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Current Topics in Medicinal Chemistry, 2015, 15, 631-637
631
Ion Channels as Medicinal Targets of Biological Toxins: The Impact of
Automated Patch-Clamp Electrophysiology
Arturo Picones*
Laboratory of Channelopathies. Unit of Biophysics and Pharmacology of Ion Channels. Instituto de
Fisiología Celular Universidad Nacional Autónoma de México
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Abstract: Patch-Clamp electrophysiology, the “gold standard” for the functional study of ion channels has become automated. This innovative technology, already over a decade old, has revolutionized
the strategies for the search of medicinal compounds which now can be screened at unprecedented
speed, approaching the high throughput standards required by primary screening campaigns emblematic of the pharmaceutical and biotechnology industries. Consequently, an acceleration of the discovery and development of new drugs targeting ion channels is expected. These pore forming membrane
proteins had been relegated as crucial therapeutic drug targets due to the difficulty of their experimental analysis. This new technological approach has begun to impact the finding of new toxins which are conspicuously
relevant as medicinal agents given their extraordinary potency and specificity when acting upon ion channels. The introduction of automated patch-clamp instrumentation to academic labs and institutions pursuing the finding of new pharmacological agents, peptide toxins in particular, will certainly enrich these scientific and technological fields by contributing
with their always prolific generosity of originality and innovation.
Keywords: Automated patch-clamp electrophysiology, Ion channels, Toxins.
1. INTRODUCTION
ION CHANNELS AS MEDICINAL TARGETS
Be
Contrary to what would be expected, given their relevance as major “drugable” targets in key therapeutic areas,
ion channels have been historically relegated to a level of
limited success in the discovery and development of new
therapeutic agents, which could help normalize their dysfunctions associated with pathological conditions. Such
conditions range from diarrhea to degenerative diseases of
the nervous system, and remarkably include cardiac arrhythmias, immunologic deficiencies, and metabolic dysfunctions. This demoted status has been mainly due to the
difficulties that their direct experimental study entails, requiring an amount of effort and resources, in terms of
work, time and financial investment, deemed prohibitive by
the pharmaceutical and biotechnological industries. The
workflow of these industries relies on high throughput
screening (HTS) of large chemical compound libraries to
discover potential drug candidates.
The present review centers on how the application of
automated patch- clamp (APC) electrophysiology, is impacting the discovery and development of new peptide-like drugs
targeting ion channels, particularly toxins. In essence, APC
accelerates the pace at which the action of potential or actual
therapeutics on ion channels proteins is detected, analyzed
and understood.
*Address correspondence to this author at the Instituto de Fisiología Celular. Universidad Nacional Autónoma de México;
Tel: 5622 9250 ext. 44 630
E-mails: apicones@email.ifc.unam.mx or arturopicones@yahoo.com
1873-5294/15 $58.00+.00
The molecular location of the vast majority of ion channels, the plasma membrane, makes them readily available for
the action of pharmacological agents. The experimental approach to the study and understanding of the genetics, structure and function of these complex macromolecules has accomplished colossal achievements. 450 genes coding for ion
channels are currently known and the structure of several of
them has been resolved at a resolution of few Angstroms.
The experimental examination of ions channels has achieved
the most remarkable level of resolution, being able to detect
the activity of a single macromolecule. This is done as it
occurs in real time, under tight regulation of their electrochemical vicinity and, essential to their functionality as
bioelectrical conductance devices, commanding masterfully
the electric field that governs their conformational operating
states. Despite the functional and structural knowledge available, only 13% of the approved drugs in the market exert
primary action on ion channels [1-4], clearly pointing out to
the vast number of opportunities for addressing a long list of
unmet therapeutic needs.
BIOLOGICAL TOXINS, CHEMICAL EFFECTORS
PROVIDING EXTRAORDINARY MEDICINAL OPPORTUNITIES
Toxins are noxious or poisonous substances which are
the product of the metabolism of a living organism. Because
of this the terms biological toxin and biotoxin are more properly used. Toxins can be small molecules, peptides or proteins.
© 2015 Bentham Science Publishers
632 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 7
Effectors in behaviors such as protection from predators
and catching of prey, toxins have evolved in a diversity of
venomous plants, animals and microorganisms. By the beginning of the 1990s, thanks to the seminal studies of B.
