M
equilibration of the dye. Fluorescence measurements were KCl. Theoretical reversal potential was estimated using the
done at an emission wavelength of 510 nm and an excitation Nernst Equation and compared with the experimentally
wavelength of 460 nm.
derived value.
Preparation of vesicles for Lucigenin Assay: Cholesterol (1.6
mg, 4.1 µmol, 1 equiv) was added to a solution of EYPC lipids
(28.4 mg, 36.9 µmol, 9 equiv) in chloroform. Chloroform was
removed under a stream of nitrogen and further in vacuo at 0
°C for 2 h to give a lipid film. The lipid film was hydrated with 1
mL of a solution containing Lucigenin dye (1.0 mM Lucigenin,
225 mM NaNO3). The resulting suspension was allowed to stir
at room temperature for 5 minutes and then subjected to five
freeze-thaw cycles. The vesicle solution was placed in an ice
bath and subsequently sonicated in a bath sonicator at 0-4 °C
for a total time of 2 minutes (30 s on & 15 s off in degas
mode), following which they were extruded 20 times through
400 nm polycarbonate membranes using a mini-extruder from
Avanti Polar Lipids. The extra-vesicular dye was removed by
size exclusion chromatography (SEC) using Sephadex G-50
(eluent: 225 mM NaNO3). (Note: Carrying out the SEC twice
ensured removal of majority of the extra-vesicular dye). The
milky white solution containing vesicles were collected and
used for further study.
ACKNOWLEDGMENT
This research was supported by DST (SR/S1/OC-41/2009), New
Delhi, India. D.B thanks UGC for his fellowship. We thank Dr. E.
Prasad for the use of his dynamic light scattering instrument.
We thank S. Chakraborty for her contribution towards the
synthesis of peptide 2. We thank the Sophisticated Analytical
Instruments Facility (SAIF) and Dept. of Metallurgical and
Materials Engineering, IIT Madras for SEM and TEM images.
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