Tải bản đầy đủ - 0 (trang)


Tải bản đầy đủ - 0trang


R. Guidelli and L. Becucci

Rolando Guidelli

Rolando Guidelli was born in Florence on December 27, 1938. He was promoted to

full professor of Electrochemistry at Florence University in 1971. His scientific

interests have been focused to electrode kinetics, structure of the metal/water interface and bioelectrochemistry. Professor Guidelli is the recipient of the 2005

“Katsumi Niki” Prize for Bioelectrochemistry of the International Society of Electrochemistry (ISE), of the 2009 “Giulio Milazzo” Prize of the Bioelectrochemical

Society, and of the “Sigillo d’oro” of the Italian Chemical Society. He is a fellow of

the ISE, honorary member of the Bioelectrochemical Society, and associate editor

of the journal Bioelectrochemistry.


Electrochemical Analysis of Ion Channels

and Transporters in Pore-Suspending


Eva K. Schmitt* and Claudia Steinem**


Nuffield Department of Clinical Laboratory Science, John Radcliffe Hospital, University of Oxford, Oxford OX3 9DU, United Kingdom


Institute of Organic and Biomolecular Chemistry, University of Göttingen, Tammannstr. 2, 37077 Göttingen, Germany



Ion channels represent a class of membrane spanning protein pores

that mediate the flux of ions in a variety of cell types. They reside

virtually in all the cell membranes in mammals, insects and fungi,

and are essential for life, serving as key components in inter- and

intracellular communication.1 They are also of major importance

for the human physiology and, thus, are highly attractive molecular

drug targets.2 However, investigation of ion channels and their

pharmacological modulation is by no means an easy task, as their

function relies on a laterally mobile and highly insulating lipid bilayer. Functionality of an ion channel is synonymous with the detection of ion currents. Upon the opening of one channel, typically

107–108 charges are transferred across the membrane per second

and per channel, which gives rise to a current of around 2–20 pA

N. Eliaz (ed.), Applications of Electrochemistry and Nanotechnology


in Biology and Medicine II, Modern Aspects of Electrochemistry 53,

DOI 10.1007/978-1-4614-2137-5_5, © Springer Science+Business Media, LLC 2012


E.K. Schmitt and C. Steinem

that can only be monitored with high gain amplifiers. The patch

clamp technique is the state-of-the-art technology for the study of

ion channels,3 but patch clamping is a laborious process requiring

skilled and highly trained scientists. Owing to its ultra-low

throughput, conventional patch clamp is frequently applied only in

the later stages of drug discovery and development. To be able to

search for new drug candidates at an earlier stage of drug development, high throughput screening assays are required. In recent

years, new methods have been developed, which are a trade-off

between high throughput and high information content. These socalled automated patch clamp systems4-6 provide a platform, which

allows for the investigation of ion channels from a functional perspective, i.e., the electrical characteristics of ion channels and their

modulation by drugs can be monitored in great detail. Automated

patch clamp, however, relies on currents in the picoampere regime,

which are generated by a single ion channel, and data workup of

these single events is rather tedious.

Hence, integral methods such as impedance spectroscopy, cyclovoltammetry or chronoamperometry are in some cases advantageous. For example, electrogenic transporter proteins exhibit

transport rates of only 10–1000 s-1 and, thus, the electrical activity

of a single protein cannot be detected. To obtain a detectable electrical current in the range of picoamperes, at least 104 transporters

have to work in concert. Some of the most profitable drugs are inhibitors for transport proteins, and many transporters are possible

targets for new drug developments. For example, ouabain and digoxin act on the cardiac muscle cell Na+/K+-ATPase in patients

with congestive heart failure, while fluoxetine and imipramine are

inhibitors of the sodium coupled serotonin transporter that are used

as antidepressants. The gastric proton pump of the stomach mucosa is a drug target to treat ulcers and gastric reflux, which is medicated with omeprazole, lanosprazole, pantoprazole, and pariprazole. It is obvious that new approaches are necessary to develop

bioanalytical devices and drug screening techniques for the pharmaceutical industry, targeting transport proteins as well as ion

channels in an integral manner.

A number of approaches are based on lipid membranes that

are attached to a conducting solid substrate such as gold, platinum

or indium tin oxide. To establish solid supported membranes, either two separate monolayers can be deposited on the material by

Ion Channels and Transporters in Pore-Suspending Membranes


means of the Langmuir-Blodgett and/or Langmuir-Schäfer technique, or, more easily, self-assembly processes are exploited.