Olivera, M. Adams, and L. Possani, among others, it was
clear that most animal venoms constitute a complex combination of peptides and proteins with only a minority of them
being lethal [5].
and biotechnology industries. Implementation of APC technology in the industrial setting undoubtedly helped to demonstrate that this technology was real and very effective for
the pharmacological screening of a much larger number of
compounds acting on ion channel currents recorded under
voltage clamp. APC technology and instrumentation is now
firmly established within the pharmaceutical and biotechnology industries. In direct linkage with this, the number of private Contract Research Organizations (CROs) offering
screening and pharmacological profiling services with APC
instrumentation has increased significantly [16].
Other types of automated instrumentation based on different technologies, such as ligand-binding, fluorescencebased readouts and ion-flux measurements have also appeared and been marketed [17], their higher throughput was
initially attractive from the cost-effective point of view, but
their obvious lack of membrane voltage control, essential for
the regulation of channel functional states, with the concomitant generation of false positives and false negatives, made
them languish in comparison to automated electrophysiology, particularly when the throughput of the latter became
even higher . In this respect it should be mentioned that there
are reports of well-controlled fluorescence-based thallium
flux functional assays for different ion channels that show a
good correlation with manual and automated electrophysiology determinations and are capable of meeting industrial
HTS standards by performing at a capacity of 384- or 1536well formats [18-20].
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Discussing venom toxins targets is virtually equivalent to
speaking of ion channels. Since long ago, venoms and their
peptide components have been recognized as very potent and
highly selective modifiers of ion channel function. A number
of biotoxins not only have been tested as chemical substances with potential as drug candidates but also utilized as
pharmacological tools assisting in revealing the structure and
function of fundamental biophysical features of the ion
channels they bind. Given the aforementioned properties,
many peptide toxins are in clinical trials and even one has
been already marketed (ziconotide/Prialt, [6-9]), it is now
clear the significant advantage of applying APC technology
and instrumentation to elevate the screening of novel potential toxin-derived medications to unprecedented rates. The
application of this technology is already transforming the
strategy of how to approach the search for innovative medicinal chemicals capable of modulating ion channel activity.
Arturo Picones
Many excellent reviews have been published recently addressing the richness of pharmacologic action, structure interaction and therapeutic potential of toxins and toxinderived compounds with recognizable activity on ion channels. In several of those publications their analyzed contents
have been cleverly presented in comprehensive and welldesigned summarizing tables, covering a vast range of
biotoxins and the different types of ion channels they act
upon [1-12].
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REVOLUTIONIZING THE SCREENING OF SUBSTANCES ACTING ON ION CHANNELS
Electrophysiology, remarkably since the inception of the
patch-clamp (PC) technique [13], is undoubtedly considered
the “gold standard” for the study of ion channels functionality. Any claim regarding an ion channel, starting by its mere
existence, has to be proven ultimately by demonstrating the
recording of the corresponding ionic current going through
such putative protein entity. The already mentioned capabilities of exquisite resolution of ionic current detection, precise
electrochemical manipulation, and control of transmembrane
voltage, endow this technique with such privileged status.
Despite all these advantages, the low throughput and high
personnel cost, both requirements derived from the labor
intensive evaluation of individual compounds, had prevented
its implementation in industrial settings.
In 2003 a revolution began with the advent of the automation of the PC technique, a technological expansion that
very few believed possible ever to achieve or even to conceive. As could be expected, a good deal of resistance arose
from within the field (see for instance [14, 15]). Since the
beginning, given the nature of the initial scope of this novel
technology and, equally important, the inevitable high cost
of the instrumentation and consumables to keep it working,
the natural place for this novelty has been the pharmaceutical
Some initial PC automation attempts were made with
systems designed and constructed to closely emulate the operational maneuvers performed in a classic manual PC rig.
This notion led to somehow cumbersome designs and instrumentation assembles.
Arising from a completely different approach, the concept of the “planar patch-clamp” implemented in a “chip”
produced a basic, simple and practical, as well as effective
and even elegant solution. The requirements of micromanipulation, mechanic isolation (anti-vibration table) and optical visualization and control (microscope) became completely unnecessary, indeed irrelevant.
