ARTICLE
Auteur(s) : Beata Paczosa-Bator1, Milena
Stepien1,2, Magdalena Maj-Zurawska3, Andrzej
Lewenstam1,2
1Faculty of Material Science and Ceramics,
AGH-University of Science and Technology, Cracow,
Poland
2Center for Process Analytical Chemistry
and Sensor Technology (ProSens), c/o Process Chemistry Center
of Excellence, Åbo Akademi University, FIN-20500 Turku-Åbo,
Finland
3Department of Chemistry, University
of Warsaw, Warsaw, Poland
Membrane potential is rudimentary in biological life and sensor
technology. Recently, we proposed taking advantage of processes
common for those areas which lead to membrane potential formation
[1-4]. Namely, this approach employed the redistribution of ions in
the vicinity of the membrane and coupled dynamic potential change.
From the biological point of view, we are interested in the
mechanism of voltage-dependent channel block and related ionic
antagonism, such as divalent calcium-magnesium ion competition in
the case of NMDA [4-7], or monovalent ion effects such as
potassium-sodium/lithium/TEA in the case of potassium and sodium
channels [8]. From the electrochemical perspective it means that we
are concerned with the time-dependent, characteristics and those
resulting from a competitive ion-exchange of ion-selective sensors
such as magnesium [9] or any other ion-selective electrode [10,
11].
Our deliberate biomimetic concept allows for studying of a
competitive (“antagonistic”) ion exchange and its coupling to
membrane potential formation on biologically active sites. The
novelty is in using conductive polymers as the membranes in which
the sites are dispersed.
In this report, recent results on calcium-magnesium antagonism,
and for the first time, the data on potassium-lithium/sodium are
shown.
Experimental
Reagents
Pyrrole, N-methylpyrrole (Merck) was purified by double
distillation under argon and then stored under argon at low
temperature and protected from light. The L-asparagine,
L-glutamine, adenosine 5′- triphosphate sodium salts were obtained
from Fluka. The other reagents were also purchased from Fluka. All
the chemicals used were of analytical grade. Doubly distilled and
freshly deionized ELGA water (resistivity 18.2 MΩ cm) was used
throughout the work.
Apparatus
The potentiostatic synthesis of polymer films on GC and ITO
electrodes was carried out using an Autolab general Purpose System
(AUT20.Fra2-Autolab, Eco Chemie, B.V., Utrecht, The Netherlands)
connected to a conventional, three-electrode cell. The working
electrode was a glassy carbon (GC) disk with an area of
0.07 cm2 or conducting glass pieces with an area of
about 1 cm2 (ITO, Lohja Electronics, Lohja,
Finland, used for the EDAX, XPS and LA-ICP-MS experiments). The
reference electrode was an Ag/AgCl/3M KCl electrode connected to
the cell via a bridge filled with supporting electrolyte solution,
and a glassy carbon (GC) rod was used as the auxiliary electrode.
The solutions used for polymerization contained 0.1 M monomer of
polymer and an electrolyte that provided the doping ion.
Electropolymerization was performed in solutions saturated with
argon at room temperature.
The chrono-amperometric measurements were carried out also using
an Autolab general Purpose System (AUT20.Fra2-Autolab. Eco Chemie.
B.V.) connected to a conventional, three-electrode cell where a
glassy carbon electrode prepared with PMPy-ATP-Na polymer was used
as the working electrode, and as the reference electrode - an
Ag/AgCl/3M KCl electrode, and the auxiliary electrode - a glassy
carbon electrode.
The potentiometric measurements were made with a homemade
multi-channel set-up. The reference electrode was an Ag/AgCl/3M KCl
electrode. All experiments were performed at room temperature.
The elemental analysis of the polypyrrole films was performed
using X-ray photoelectron spectroscopy (XPS). The XPS analysis was
performed with a Physical Electronics Quantum 2000 XPS-spectrometer
equipped with a monochromatized Al-X-ray source to assess
qualitatively the influence of soaking on the composition of these
films. The size of the analyzed area was 100 μm in diameter
and the analysis depth was about 2-5 nm depending on the
investigated element.
The Energy Dispersive Analysis of X-ray (EDAX) measurements were
performed using a Scanning Electron Microscope, SEM model LEO 1530
from LEO Electron Microscopy Ltd, which was connected to an Image
and X-ray analysis system – model Vantage from ThermoNoran.
