screening technologies for ion channel drug discovery€¦ · it is modulated by a variety of...

1ISSN 1756-8919Future Med. Chem. (2010) 2(5), xxx–xxx 10.4155/FMC.10.180 © 2010 Future Science Ltd

Review

Ion channels are pore-forming membrane pro-teins that allow the passage of ions across cell membranes. Their ion conductivity is typically highly specific and has been used for their clas-sification into sodium, potassium, calcium, chloride and nonselective cation channels. In contrast to active transport mediated by mem-brane pumps and transporters, ion channels allow only passive transport of ions down their electrochemical gradient. The opening and closing (‘gating’) of ion channels is regulated by a variety of different stimuli including trans-membrane voltage, ligand binding, mechanical stress and temperature (Box 1). As the first two stimuli are the most common, these membrane proteins are also broadly grouped into voltage-gated and ligand-gated ion channels. Ion chan-nels are important for regulating the electrical properties of excitable cells such as neurons and myocytes. In many other cell types they con-tribute to important physiological processes such as hormonal secretion and blood pressure regulation (Box 1).

Ion channels constitute a complex gene family, and most are characterized by a pore-forming region (contained in the so-called a-subunit), which determines ion selectivity and mediates ion flux across cell membranes. In the human genome there are about 400 ion channel genes that correspond to about 1.3% of the genome [1]. Further complexity is generated as functional ion channels often are homo- or heteromeric protein complexes, which can co-assemble with accessory (b- and other) subunits thus creating a large number of physiological ion channel complexes with different functions

and pharmacology. Large numbers of disease-relevant ion channels have been identified that are often expressed in a tissue-specific manner. Novel assay technologies in conjunction with structural and mechanistic insights into channel function enable the discovery and development of selective and state-dependent drugs, which are on the horizon.

Ion channel diseases & drugsAlthough ion channels have long been implicated in many diseases [2,3], a causal link between ion channel dysfunction and disease was first estab-lished in 1989 with the identification of the cystic fibrosis transmembrane conductance regulator (CFTR) [4]. The term ‘channelopathies’ to define inherited ion channel disorders was introduced in the early 1990s with the discovery that sev-eral diseases associated with alterations in muscle membrane excitability were caused by missense mutations in voltage- and ligand-gated ion chan-nels [5]. Currently about 50 channelopathies in man are known [6] – ranging from neuromuscular disorders to kidney diseases – that have provided unique insights into the important role of ion channels in cellular function. In addition, many more disease conditions exist such as hyperten-sion, pain and insomnia, in which ion channels are critically involved. Most of the channelo-pathies are not targeted by current ion channel drugs since these drugs originated mainly from empirical pharmacology and represent symptom-atic rather than disease-modifying treatments. Between 1980 and 2004 a total of approximately 2416 drugs were launched in the major markets of which 171 act on ion channels. Today, ion

Screening technologies for ion channel drug discovery

For every movement, heartbeat and thought ion channels need to open and close. It is therefore not surprising that their malfunctioning leads to serious diseases. Currently, only about 10% of drugs with a market value in excess of US$10 billion act on ion channels. The systematic exploitation of this target class has started, enabled by novel assay technologies and fundamental advances of the structural and mechanistic understanding of channel function. The latter, which was rewarded with the Nobel Prize in 2003, has opened up an avenue for rational drug design. In this review we provide an overview of the current repertoire of screening technologies that has evolved to drive ion channel-targeted drug discovery towards new medicines of the future.

Georg C Terstappen1†, Renza Roncarati1, John Dunlop2 & Ravikumar Peri2†

†Authors for correspondence1Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, 53100 Siena, ItalyTel.: +39 0577 381302Fax: +39 0577 381208E-mail: [email protected] Research Unit, Pfizer, Eastern Point Road, Groton, CT, USATel.: +1 215 378 0727E-mail: ravikumar.peri@ gmail.com

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Review | Terstappen, Roncarati, Dunlop & Peri

Future Med. Chem. (2010) 2(5)2 future science group

Box 1. Physiological functions and gating of ion channels.

Na+ channels

Voltage-gated Na+ channels (Nav) are responsible for membrane depolarization leading to action potential initiation and propagation

in muscles and nerves.

The amiloride-sensitive epithelial Na+ channel (ENaC) mediates transport across the apical membrane of epithelial cells and is involved in Na+ reabsorption and regulation of blood volume and pressure. It is modulated by a variety of hormones that control activity via phosphorylation, GTP-binding proteins and lipid metabolites.