The planar PC conception was implemented into ad hoc
microtiter plate-like devices. This multiwell design allows
assay miniaturization and integration of microfluidics to the
now distinctive “multi-recording chamber” (a non-reusable
consumable) of these automated systems, permitting the parallel recording of the electrical currents from ion channels
present in cells patch-clamped after being gently attracted,
by negative hydrostatic pressure (suction) and electrically
sealed, to a 1-2 µm diameter apertures perforated on the bottom of each well. This well bottom, if made out of glass or
Silicon Dioxide (SiO2), is capable of forming Giga Ohm
(GΩ, 109 Ω) electric seals on the cell membrane, the required
condition for a truly high quality resistance PC recording, in
all respects comparable to those obtained by classic/standard
manual PC.
In 2005, Molecular Devices introduced the concept of
Populated Patch Clamp (PPC) consisting of recording from
multiple cells each patched in one of 64 different apertures
residing in each well of the 384-well format of the so called
Ion Channels as Medicinal Targets of Biological Toxins
Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 7
633
not surprising, most of those results document assay validations studies with well-known hERG channel inhibitors, utilized as reference compounds and providing positive controls.
The somehow more exciting results, those related to the proprietary chemicals of the companies, are well kept undisclosed, at least until they have to be made public in the
documentation presented to a government regulatory agency
(for instance as part of an Investigational New Drug, IND,
application to the US Food and Drug Administration, FDA).
At present, the most successful instruments and their
manufacturers are QPatch, manufactured by Sophion Biosciences, PatchLiner by Nanion Technologies, IonFlux by
Fluxion Biosciences, and PatchXpress and IonWorks Quattro by Molecular Devices. Although not parallel multichannel high throughput instruments, Nanion’s Port-a-Patch
and CytoPatch from Cytocentrics, have also made their
mark, producing high quality studies in the field of automated electrophysiology. The newest generation of APC
instrumentation, launched in 2014, is already reaching HTS
capabilities compatible with the requirements of industryclass primary screening campaigns: Sophion’s Cube, Nanion’s Synchropatch, and Molecular Devices’ IonWorks Barracuda. Except for IonWorks Quattro and IonWorks Barracuda, all the mentioned instruments are capable of producing
real tight Giga-Ohm seals, in all respects comparable to classic standard manual PC. Despite the limitation of not forming Giga-Ohm seals, IW Quattro and IW Barracuda record
ion currents utilizing the perforated PC technique (by means
of amphotericin B, an antifungal drug forming permeable
pores in membranes), then offering the advantage of better
preserving the cytoplasmic content of the recorded cells.
Although not offered by all platforms, compensation for series resistance and capacitance further assures the high quality and reliability of the obtained experimental results.
A straightforward analysis performed on the number of
publications devoted to automated electrophysiology that
have appeared in PubMed since 2003, when the first APC
instruments became commercially available [40], reveals a
close exponential growth in the number of these publications
in the past twelve years (Fig. 1). Among those publications,
there is a number of noteworthy reviews that have expertly
highlighted the prominent stages and accomplishments, and
also weaknesses and deficiencies (many of the latter successfully resolved), during this still short and thrilling story of
the origins and progression of the APC revolution [41-49].
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PatchPlate. IonWorks Quattro became the first instrument
based on this new technology [21]. The advantages of these
multi-hole systems are notable by improving consistency and
success rate of the experimental results. The first of these
improvements is obtained by measuring the average membrane current of many cells in parallel, thus the cell-to-cell
variability in the amount of whole-cell currents is minimized. The second is confirmed by regularly having more
than 95% of the wells getting useful data.
