Case study

Developing new therapies targeting
P2X receptors

Tools:
Port-a-PatchProf. Samuel Fountainfeatured by Nanion Technologies
Samuel Fountain is chair of Pharmacology andP2X receptors are ligand-gated non-selective cation channels

Associate Pro-Vice-Chancellor at the University(permeable to Na+, K+, and Ca2+) that open within millisecondsof East Anglia. His research focuses on theof ATP binding. They have a widespread tissue distribution andfunction and pharmacology of P2X receptors,

play roles in a variety of essential cellular functions. Numerous

a family of ligand-gated cation channels

studies have shown the involvement of different members ofactivated by extracellular ATP. Known for his

the P2X receptor family in pain, irritation, hypersensitivity, andcontribution to the pre-clinical development

inflammation, and thus, they are now regarded as promisingof Gefapixant, a P2X3 receptor antagonist,

drug targets.

recently approved as a new medicine.

The human genome encodes seven P2X receptor subtypes(P2X1 – P2X7) capable of forming both homomeric and
ATP is widely recognized as the “universal energy currency”
of cells, providing a readily accessible source of energy for
all cellular processes. However, ATP is not only present inside
cells. Many cell types release ATP into the extracellular space.
Although the mechanisms and physiological reasons for suchrelease are sometimes not very well understood, it is known
that, for example, under inflammatory conditions, ATP canbe released both passively from cells undergoing lysis andactively via vesicular and channel-mediated processes.
ATP is also known to be released alongside a range of otherneuromediators, such as acetylcholine, norepinephrine,
glutamate, GABA, neuropeptide Y, and thus, ATP is currently
recognized as an important autocrine and paracrine
transmitter.
Extracellular ATP exerts its signaling function via specialized cellsurface receptors, called P2 purinoceptors (for reference, P1
receptors are metabotropic adenosine receptors). These P2
receptors are of two types – metabotropic P2Y receptors andionotropic P2X receptors. P2Y receptors typically mediate slow
responses to ATP or other nucleotides, whereas P2X receptors
mediate fast responses solely to ATP.

Nanion's Port-a-Patch platform
is a turn-key miniaturized patch clamp systemenabling the user to rapidly generate high quality
data, regardless of experience. Behind its small
and compact appearance lies sophisticatedtechnology, producing high-quality measurementswith giga-seals and high success rates.

heteromeric trimers. Native P2X receptors are characterized bysignificant heterogeneity in functional properties. In most cell
types, under standard physiological conditions, P2X receptors
carry a depolarizing cationic current, although the kineticsof this current, as well as the involvement of different cations,can vary greatly depending on the cell type. In some cells
however, P2X channels are also significantly permeable toanions.

“The Port-a-Patch is used by those in my group whose main
focus is electrophysiology, but also those who need to undertake
shorter-term electrophysiological studies. The latter is enabledby the instrument’s accessibility and limited training requirementcompared to manual patch-clamp.”
Prof. Samuel Fontain, University of East Anglia

Typically, the biophysical properties of P2X receptors, like otherassociated with the manual patch clamp technique.

ion channels, are studied using the patch clamp technique.

Modern APC systems can measure currents from 1 to 384

This powerful Nobel Prize-winning technique allows for thecells simultaneously. These systems are designed for ease-measurement of ion channel currents. However, despite its

of-use and frequently offer automated data analysis, saving

advantages, the patch clamp technique is often viewed as a

researchers a significant amount of time.

complex, time-consuming, labor-intensive, and low-throughput
approach. It demands significant training and technical

Professor Samuel Fountain is one of those visionary groupexpertise and involves the use of large, complex, andleaders who employs the latest APC technologies in hisexpensive equipment. Nevertheless, despite these limitations,research, which focuses on the function and pharmacology ofmanual patch clamp has remained the gold standard forP2X receptors. He has equipped his lab with the Port-a-Patch

studying ion channels, with little alternative to gather the same

system, a compact and user-friendly semi-automated patch

information-rich data content.

clamp setup from Nanion. The Port-a-Patch enables users torapidly generate high quality data, regardless of experience.

Thankfully, over the last two decades, several companies

This makes it ideal as an easy-to-use tool for learning patch

have developed a technique to automate patch clamp.

clamp electrophysiology and it can be implemented in many

Recent advancements in automated patch clamp (APC)

aspects of drug discovery and basic ion channel research.

instrumentation have mitigated many of the limitations

David Richards performs patch clamp experiments using Port-a-Patch. The adapted figure
shows MK-7264 activity with representative whole cell patch clamp recordings for human P2X3(a), P2X2/3 (b) currents in 1312N1 stably transfected cells. Fully recovered P2X3 currents wereevoked by 10 μM α,β-meATP applied for 1 s every 4 min and 10 μM α,β-meATP applied for 2 sat 30 s intervals for P2X2/3 currents.1