The LA-ICP-MS measurements were performed using a model 6100
Elan DRC Plus of ICP-MS from Perkin Elmer SCIEX (Waltham, USA) and
UP-213 of Laser Ablation from “New wave Research” Merchantek
Products (Fremont, USA). The LA-ICP-MS measurements for the
PMPy-ATP sample were investigated by vaporization in laser plasma
with a spot size of 15 μm and a scan speed of 10 μm per
second. The repetition rate was 10 Hz and output 40% in every
case during the measurement.
Procedures of CP-BL-Me electrode preparation
Poly(N-methylpyrrole) and poly(pyrrole) deposition
The electrodeposition of the poly(N-methylpyrrole) and
poly(pyrrole) films was carried out potentiostatically from a
solution that contained 0.1 M ATP as dopant and 0.1 M
N-methylpyrrole or pyrrole. Potentiostatic growth was achieved by
holding a potential at + 0.8 V vs Ag/AgCl/3M KCl for different
times in order to obtain different amounts of charge density
(510-750 mC cm-2).
The potentiostatic method was also used to grow poly(pyrrole)
films doped with amino acids. PPy-Asn(Gln) films were grown on the
working electrode at a potential of +1.0 V vs Ag/AgCl/3M KCl and
charge density of 240 mC cm-2. Besides 0.1 M pyrrole,
the solution contained 0.1 M Gln (or 0.1 M Asn).
The process of making cation-sensitive CP-BL films
After synthesis, the polymer membranes were washed with deionized
water and then the electrodes were soaked and stored in a mixture
of 0.1 M MeCln and Me(OH)n (pH of about 9-10)
where Me was a main ion. As a rule, a cationic response with a
linear range within the K+, Na+,
Li+ activities from 10-1 M to 10-4
M or Ca2+, Mg2+ activities from
10-1 M to 10-5 M and with a
close-to-Nernstian slope was observed for the CP-BL films after 1
week of soaking. Only conditioning in the alkaline solution of main
ions was effective. The cation complexes with BL were formed after
PPy-BL film deprotonation in alkaline solutions (protons were
substituted with other cations) [1, 2].
Results and discussion
The results described below reflect a methodological pattern used
to explore the biomimetic properties of CP-BL membranes and the
mechanism of their membrane potential formation. In the first
stage, the films were electrosynthesized and doped with respective
biological ligands. Next, a potentiometric sensitivity for the main
ion of interest was induced. After this, the influence of other
ions, i.e. the selectivity of interest was studied.
Only properly behaving membranes, characterized with a
time-independent response for both the main and other than main
ions, were used in the studies of a dynamic (time-dependent)
response for changes to the electrochemical membrane potential
driving forces, i.e. changes in bulk ion concentrations in the
bathing solutions and influence of the external electric potential
[1-4].
Potentiometric response of CP-BL membranes
The potentiometric response of all membranes was studied in a
solution of chloride salt of main ions in the concentration range
from 10-6 M to 10-1 M.
The first calibration was performed before conditioning and then
after a different time of soaking. Exemplary calibration and
dynamic response curves are shown in figure 1: for a membrane
sensitive towards monovalent potassium cation PMPy-ATP-K (figure 1A) and a
membrane sensitive to divalent magnesium ion PMPy-ATP-Mg (figure 1B). Figure 1 illustrates a
need for conditioning of the films to achieve a close-to-Nernstian
potentiometric response, i.e. the slope close to 59.2 mV/pNa, (pLi
or pK) and 29.6 mV/pMg or pCa in the range of 10-1 M to
10-4 M for Na+, Li+, K+
and 10-1 M to 10-5 M for Ca2+ and
Mg2+ activities. It is shown that, after one week of
conditioning in the alkaline solution of the main ion, both
potentiometric sensitivity and a short potential response time
(less than 10 s) were obtained. This applies to all the
membranes studied.
Potentiometric selectivity of CP-BL membranes
After inducing a proper sensitivity, the selectivity of the films
to other ions was studied by a separate solution method (SSM) [12].