Be
Of exceptional importance, arguably the central driving
force in the development of this technology, has been the
pharmaceutical industry’s requirement to comply with safety
pharmacology standards, prominently those in direct relation
to the acquired, drug-induced Long QT liability associated
with the blockade of the human Ether-a-go-go Related Gene
(hERG) channel. This potassium permeable channel, responsible for the most part of the repolarizing phase of the cardiac action potential (AP), is the molecular entity accountable for the inherited Long QT syndrome type 2 and constitutes a well-known pharmacologically promiscuous protein
that can be blocked by an impressive diversity of compounds. Blockage of the hERG channel by these compounds
prolongs the cardiac AP provoking such risky events as the
Torsades de Pointes, a form of ventricular arrhythmia, which
can lead to cardiac fibrillation and sudden death. It is clear
that drug development companies have shown a determined
interest in detecting drug candidates with such undesired
potential as early as possible in the drug development process. Extreme occurrences of cardiac toxicity events of this
kind have led to the withdrawal of medications already in the
market [22]. APC electrophysiology instruments have been
extensively committed to hERG channel data production and
more recently also to other ion channels relevant to cardiotoxicity issues. This has generated a significant amount of
publications [examples of reviews on the subject are: 23-32],
and some extensive studies: [33-39]. Nevertheless, as it is
Fig. (1). Number of publications on automated patch-clamp electrophysiology since its inception.
APC technologies have already contributed to the field of
small molecule pharmaceuticals for most types of ion channels: sodium channels [50-53]; potassium channels (Herrington et al., [54], using IonWorks Quattro 384-well automated
platform, screened approximately 200,000 compounds and
identified two which potently inhibited Kv2.1 and Kv2.2
channels; and [55-58]); calcium channels [59-62]; ligandgated channels [63-68]; TRP channels ([69], reviewed in
[70]).
ACCELERATING THE IDENTIFICATION OF MEDICINAL TOXINS TARGETING ION CHANNELS
The search for new therapeutic drugs acting on ion channels has itself distinctively shown a more efficient strategy
by the adoption of screening campaigns based on targetfocused compound libraries. Such libraries are collections of
chemicals deliberately designed or gathered on the basis of
detailed knowledge of the type of targeted proteins in question. In the case of ion channels, where the amount of structural information is not particularly abundant, the strategies
to form focused libraries has arisen from broader chemogenomic principles, relying on sequence, mutagenesis and his-
634 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 7
torical biological data [71, 72]. In order to become viable
pharmaceutical products, toxins, as in the case of any other
peptide substance with potential medicinal purposes, have to
overcome well known limitations such as: short duration of
action, inadequate receptor subtype selectivity and lack of
oral bioavailability [73].
Of manifest relevance for this review, experimental
works have been published documenting the successful implementation of diverse pharmacological assays for the
screening of biotoxins, tried on a variety of ion channels,
taking advantage of the higher throughput capabilities of
different APC platforms. The following paragraphs describe
examples of this increasing interest and production in this
particular field.
voltage-gated potassium channels. Probes based on these
polypeptide prototypes could have a promising future as reporters for the action of therapeutic drugs [79].
In a recent publication, the IonFlux instrument was successfully verified as a fast and reliable technology for the
screening of nine different marine toxins, all of them analogs
of saxitoxin (STX), tested on seven different cloned sodium
channels expressed heterologously [80]. This study has very
interesting commercial connotations since it was developed
within the framework of introducing a faster and much more
reliable assay for the evaluation of levels of toxicity in marine products for human safe consumption.
The aforementioned examples clearly demonstrate the effectiveness of the use of APC technology and instrumentation in the characterization of the action of a diversity of
toxins on ion channel activity.
The actuality of extending the automation of the PC
technique to the recording of APs under current clamp mode
in optimized preparations of human cardiomyocytes and
neurons, both derived from induced pluripotent stem cells
[27, 81-86], will certainly widen the investigation of the effects of biotoxins into the realm of a more physiological cellular manifestation (the AP) and cell preparations.
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Randall et al. [74] optimized the recording of Na+ currents of the human rhabdomyosarcoma cell line SJ-RH30 for
an APC system. They found that blockage of those currents
with tetrodotoxin (TTX) and the toxin m-CTx-GIIIB was
consistent with the pharmacological phenotype of Nav1.4
channels. Their observations were very similar in both automated and conventional PC.
Arturo Picones
In addition to showing that the recording of Kv2.1 channel currents by the IonWorks Quattro system was highly
resistant to serum concentration as high as 33% and relatively insensitive to plasma, Ratliff et al. [75] also presented
the quantitative effects of the gating modifier guangxitoxin1E, demonstrating the effectiveness of such APC instrument.