Related channels are the acid-sensing ion channels (ASIC), which are activated by pH 6.9 or higher H+ concentrations. These channels are expressed in brain and sensory neurons.

K+ channels

K+ channels represent the most widespread group of ion channels.

Voltage-gated K+ channels (Kv) define the resting membrane potential and modulate secretion of endocrine cells. In conjunction with

calcium-activated K+ channels (KCa

) (e.g., SK channels) they are involved in membrane repolarization following action potentials, thereby controlling the excitability of nerves and modulating synaptic transmission.

Ca2+-activated K+ channels (KCa

) represent the second major group of potassium-selective channels. SK and IK channels are voltage-insensitive and are activated by low concentrations of internal Ca2+ (<1.0 µM). In contrast BK and Slo channels are activated by voltage and internal Ca2+. K

Ca channels play important roles in many processes including excitability and Ca2+-dependent signaling in

electrically excitable and nonexcitable cells.

ATP-sensitive K+ channels (KATP

) couple cellular metabolism to electrical activity since elevation of intracellular ATP levels inhibits these channels. They are present in cardiac smooth and skeletal muscle, neurons, epithelial and pancreatic b-cells.

Inwardly rectifying K+ channels (Kir) comprise ROMK, IRK, GIRK and BIR. K

ir channels are a class of K+ channels that can conduct

much larger inward currents at membrane voltages negative to the K+ equilibrium potential than outward currents at voltages positive to it, even when K+ concentrations on both sides of the membrane are equal. Gating is regulated by ligands such as ATP, G proteins and phosphatidylinositol 4,5-bisphosphate. These channels govern the resting membrane voltage and play important physiological roles in many cells and organs, including brain, heart, kidney, endocrine cells, ears and retina.

The two-pore K+ channels (e.g., TWIK, TASK, TREK) are a unique structural subclass of K+ channels with twin pore-forming sequences (2P). These channels are activated by various physical and chemical stimuli including membrane stretching, temperature, acidosis, lipids and inhaled anaesthetics and are controlled by membrane receptor stimulation and second messenger phosphorylation. Several members are highly expressed in the central and peripheral nervous systems where they underlie background or ‘leak’ K+ currents.

Ca2+ channels

Voltage-gated Ca2+ channels (Cav) transduce membrane depolarization in a range of intracellular functions and mediate muscle

contraction, secretion, neurotransmission and gene expression.

Ligand-gated Ca2+ channels comprise the ryanodine receptor (RyR) and the IP3 receptor (IP

3R). Both channels mediate Ca2+

release from intracellular stores. Whereas RyR is predominantly expressed in muscle, IP3R is ubiquitously expressed and involved

in excitation–contraction coupling in smooth muscles, olfaction, synaptic transmission and neurotransmitter-mediated hormone secretion.

Store-operated channels (SOC) carry a highly Ca2+-selective, nonvoltage-gated, inwardly rectifying current, termed the Ca2+ release-activated Ca2+ current (I

CRAC) in many cell types. The activation of SOCs mediates longer term cytosolic Ca2+ signals and

replenishment of intracellular stores. The CRAC channel is the best characterized SOC.

Cl- channels

Voltage-gated Cl- channels (CLC) are primarily expressed in muscles or the kidney where they regulate excitability, transepithelial transport and regulation of cell volume and intracellular pH.

The cystic fibrosis conductance regulator (CFTR) functions as a cAMP-activated, anion-selective channel. It is expressed in epithelial cells in the airways, pancreas, intestine and other fluid-transporting tissues.

Ligand-gated Cl- channels comprise the glycine and GABAA receptors, which both mediate inhibitory synaptic transmission in the

adults. Glycine receptors are predominantly expressed in the brain stem and spinal cord, whilst GABAA receptors are present in the

brain and spinal cord.

Proton channels

Proton channels are characterized by high proton selectivity, strong pH and voltage dependence, and high sensitivity to Zn2+. Proton channels open only when the electrochemical gradient for protons is directed outward; hence, they carry only outward current in the steady state. Proton channels have been found in a variety of cells and are best characterized in phagocytes.