While Langmuir-Blodgett films offer the control over the composition and lateral pressure of both leaflets, self-assembly techniques are more versatile and the bilayers are easier to prepare. In

the latter case, lipids are attached via covalent or quasi-covalent

linkage, which appears to be an attractive solution to the problem

of membrane immobilization. However, at the same time, the lateral mobility and a sufficient distance between solid support and

the surface-facing membrane leaflet needs to be maintained, which

is a prerequisite for a functional reconstitution of membrane proteins. Only few examples are given that demonstrate the feasibility

of incorporating transmembrane proteins in solid supported membranes owing to the close proximity of the membrane to the solid

substrate.7-9 To overcome these difficulties, other strategies comprising the separation of a solid supported membrane by a thin,

water-swollen polymer cushion or anchoring of lipids to the support by spacer units (tethered bilayers) to increase the distance between the membrane and the substrate have been developed,10,11

which allowed monitoring of ion flows through reconstituted

channels.7,12-15 However, the ion currents are hampered by the capacitive coupling of the membrane to the substrate and, thus, single channel ion currents cannot be detected.

To combine the merits of robustness of membranes attached

to a support with the advantage of freestanding bilayers, which allow an easy insertion of transmembrane proteins without a capacitive coupling, substrates with pore arrays or even porous materials

have been introduced as membrane supports.16-19 Membranes

spanning these holes with diameters between 50 nanometres and

several tens of micrometres take advantage of the natural properties of a cell membrane, where integrated ion channels are mobile,

and transport of ions from one side of the membrane to the other is

not hindered by the vicinity of a substrate.

Here, we give an overview of the potential of pore-suspending

membranes for electrical monitoring of ion channel and transporter

activities. We will focus on our established pore-suspending membrane systems based on functionalized porous alumina substrates

with pore diameters of only 60 nm. The presented systems exhibit

membrane resistances which allow for integral electrical readouts


E.K. Schmitt and C. Steinem

by impedance spectroscopy and, in part, voltage clamp experiments on single ion channel forming peptides and proteins.





A couple of years ago, we developed an artificial membrane system based on porous alumina substrates that can be considered as a

hybrid between a solid supported membrane and a freestanding

lipid bilayer. As each of the bilayers spanning the pores of the substrate resembles a black lipid membrane (BLM), a membrane that

was first invented by Müller and Rudin in the 1970's, but of nanometre size, the system was called nano-BLMs.

To investigate the electrical properties, such as capacitance

and resistance of lipid membranes, electrochemical impedance

spectroscopy (EIS) is a powerful technique.12,20,21 In general, this

method is well suited to elucidate the electrochemical properties of

sensitive biological interfaces in a non-invasive manner by the application of an alternating voltage of small amplitude.

(i) Formation and Impedance Analysis of Nano-BLMs

Porous alumina can be prepared easily, reproducibly and rather cheaply in almost arbitrary lateral dimensions, making this

material well suited for routine applications as well as for biosensor devices. The substrates are formed by anodisation in the presence of acids.22,23 To use them as an underlying substrate for nanoBLMs, the pore-bottoms need to be removed, which results in a

sieve-like structure.24 For the formation of nano-BLMs we adapted

a method, which was originally established to prepare solid supported membranes on gold electrodes.9 The strategy is to utilize

the strong, quasi-covalent interaction between gold and sulfur to

chemisorb alkane thiols or thiol-functionalized lipids such as the



(DPPTE) on gold surfaces. Thus, the upper part of the alumina

substrates is covered with a 60 nm gold layer followed by the

chemisorption of a thiol compound that renders the upper surface

Ion Channels and Transporters in Pore-Suspending Membranes


hydrophobic. In order to allow for nano-BLM formation in an

aqueous environment, the substrate is fixed in a Teflon cell consisting of two identical compartments separated by the porous

alumina substrate with an area of 7 mm2 sealed by two O-rings

(Fig. 1A).

For impedance analysis, platinised platinum electrodes, immersed in the electrolyte solution on both sides, serve as working

(cis compartment) and counter electrode (trans compartment) and

are connected to an impedance/gain-phase analyser SI 1260 from

Solartron Instruments (Farnborough, UK). Owing to the sieve-like

alumina structure, solely the electrolyte resistance Rel is monitored

in impedance measurements, with the capacitance of the platinum

electrode occurring at low frequencies (Fig. 2 A/B, filled squares).