This method allows determining a potentiometric selectivity
coefficient () which is a quantitative measure of the ability of
the ion-sensitive film to discriminate the main ion from the other
ions. The coefficient is dictated by the thermodynamic properties
of ions in the phases submitted for ion exchange and should
correlate with a corresponding ion-exchange equilibrium constant at
the solution-membrane interface [11].
The values of the potentiometric selectivity coefficients
obtained were in excellent agreement with the stability constants
of BL complexes with ions, as shown in figure 2 for two chosen
membranes, PMPy-ATP-Na and PMPy-ATP-Mg. The potentiometric
selectivity coefficients follow the same sequence as the stability
constants of ATP complexes with cations.
As expected, in the case of membranes sensitive towards
monovalent cations, strong interferences of divalent cations were
observed. By subsequently soaking the CP-BL-Na(Li)(K) films in the
solution of Ca2+, or Mg2+, forming a stronger
complex with BL, it was possible to obtain calcium- or
magnesium-sensitive films by competitive ion exchange.
The calcium- and magnesium-sensitive CP-BL films were
insensitive towards sodium, potassium or lithium. Importantly, the
selectivity coefficient values for the membranes sensitive to
divalent metal ions and monovalent ions were close to = 1. This
manifestation of similar thermodynamic properties of ions (in the
groups) studied, makes any dissimilarity of the response
attributable to the kinetic properties of these ions in the
membrane systems studied.
Chemical analysis of CP-BL membranes
The elemental analysis of poly(N-methylpyrrole) and polypyrrole
films was performed using three different methods, X-ray
photoelectron spectroscopy for membranes doped with amino acids,
energy dispersive analysis of X-ray for CP-ATP films sensitive to
divalent ions, and laser ablation inductively coupled plasma mass
spectrometry for CP-ATP films sensitive to monovalent ions. For the
chemical and morphological analysis, two kinds of samples were
prepared, namely: CP-BL without soaking and CP-BL after 2 weeks’
soaking in the solution of main ions.
The presence of the phosphorus signal in the case of PPy-ATP and
PMPy-ATP films in the EDAX and LA-ICP-MS spectra, as shown in figure 3 and 4, proves
that counter-ions dope the films formed during
electrodeposition.
The EDAX analysis of PPy-ATP films showed that, after the
conditioning process, calcium or magnesium peaks had also appeared
on the spectrum. The LA-ICP-MS measurements for the PMPy-ATP sample
sensitive to monovalent ions proved that, after the conditioning
process, ions that were present in the solution also appeared in
the membranes.
Changing the composition of the conditioning solution from
sodium to lithium (or potassium) gave membranes sensitive to these
ions and the signal from the new ions appeared on the spectrum as
shown in figure
3.
Depending on the composition of the solution used during
conditioning CP-ATP membranes, different potentiometric sensitivity
was observed. After conditioning in alkaline lithium solution, the
potentiometric sensitivity to these ions had been induced and the
LA-ICP-MS spectrum showed a signal from Li (which was not observed
before the conditioning process) as presented in figure 3A. The same
behaviour was observed for calcium, magnesium and potassium ions.
The chemical composition of the PPy-Asn(Gln) films was investigated
by XPS. Figure
3B (curve i) shows on overview spectrum of a freshly
deposited PPy-Asn sample on which the following peaks were observed
at the given binding energies: O1s at 532.8 eV, N1s at 400 eV and
C1s at 284.8 eV. The peaks in the region of 88.8 eV binding energy,
as shown in figure
3B (curve ii), are identified as an Mg2s signal, which was
observed only after conditioning in alkaline magnesium solution.
Similarly, the XPS analysis taken after soaking of the PPy-Asn(Gln)
films in an alkaline calcium solution proved to peak Ca2p3 at 347.2
eV (figure 3B,
curve iii).
In the case of sodium-sensitive membranes, the signal from Na
was observed after soaking with EDAX, providing good evidence for
introducing sodium to the membrane during soaking, as shown in
figure 4.
The chemical analysis provides the evidence that the sensitivity
for a particular ion is always associated with the presence of this
ion in the membrane.
Ion competition during open-circuit response
In spite of similar sensitivity and selectivity of both groups of
polymer films (namely CP-BL-Ca(Mg) or CP-BL-K(Li)(Na)) towards
divalent (calcium and magnesium) ions or monovalent (sodium,
potassium and lithium) ions, the transitory potential provoked by
the changes in bulk concentrations of these groups of ions was
strikingly different.