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Extracellularenvironment

[Na+]145 mM

[Ca2+]1.5 mM

[K+]4 mM

[Na+]12 mM

[K+]155 mM

[CI-]123 mM

[Ca2+]100 nM

[CI-]4.2 mM

Intracellularenvironment

Transmembrane potential (Vm)

Ion flux(current)

E = InRTZF

[Ion]out

[Ion]out

Screening technologies for ion channel drug discovery | Review

www.future-science.com 3future science group

channel drugs constitute approximately 10% of drugs currently on the market and around 15% of the top 100 best-selling drugs target ion channels. Examples are the Ca2+ channel blocker norvasc (Pfizer), the GABA

A blocker zolpidem

(Sanofi-Aventis), the nicotinic receptor modu-lator varenecline (Pfizer) and the Na+ channel blocker lamotrigene (GlaxoSmithKline). Still, only a small fraction of ion channels have been explored in terms of drug discovery. In fact, based on a recent ana lysis [7] it can be assumed that only about 15 ion channels are targeted by the current drugs on the market. Thus, there is an enormous potential for novel ion channel therapeutics.

Ion channel screeningWith the rational design of ion channel modula-tors still in its infancy, the emphasis for ion chan-nel drug discovery programs remains random or focused screening. Whereas high throughput screening (HTS) assays are well established for target classes such as G protein-coupled recep-tors (GPCRs) and enzymes, ion channel drug discovery is less developed due to the technical difficulties in developing such assays. In fact, the ‘gold standard’ for functional ana lysis of ion channels (patch clamp electrophysiology) [8] tra-ditionally required mechanically fabricated glass capillary electrodes and highly skilled microma-nipulation allowing acquisition of only tens of data points per day. Advances in the develop-ment of functional ion channel assays over the last five to ten years are currently enabling a more systematic exploitation of this important target class. All of these assays capitalize on the fact that activation of ion channels results in a move-ment (flux) of charged molecular species across a

cell membrane that is associated with a concomi-tant change in membrane potential, since both features can be measured as functional correlate of channel activation (FiguRe 1).

Two major and highly complementary strate-gies have emerged to support ion channel screen-ing in higher-throughput modalities. First, non-electrophysiological approaches such as direct ion flux measurements and optical approaches to ion flux and membrane potential measurements were established and validated in the field. Second, a number of higher-throughput approaches to the

Box 1. Physiological functions and gating of ion channels.

Nonselective cation channels

Cyclic nucleotide-gated ion channels are activated by cAMP or cGMP and are weakly voltage-dependent (CNG channels) or require hyperpolarization (HCN channels). CNG channels play a fundamental role in visual transduction, whereas HCN channels participate in the slow pacemaker depolarization of neurons and the sino-atrial node in the heart.

Nicotinic acetylcholine receptors (nAChR) mediate fast synaptic transmission in the nervous system and the neuromuscular junction.

Ionotropic glutamate receptors are classified based on their ligands as AMPA, NMDA, and kainate receptors. They mediate excitatory synaptic transmission in the brain. In particular, NMDA receptors mediate slow synaptic transmission, which is involved in synaptic plasticity.

Purinergic receptors (P2X) are ATP-gated ion channels that mediate postsynaptic potential in smooth muscle and in several brain neurons.

5-hydroxytryptamine 3 (5-HT3) receptors are localized in presynaptic nerve terminals where they regulate release of neurotransmitters

such as dopamine and neuropeptides.

Transient receptor potential (TRP) channels comprise six related protein families whose mechanism of action is currently poorly understood. These channels are ubiquitously expressed and underlie diverse functions responding to stimuli such as temperature, touch, osmolarity and pheromones. Polycystin kidney disease proteins and the vanilloid receptors belong to this superfamily of ion channels.

Figure 1. Activation of ion channels leads to ion flux down an electrochemical concentration gradient and concomitant changes in transmembrane potential. The quantitative relationship of these parameters, which can both be exploited for the configuration of functional ion channel assays, is defined by the Nernst equation.

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Strategies for the implementation of multiple assay platforms in ion channel drug discovery

Nonelectrophysiological assays• High-throughput (96/384/1536 well)• HTS and hit to lead optimization

Automated electrophysiology• Medium throughput (16/48/96/384 well)• HTS & hit to lead optimization

IonWorks HT & quattro (384 well)• Higher throughput• Intermediate content and quality

Giga seal platforms (16/48/96 well)• Intermediate throughput• High content and quality

Primary screeningCounter screeningSelectivity

Functional correlation

Optical

Radioligand binding

Atomic absorption

Advanced leads

Manual EP

Good

Bad

Confirmation



traditionally low-throughput and labor-intensive electrophysiological screening of ion channel-targeted compounds have now been estab-lished. Used in combination, these approaches permit a highly integrated and efficient process for ion channel drug discovery (FiguRe 2) that was not possible just a decade ago. As such this approach, the elements of which are discussed below, offers much promise for the development and introduction of the next generation of ion channel-targeted therapeutics.