In a theoretical approach, we previously addressed the problem of

the conducting or so-called “active” area of the porous substrates.25

We concluded that only the electrolyte-filled pores contribute to

the electrical characteristics of the material, and only the porous

area of the substrate needs to be taken into account when area dependent parameters are calculated.

Nano-BLMs are established on the functionalized porous

alumina substrates by applying a droplet of a lipid dissolved in a

non-volatile solvent such as n-decane. Owing to the hydrophobicity of the functionalized porous alumina the droplet spreads evenly, leading to an insulating layer. The formation of lipid bilayers

can then be followed by means of impedance spectroscopy in a

frequency regime of 10-2–106 Hz. In Fig. 2A/B, typical impedance

spectra of a functionalized porous alumina substrate before (filled

squares) and after nano-BLM formation (open squares) are shown.

Figure 2A depicts the absolute value of the impedance |Z|, whereas

Fig. 2B presents the phase angle I as a function of the applied frequency. The absolute value of the impedance of a fully established

bilayer is determined by the electrolyte resistance at 106 Hz,

whereas immediately after lipid-solvent application a capacitance

is predominant and, thus, a phase shift of around –90° is detected.

By measuring I at a frequency of 106 Hz, the kinetics of the thinning process, a process that is known from classical BLMs, can be

monitored with high time resolution. Figure 2C illustrates the shift

of the phase angle I(106 Hz) from –85° after the lipid-solvent

droplet has been applied to almost 0° after 150 s, indicating the


E.K. Schmitt and C. Steinem

Figure 1. A) Teflon cell that is used for impedance analysis and

voltage clamp experiments on nano-BLMs. The porous alumina

substrate is clamped between two O-rings, which define the active

area. An electrode is placed in each compartment (two-electrode

configuration), which are connected to either an impedance analyser or the headstage of the patch clamp amplifier. B) Setup used

for electrical measurements on pore-spanning membranes obtained

from vesicle spreading. The porous substrate is located between

the two upper parts of the Teflon chamber by means of sealing

rings. The single pieces of the setup are tightly secured via the

third part of the setup, which serves as the trans compartment,

whereas the chamber on top is the cis compartment. Platinised

platinum electrodes in the cis and trans compartment (twoelectrode configuration) serve to detect the signal in impedance

and photocurrent measurements.

|Z| / W

Ion Channels and Transporters in Pore-Suspending Membranes





























f / Hz























Figure 2. Bode representations of the impedance

data [A) Magnitude of the impedance, B) phase

angle between current and voltage] of goldcovered porous alumina membranes functionalized with a DPPTE-monolayer before (Ŷ) and one

day after (Ƒ) the application of DPhPC dissolved

in n-decane on the porous alumina substrate to

form a nano-BLM. The solid lines are the results

of the fitting procedure to (Ƒ) using the equivalent

circuit shown in the inset: Cm = 11.7 nF, Rm = 4.7

Gȍ. Inset: Equivalent circuit composed of a parallel RC-element (Rm and Cm) representing the electrical behaviour of a lipid bilayer in series to an

Ohmic resistance Rel that represents the electrolyte

solution and the wire connections.24 C) Timeresolved increase in I(106 Hz) during the membrane formation process, allowing to follow the

kinetics and success of the thinning process. Electrolyte: 1 M KCl, 1 mM CaCl2, pH 6.0.26



E.K. Schmitt and C. Steinem

removal of the solvent, leaving an about 5 nm thin bilayer behind.

This procedure allows to quickly assess whether a nano-BLM has

been successfully formed.26

In order to elucidate if a lipid bilayer is fully covering the porous substrate, characteristic membrane parameters are extracted

from the impedance spectra. The data analysis is based on an electrical model (Fig. 2 A, inset). The very simple equivalent circuit

comprises three elements, which represent the different components of the electrochemical system. It is composed of a parallel

RC-element, namely an Ohmic resistor Rm and a capacitor Cm,

which represent the electrical behaviour of a lipid bilayer, in series

to another Ohmic resistor Rel that represents the electrolyte solution and the wire connections. The obtained impedance spectra are

characterized by the electrolyte resistance Rel in the high frequency

regime (5·104–106 Hz), and the capacitance Cm at frequencies below 5·104 Hz. At frequencies below 3·10-1 Hz a second Ohmic resistance is discernable, which is attributed to the membrane resistance Rm. Fitting the parameters of the equivalent circuit to the

data results in good agreement between data and fit with a membrane capacitance of Cm = 11.7 nF and a membrane resistance of

Rm = 4.7 Gȍ. Taking the porosity of the alumina substrate of 33 %

into account, which was determined by scanning electron microscopy,24 an active area of A = 2.3 mm2 is calculated. Thus, the mean

capacitance of Cm = (14.9 ± 3.3) nF (n > 50) translates into a specific capacitance of Cm,sp = (0.65 ± 0.2) μF cm-2. Cm,sp is defined as

Cm A-1. This value agrees well with those obtained for classical

BLMs27 and supports the idea that single lipid bilayers have been

formed across the pores.