Be
Also employing automated electrophysiology, Revell et
al. [76] confirmed the high antagonistic potency of the spider
venom peptide Huwentoxin-IV against hNav1.7, a voltagegated sodium channel involved in the generation and conduction of neuropathic and nociceptive pain signals. Interestingly, single residue mutations in four distinctive positions
of this toxin molecule were revealed to be important modulators of its pharmacological potency without affecting the
original selectivity profile on the related channel hNav1.5.
The adequacy of the QPatch was validated by Jenkins et
al. [58] as a comparable but much faster approach to study
the action of Charybdotoxin (ChTx, a 37 amino acid neurotoxin from the venom of the scorpion Leiurus quinquestriatus hebraeus) and other inhibitors and activators of the intermediate-conductance Ca2+-activated K+ channel KCa3.1,
stably expressed in the human embryonic kidney cell line
HEK293.
Experimental work successfully carried out with the single recording portable APC system Port-a-Patch, confirmed
the predicted higher potency and selectivity of a mutation of
the ShK peptide (from the sea anemone Stichodactyla helianthus) acting on the voltage-gated potassium channel
Kv1.3, a well-established target for treatment of autoimmune
diseases [77]. This same group of investigators, again utilizing the same APC system, also has characterized the therapeutic potential of the scorpion toxin HsTX1 as a potent and
selective blocker of Kv1.3 [78].
A QPatch system assisted in the electrophysiological testing of a number of variants of synthesized chemoselective
derivatives of the tarantula toxin guangxitoxin-1E (GxTX),
an inhibitory cystine knot peptide that binds to Kv2-type
On the other hand, the successful application of APC
technology to the experimental recording of the electrophysiological properties of neuronal circuits, specifically
those preserved in brain slices, still represents an ambitious
and challenging project (see for instance [87]). The expectation is indeed great, given the possibility of automating the
study of neuronal function at the synaptic and network levels, providing significant advantages in the search of improved therapeutic drugs. Nevertheless, the efforts along this
direction have only yielded quite complex apparatuses that
reproduce, by mechanizing and multiplying in parallel instrument arrangements, the operations of a manual PC rig
with still limited impression on the field [88-91].
PRESENCE OF APC INSTRUMENTATION IN ACADEMIA
In recent years a number of academic institutions, sensing the influence that the new technology of automated electrophysiology is exerting on the future of ion channel basic
science research and its evident practical and commercial
applications, have created laboratories and core facilities
dedicated to implementing and developing multidisciplinary
fundamental and applied science projects within the framework of joint efforts and resources by multiple academic
groups and even offering services on a fee-for-service basis
to external institutions and private sector companies [92-96].
Such development has been catalyzed by the offering of discounts and incentives by the manufacturing private companies, realizing that this type of policy will result in the commercial advantage of their business development. Given the
historic role played by academia one can expect a significant
enrichment of the field. Academic institutions have been the
generous source of most fundamental science and technological ideas, most of them released to the public almost as
soon as they are discovered with almost unrestricted openness and accessibility (at least until quite recently). This has
Ion Channels as Medicinal Targets of Biological Toxins
been lately recognized by the industry with the creation of an
increasing number of alliances between universities and private sector companies, big and small.
Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 7
[12]
[13]
CONCLUSION
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[16]
[17]
[18]
[19]
CONFLICT OF INTEREST
The author confirms that this article content has no conflict of interest.
ACKNOWLEDGEMENTS
The author is indebted to Dr. Arlet Loza-Huerta for her
valuable assistance in the compilation and edition of the bibliography for this review. This work was supported by the
Secretaría de Ciencia, Tecnología e Innovación del Distrito
Federal (SECITI) grant 039/2013 to AP.
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APC technology and instrumentation has already established a definite place in the study of ion channel function,
conquering acceptance and affordability beyond its initial
enclave within private companies of the pharmaceutical
and biotechnology industries, with an increasing presence
in academic institutions. This innovative technology has
not only made its mark by significantly expediting experimental data production but also influencing the strategy to
approach the research and development of new drugs targeting ion channels. APC technologies have already generated important initial contributions to the investigation of
the action on ion channels by known biotoxins and identify
new ones. Undoubtedly this new automated instrumentation is impacting the future of such rich source of potential
medicinal compounds.
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