Nonelectrophysiological screening technologies Assays measuring changes in

membrane potentialFluorescence spectroscopy-based assays with membrane potential-sensitive dyesChanges in membrane potential, as a con-sequence of ion channel activation, can be measured optically using fluorescence probes

(Box 2) that function as membrane potential indicators. Fast electrochromic probes such as Di-4-ANEPPS can detect voltage changes with microsecond resolution but display only low voltage sensitivity (~1–10% DF/F

0 per 100 mV)

[9,10] and are thus not suitable for the configura-tion of robust screening assays. Bis-barbiturate trimethine oxonols, on the other hand, have found widespread application for the develop-ment of ion channel screening assays. These flu-orescence dyes partition across cell membranes according to the membrane potential [11] and exhibit good voltage sensitivity (~10–100% DF/F

0 per 100 mV). If cells expressing an ion

channel under study are loaded with an oxonol probe, it will partition between the inside and outside to reach a partitioning equilibrium, which depends on the actual transmembrane potential (FiguRe 3). Upon channel activation the membrane potential change leads to repar-titioning of the dye until a new equilibrium is

Figure 2. Ion channel drug discovery employs multiple technology platforms. High precision, reproducibility, functional correlation and throughput are important prerequisites for all ion channel assays and need to be thoroughly validated while configuring screens. Throughput is the most important requirement that dictates the choice of a screening platform and a tiered approach is employed in most ion channel screens. The need for high throughput to screen large libraries of compounds for identifying hits employs indirect higher-throughput optical methods, radioligand binding and atomic absorption spectroscopy. The lead optimization phase employs a combination of indirect platforms as well as direct electrophysiology platforms. The choice of electrophysiology platform is determined by the need for voltage control, perfusion and throughput. Selected compounds can then be screened and processed in ‘gold quality’ by manual patch clamp assays. EP: Electrophysiology; HTS: High throughput screening.

Key TeRm

Random or focused screening: ‘Random’ compound libraries typically consist of 100,000s of chemical entities, whereas focused (often also called targeted or biased) compound libraries usually contain 1000–10,000s of compounds that are selected based on knowledge of the target protein and/or ligands binding to it. If no such information is available, typically random screening is performed in order to identify chemical molecules as entry point (‘hits’) for subsequent chemical optimization programs

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reached; hyperpolarization leads to a net extru-sion of the fluorescence probe from the cells, whereas depolarization results in an increase in cytoplasmic dye. In the screening practice, fluorescence changes are typically measured through the bottom of microplates as changes of cellular fluorescence and the fluorescence of the dye in the supernatant is either ‘opti-cally’ reduced as in the case of the often-used FLiPR® system (Molecular Devices, now MDS Analytical Technologies [12]) or completely eliminated by addition of a noncell-penetrating quenching dye such as Brilliant Black (reagent kits containing a mix of fluorescence indicator and quenching dye in one solution are com-mercially available). The latter allows measure-ments employing simple and less expensive fluo-rescence plate readers. One of the first such ion channel screening assays used the oxonol probe

DiBAC4(3) for the identification of openers

of an ATP-sensitive potassium channel [13]. DiBAC

4(3) was subsequently broadly applied

for the configuration of screening assays [14–18] despite the major limitation of its having a slow response time in the range of minutes. More recently, nondisclosed membrane potential-sensitive fluorescence dyes were introduced in form of assay kits (e.g., FMP, MPAK; MDS Analytical Technologies), which display faster response times (in the range of tens of seconds) while good voltage sensitivity is maintained. For a variety of different ion channels (e.g., sodium, potassium, chloride (GABAA) and nonselective cation channels) the successful development of screening assays using such kits has been described [19–22]. Fluorescence assays with membrane potential-sensitive dyes are fully compatible with the requirements of

Key TeRm

Quenching dyes: The concept of the addition of a noncell-penetrating quenching dye such as Brilliant Black in order to eliminate ‘background’ fluorescence of excessive fluorescence dye in cell supernatants of assays employing membrane potential-sensitive dyes was originally introduced in order to enable such measurements with conventional fluorescence plate reader set-ups. Subsequently, the use of a quenching dye was also employed for assays using FLiPR® systems. Currently these quenching dyes are part of commercially available kits for such assays

Box 2. Chemical structures of commonly used fluorescence probes for optical ion channel screening assays.