The representative plots for the measurements made for
monovalent ion-sensitive membranes are shown in figure 5A-C, and the
membranes sensitive towards divalent ions are shown in figure 5D.
As can be seen from figure 5, potential-time
(E-t) response strongly depends on the kind of ion that was
involved in the competitive ion-exchange equilibration process.
Lithium ion-exchange with sodium-rich CP-ATP-Na membranes results
in a monotonic response (figure 5A), while if
potassium ions are engaged in the ion exchange, instead of lithium,
a non-monotonic (overshoot-type) response is observed (figure 5B). If a
sodium-rich membrane is converted to a potassium-rich one, then
both the lithium and sodium responses, as expected, are monotonic
(figure 5C).
A similar pattern in observed for the CP-ATP-Mg membrane (figure 5D). Changes
in bulk concentrations of magnesium ion are always associated with
monotonic potential changes, while changes in the concentration of
calcium are associated with overshoot-type responses. These
characteristic differences between potassium, sodium and lithium,
as well as magnesium and calcium can be called “ionic antagonism”.
Interestingly, and most probably not coincidentally, the same pairs
of ions, i.e. Ca2+-Mg2+,
Na+/Li+-K+, are indeed considered
as antagonistic in real biological membrane systems, and
specialized voltage and/or ligand-gated ion channels engage these
ions, e.g. NMDA.
As stated above, when discussing the selectivity of the
membranes used in our study, thermodynamically, the differences do
not allow prediction of a striking difference in membrane
responses. However, this fact lends credence to kinetic aspects in
signal formation. A different rate in the transport of ions to
(the source of ion exchange) the interface of the solution and
membrane could be a primary source of the “antagonism” observed
[4]. The hypothesis is that faster ions, (Ca and K characterized by
the mobility 6.17 and 7.62 10-8 m2
s-1 V-1, respectively [13]) coming from the
solution bulk and substituting via ion exchange slower ions from
the film (Mg, Na and Li, characterized by the mobility 5.49, 5.19
and 4.01 10-8 m2 s-1
V-1, respectively [13]) sites, allow for local
accumulation of slower ions in the solution bathing the film. And
vice-versa, if slower ions come from the solution to the film
containing faster ions, a deficit of this ion can be observed. This
mechanism is schematically illustrated for Mg2+ -
Ca2+ pair in figure 6
Figure 6
clearly shows the limited value of our approach when confronted
with realistic biological cell membranes, which contain a lipid
environment and complicated 3D protein-channel structures.
Undoubtedly, our CP membrane serves only one aspect of many
relevant to real potential gated channels. It allows addressing and
controlling electrical potential on the site, and observing
resulting ion redistributions and fluxes.
Ion competition during amperometric response
As shown in figure
5, the changes in bulk concentration of ions result in
characteristic changes of potential vs. time, and are attributed,
as shown in figure
6, to local redistributions of ions in the vicinity of the
membrane-solution interface. It is of great interest to convert the
problem and ask whether one could observe any manifestation of this
process in an experiment where the membrane redistribution is
provoked by an external electric potential impulse. In this
respect, in the absence of a method for direct visualization of the
ionic concentration changes in the vicinity of a membrane
interface, a chrono-amperometric method was used. In this method,
the external potential (+ 5/-5 and + 10/-10 mV from the
open-circuit potential) is applied to provoke ion fluxes to and
from the membrane, and the fluxes are characterized by (ionic)
current over time. It would be expected that faster ions (potassium
or calcium), in response to the potential applied, would produce
currents that come to a base-line faster than ions of lower
mobility (sodium/lithium or magnesium) do. The current response of
the PMPy-ATP-Na electrode with time was measured in solutions of
chloride salts of sodium, lithium and potassium with concentrations
equal to 0.0001M under different values of potential. In figure 7, the
current-time (I-t) responses for a sodium sensitive PMPy-ATP
membrane are shown. The plots indeed prove the interrelation
between the size of ions (resp. mobility of ions) and the I-t
signal measured.