(ii) Long-Term Stability of Nano-BLMs

The achieved membrane resistances of the nano-BLMs are

similar to those of traditional BLMs and are obviously sufficient to

perform single channel recordings. However, the suitability of

classical BLMs in biosensor applications is limited, as the BLM

ruptures at a certain point, i.e., owing to mechanical distortion,

leading to the loss of the insulating membrane in one single event.

In contrast, in nano-BLMs each bilayer, suspending a single pore,

is decoupled from the others and can therefore rupture separately,

as proven by visualizing the process by fluorescence microscopy28

Rm / W

Ion Channels and Transporters in Pore-Suspending Membranes

























Figure 3. Time course of the membrane resistance Rm obtained by EIS analysis of a

nano-BLM in a frequency range of 10-2-106 Hz. The membrane resistance was extracted from the impedance data by fitting the parameters of the equivalent circuit

shown in the inset of Fig. 2A to the data.24

and scanning ion conductance microscopy (SICM).29 Monitoring

this process by EIS results in a decreasing membrane resistance Rm

over time as shown in Fig. 3.

In the first 48 hours after membrane formation, Rm drops from

7 Gȍ to 1 Gȍ. During this time period, the membrane is well suited for single channel measurements. Then, the membrane resistance decreases further leading to membrane resistances of

around 150 Mȍ after 72 hours and 2 Mȍ after 120 hours. After

132 hours, the membrane resistance has reached 1 Mȍ. From a

statistical analysis, the lifetime of the nano-BLM with membrane

resistances larger than 1 Gȍ was calculated to be (1.5 ± 0.5) days

(n > 30). These data prove that the long-term stability of nanoBLMs is by far greater than the one of classical BLMs. Notably, in

some cases it happened during the time course of the experiment

that the membrane resistance increased again, which indicates that

a self-healing process might occur within the membrane. This implies that nano-BLMs underlie permanent fluctuations affecting

the resistance and capacitance of the membrane.


E.K. Schmitt and C. Steinem

These data show that nano-BLMs are a powerful membrane

system that overcomes the disadvantage of poor long-term stability

of classical BLMs. However, classical BLMs have also been developed further in recent years in order to diminish their drawbacks such as long-term stability and applicability in chip based

assays. Glass supports with a single pore of 100-400 nm in diameter served as supports for BLMs, which are stable for up to two

weeks.30 In another approach, giant unilamellar vesicles are sucked

on a single hole by means of low pressure, which ruptures the vesicle and generates a bilayer within seconds.31 Nevertheless, these

systems still only provide a bilayer covering one single hole,

whereas nano-BLMs are composed of more than 109 individual



Pore-Suspending Membranes on CPEO3

(i) Impedance Analysis of Pore-Suspending Membranes on

Porous Alumina with Fully Opened Pore Bottoms

Nano-BLMs proved to be a robust system for the investigation

of ion channels on a single channel level (see Section III). Due to

the method of preparation, nano-BLMs still contain some organic

solvent, which is reflected in the mentioned fluctuations in membrane resistance and the obtained lateral diffusion coefficients.28

Several membrane proteins lose activity in the presence of organic

solvents such as n-decane and hence bilayers prepared without addition of solvent are highly desirable. For BLMs this has been realized by the method of Montal and Mueller, who established solvent-free lipid bilayers by the membrane folding method.32

Already some years ago, we followed a strategy to form poresuspending bilayers starting from large unilamellar vesicles

(LUVs).22,33,34 With this method, it is not only possible to gain solvent-free pore-spanning membranes, but in addition it holds the

potential for establishing lipid bilayers with high protein density.

As a starting point, porous substrates were functionalized with a

thiol-component to chemically distinguish between the upper surface and the inner pore walls, which should prevent the fusion of

vesicles within the pores. Yet, these membranes proved to be rather leaky, as shown by EIS.34 To achieve highly insulating pore-

Tài liệu bạn tìm kiếm đã sẵn sàng tải về


Tải bản đầy đủ ngay(0 tr)