Since 2016, Prof. Fountain and colleagues have utilized theIn two additional recent studies, Prof. Fountain and his colleagues

Port-a-Patch to study various P2X receptors such as P2X2, P2X3,employed the Port-a-Patch to examine the mechanism of action“The Port-a-Patch has been a highly reliable instrument for

be implicated in neuropathic pain, inflammation, and arterial

P2X2/3, and P2X4. Speaking about the Port-a-Patch, he states,of various compounds on P2X4 receptors, which are known tous. We have used it to record from cell lines, but also primary

tone regulation. One study identified taspine as a unique natural

cells from human tissue. The instrument is used by those in myproduct that inhibits P2X4 activity through phosphoinositidegroup whose main focus is electrophysiology, but also those

3-kinase suppression rather than receptor antagonism2. In anotherwho need to undertake shorter-term electrophysiologicalstudy, they delved deeply into the mechanism by which the small

studies. The latter is enabled by the instrument’s accessibility

molecule 5-BDBD inhibits P2X43. By combining Port-a-Patch-based

and limited training requirement compared to manual patchclamp.”

electrophysiology with molecular modelling and other methods,
they pinpointed the essential residues for 5-BDBD binding. Greatstudies… and beautiful currents recorded with the Port-a-Patch.

In a recent project funded by Afferent Pharmaceuticals andMerck Sharp & Dohme Corp, the team made significant

The Port-a-Patch is easy to use and the solutions can becontributions to the development of Gefapixant, an oral P2X3

exchanged manually, reducing the amount of compound

receptor antagonist currently approved in Japan and Switzerland

needed for the experiment. Alternatively, for fast and accurate

for treatment of adults with refractory or unexplained chronic

exchange of solutions, the temperature-controlled Externalcough. Utilizing the Port-a-Patch, they examined Gefapixant’s

Perfusion System or the Internal Perfusion System, can be used,action, potency, and kinetics, discovering its nanomolar potency

key add-ons for Prof. Fountain’s experiments.

for P2X3 and P2X2/3 receptors, with marginal selectivity for P2X3
over recombinant P2X2/3 receptors1.

Prof. Samuel Fountain and his group at University of East Anglia. Their main research interest is the role ion channels
play in the cellular and molecular mechanisms that underlie cardiometabolic physiology and disease with anemphasis of blood vessels, adipose tissue, and the autonomic and sensory nerves that control these tissues. For
more information, visit https://research-portal.uea.ac.uk/en/persons/samuel-fountain/projects/.

“The rapid perfusion system has been key for us studying the modeof action of drug-like molecules at ligand-gated ion channels.
Working with small volumes is also useful when working small
scales of experimental compounds, natural products, animal toxins
and antibodies.”
Prof. Samuel Fontain, University of East Anglia

“The rapid perfusion system has been key for us studying themode of action of drug-like molecules at ligand-gated ionchannels. The internal perfusion system also allows for rapid

References1.

at human P2X3 and P2X2/3 receptors and in vivo efficacy in models ofexchange of intracellular components for the studies ofsensitisation. Br J Pharmacol. 2019 Jul;176(13):2279-2291.

regulator and signalling pathways. Working with small volumes
is also useful when working small scales of experimental

Richards D, Gever JR, Ford AP, Fountain SJ. Action of MK-7264 (gefapixant)2.

Nadzirin IB, Fortuny-Gomez A, Ngum N, Richards D, Ali S, Searcey M, Fountaincompounds, natural products, animal toxins and antibodies”,

SJ. Taspine is a natural product that suppresses P2X4 receptor activity via

says Samuel Fountain.

phosphoinositide 3-kinase inhibition. Br J Pharmacol. 2021 Dec;178(24):48594872.

Samuel Fountain’s lab is currently using the Port-a-Patch
to explore the heteromerization of P2X receptors and tocharacterize the action of some new drug-like molecules
targeting P2X4. We’re looking forward to exciting new results.

3.

Bidula S, Nadzirin IB, Cominetti M, Hickey H, Cullum SA, Searcey M, Schmid R,Fountain SJ. Structural Basis of the Negative Allosteric Modulation of 5-BDBD at
Human P2X4 Receptors. Mol Pharmacol. 2022 Jan;101(1):33-44

Contact Information
Prof. Samuel Fountain
Professor of Pharmacology
School of Biological Sciences
University of East Anglia
Norwich Research Park
Norwich NR4 7TJ, UK
https://research-portal.uea.ac.uk/en/persons/samuel-fountainAcknowledgments
We thank Samuel for sharing his views on theimportance of automated patch clamp in P2X ion
channel research. We are grateful for your valuable
insights and wonderful collaboration.

Nanion Technologies GmbH I Ganghoferstr. 70a, 80339 Munich, Germany+49 89 2190 95-0 I info@nanion.de I www.nanion.de