For the faster potassium ion (resp. calcium ion), after bigger
initial cathodic or anodic current values, a fast current drop was
observed, while for slower sodium and lithium (resp. magnesium)
ions, the initial currents were smaller and followed by a slower
drop in current. This amperometric behaviour can be attributed to
different mobility “antagonistic” ions. It can be concluded that
both in the open circuit and under potential, the electrochemical
behaviour of the biomimetic membrane is dictated by different
transport numbers of the ions. The kinetic difference is thus a
prerequisite of the “ionic antagonism”.
Theoretical interpretation and implications
The change in membrane potential over time provoked by bulk
concentration changes is attributed to local redistributions of
ions at the membrane-solution interface and transport to and from
this interface. If the membrane potential is changed by an external
source, resulting ionic fluxes could be observed and the ionic
currents depend on the physicochemical properties of the ions
engaged. These observations allow the development of a general
interpretation of E-t and I-t for this biomimetic system using the
Nernst-Planck-Poisson model (NPP) [11, 14, 15] or a simpler
diffusion-layer model (DLM) [2, 4, 11, 16]). Both these theoretical
models support a fundamental idea of the biomimetic membrane
concept presented and show that, when a biologically active site is
allowed for a competitive ion-exchange, the extent of the
competition is regulated by the electric potential “applied” to
this site and the transport of ions to and from the site. Different
E-t and I-t patterns have to be observed for faster ions in
comparison to slower, which is known as “ionic antagonism”. Here is
a key for applying our biomimetic approach for responses that
follow electric potential incoming to the site (e.g. as excitation
potential) and to observe ionic distributions on the sites
accumulated in 3D (i.e. three dimensional) structures, e.g.
nanotubes or 3D architectures in a form of artificial channels,
and/or on 2D nanofilms. Our study shows that a local e.g. magnesium
ion concentration increase is be expected when positive vs.
equilibrium (rest) potential is applied. In other words, it means
that magnesium ions leave the coordinating sites and the smaller
calcium ion is admitted. This is exactly what happens at the neck
of a magnesium blocked NMDA channel where this ion is attracted by
Asn and Gln. When excited by action potential, the channel gets
unblocked and allows the faster calcium ions to pass through [5,
17]. Obviously, a deficiency of magnesium in external compartments
can facilitate calcium influx and modulation of intracellular
calcium concentration. Interestingly, this mechanism and the
magnesium-calcium antagonism in relation to the NMDA receptor
channel were recently considered as one possible reason for
inflammatory responses and the metabolic syndrome [18, 19]. The
importance of the effects of Ca2+, Mg2+, ATP
and other phosphorylated species on cardiac action potentials was
recently emphasized as well [20]. A similar case of a
competitive ion mechanism could be in the interaction of the
exogenous lithium ion with negatively-charged inositol
phospholipids, which is considered to be relevant in the treatment
of bipolar disorders [21, 22].
As presented here, potential-dependent local concentration
redistribution of ions at the membrane binding sites undoubtedly
adds a new dimension in the interpretation of the above effects.We
address these issues in our present research.
Conclusion
The biomimetic membrane methodology allows visualization and
inspection of competitive and voltage-dependent ion exchange on
biologically active sites. By using selected biological membranes
and their channels (e.g. ATP, Asn, Gln), relevant to real ionic
sites, it is possible to access the ionic redistribution in the
sites in function of the bulk concentration of ions, external
potential and time. In other words, the concept presented provides
a tool to study the role of ions and the influence of ion
supplementation, ion deficiencies and ion antagonism on membrane
potential. It is, as well, a tool for investigating the nature of
bias between voltage effects (long-term potentiation (LTP), cardiac
arrhythmias, low-frequency signals in brain) on local (at/on site)
ionic or transmembrane redistributions.
Acknowledgments
This work is supported by the Polish Ministry of Science and Higher
Education, Grant No. DWM/232/MATERA/2006 and is a part of MASTRA
MATERA ERA-NET project funded under the 6th FP EU.
Financial support from the Polish State Committee for Scientific
Research (KBN), project number R15 005 03, is gratefully
acknowledged.
References
1 Migdalski J, Błaż T, Paczosa B, Lewenstam A.
Magnesium and calcium-dependent membrane potential of poly(pyrrole)
films doped with adenosine triphosphate. Microchim Acta 2003; 143:
177-85.
2 Paczosa-Bator B, Migdalski J, Lewenstam A.
Conducting polymer films as model biological membranes.
Electrochemical and ion-exchange properties of poly(pyrrole) films
doped with asparagine and glutamine. Electrochim Acta 2006; 51:
2173-81.
3 Paczosa-Bator B, Peltonen J, Bobacka J,
Lewenstam A. Influence of morphology and topography on
potentiometric response of magnesium and calcium sensitive PEDOT
films doped with adenosine triphosphate (ATP). Anal Chim Acta 2006;
555: 118-27.
4 Paczosa-Bator B, Blaz T, Migdalski J,
Lewenstam A. Conducting polymers in modelling transient
potential of biological membranes. Bioelectrochemistry 2007; 71:
66-74.
5 Nowak L, Bregestovski P, Ascher P,
Herbet A, Prochiantz A. Magnesium gates
glutamate-activated channels in mouse central neurons. Nature 1984;
307: 462-5.
6 McBain CJ, Mayer ML. N-methyl-D-aspartic acid
receptor structure and function. Physiol Rev 1994; 74: 723-60.
7 Saris NE, Mervaala E, Karppanen H,
Khawaja JA, Lewenstam A. Magnesium - an update on
physiological, pathological and analytical aspects. Clin Chim Acta
2000; 294: 1-26.
8 Hille B. Ionic Channels of excitable membanes. Sinauer
Associates Inc., 1992.
9 Maj-Zurawska M, Lewenstam A. Fully automated
potentiometric determination of ionized magnesium in blood serum.
Anal Chim Acta 1990; 236: 331-5.
10 Lewenstam A. Design and pitfalls of ion-selective
electrodes. Scand J Clin Lab Invest 1994; 54: 11-20.
11 Bobacka J, Ivaska A, Lewenstam A.
Potentiometric ion sensors. Chem Rev 2008; 108: 329-51.
12 Umezawa Y, Bühlmann P, Umezawa K,
Tohda K, Amemiya S. Potentiometric Selectivity
Coefficients of Ion-Selective Electrodes. Part I. Inorganic
Cations. Pure Appl Chem 2000; 72: 1851-2082.
13 Fraústoda Silva JJR, Williams RJP. The Biological
Chemistry of the Elements. Oxford: University Press, 2001.
14 Sokalski T, Lewenstam A. Application of
Nernst-Planck and Poisson equations for interpretation of
liquid-junction and membrane potentials in real-time and space
domains. Electrochem Comm 2001; 3: 107-12.
15 Sokalski T, Lingenfelter P, Lewenstam A.
Numerical solution of the coupled Nernst-Planck and Poisson
equations for liquid-junction and ion-selective membrane
potentials. J Phys Chem B 2003; 117: 2443-52.
16 Lewenstam A, Hulanicki A, Sokalski T. Response
mechanism of solid-state ion-selective electrodes in the presence
of interfering ions. Anal Chem 1987; 59: 1539-44.
17 Vargas-Caballero M, Robinson HPC. Fast and slow
voltage-dependent dynamics of magnesium block in the NMDA receptor:
The asymmetric trapping block model. J Neurosci 2004; 24:
6171-80.
18 Rayssiguier Y, Gueux E, Nowacki W,
Rock E, Mazur A. High fructose consumption combined with
low dietary magnesium intake may increase the incidence of the
metabolic syndrome by inducing inflammation. Magnes Res 2006; 19:
237-43.
19 Mazur A, Maier JAM, Rock E, Gueux E,
Nowacki W, Rayssiguier Y. Magnesium and the infammatory
response: Potential physiopathological implications. Archiv Biochem
Biophys 2007; 458: 48-56.
20 Michailova A, McCulloch AD. Effects of
Mg2+, pH and PCr on cardiac excitation-metabolic
coupling. Magnes Res 2008; 21: 16-28.
21 Atack JR, Broughton HB, Pollack SJ. Inositol
monophosphatase – a putative target for Li+ in the
treatment of bipolar disorder. Trends Neurosci 1995; 18: 343-9.
22 Gibbons CE, Maldonado-Pérez D, Shah AN, Riccardi D, Ward DT.
Calcium-sensing receptor antagonism or lithium treatment
ameliorates aminoglycoside-induced cell death in renal epithelial
cells. Biochim Biophys Acta 2008; 1782: 188-95.
|
Printable version
Version PDF