N

N OO

OH

N

N

OO

O

DiBAC4 (3)

O

NN

O

OOH

Cl

O

O P

O OH

O

O

O

O

O

CC2-DMPE

N

N OS

OH

N

N

SO

O

Fluo-3

O

Cl

OOH

Cl

N(CH2COOH)2

OO

N(CH2COOH)2

DiBAC2 (3)

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Extracellularenvironment

Intracellularenvironment

Ion flux Oxonol =FRET acceptor

CC2-DMPE =FRET donor

FRET

Ion flux

Oxonol =FRET acceptor

CC2-DMPE =FRET donor

∆Vm

Extracellular environment

Intracellular environment

Ion flux

∆Vm

Oxonol dye

Transmembranepotential

Oxonol dye

Oxonol dye

Oxonol dye



HTS. However, the more indirect nature of the assay readout results in a high number of ‘false positive’ hits that need to be ‘filtered out’ in subsequent assays in a screening cascade. In addition, lack of voltage control prohibits identification of bona fide state-dependent ion channel modulators, which is a particular dis-advantage for the large group of voltage-gated ion channels.

FRET-based assays with membrane potential-sensitive dyesIn order to avoid the problems associated with the slow response time of DiBAC

4(3)

f luorescence spectroscopy-based assays, a f luorescence resonance energy transfer (FRET)-based assay technology with mem-brane potential-sensitive dyes was developed [23]. In addition to the membrane potential-sensitive partitioning dye, which functions as FRET acceptor, a second fluorescent molecule is used that binds to the plasma membrane, remains stationary and functions as a FRET donor. Upon ion channel activation and con-comitant alteration of membrane potential the extent of coupling of the two dyes changes rapidly (in the range of seconds) as only fac-ile transmembrane translocation of the parti-tioning dye is necessary for FRET to occur, as compared with the slow diffusion through multiple water/membrane interfaces as in the case of fluorescence-spectroscopy-based assays (FiguRe 4) . Since fluorescence intensities are determined at the emission maxima of donor and acceptor and the calculated ratio is used as a functional measure of ion channel activ-ity, some common factors of variability (e.g., cell number variability, dye loading variability, autofluorescence of test compounds) can be eliminated or largely reduced.

The most commonly used FRET couple is a combination of the oxonol DiSBAC

2(3) as

FRET acceptor and the coumarin-linked phos-pholipid CC2-DMPE as FRET donor (Box 2), which attaches to the outer leaflet of the plasma membrane [24]. After loading of cells with this FRET couple, hyperpolarization upon chan-nel activation leads to an increase in FRET within seconds, whereas depolarization reduces

Figure 4.The principle of FRET-based assays employing membrane potential-sensitive dyes (such as oxonols) as FRET acceptor in combination with a FRET donor (such as CC2-DMPE). For details refer to the text.FRET: Fluorescence resonance energy transfer.

Figure 3. Principle of fluorescence spectroscopy-based assays employing membrane potential-sensitive dyes such as oxonols. For details refer to the text.

Key TeRm

Fluorescence resonance energy transfer: Technique based on the transfer of energy from the excited state of a donor moiety to an acceptor. Because the measurable transfer efficiency depends on the distance between donor and acceptor, FRET can be used to estimate intramolecular distances (200–1000 nm) and to investigate interactions between macromolecules

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Radioactive or nonradioactivetracer ion (e.g., Rb+)

Radioactive or nonradioactivetracer ion (e.g., Rb+)

Ion flux

Ca2+ – Fluo-3/4

Ca2+ ion

Ion flux



it. The assay sensitivity is about 1–3% ratio change per mV. For HTS purposes special-ized instrumentation is necessary, such as the originally designed proprietary platform VIPR (Aurora Biosciences, now Vertex) [25] and more recently introduced systems such as GENios Pro (Tecan) and FLIPRTetra (MDS Analytical Technologies).

The above mentioned FRET couple was suc-cessfully used for the configuration of a FRET assay on a VIPR platform for the identifica-tion of sodium channel inhibitors (Nav

1.5 and

Nav1.7

) in a 96-well plate format [26]. In order to overcome the very fast inactivation kinetics of these channels, the FRET assay was performed in the presence of a channel activator in form of an ‘agonist initiation assay’ in order to pro-long channel opening under otherwise physi-ological assay conditions. Importantly, the assay was shown to be sensitive to all known classes of sodium channel inhibitors. Using the FLIPRTetra a FRET assay for the Nav

1.8 sodium

channel was developed in a 384-well format [27]. For this channel a combination of PbTX-3 and deltamethrin was used for ‘agonist initia-tion’. Under the established assay conditions, several structurally unrelated, state-dependent Na

V1 inhibitors such as lidocaine interacted

with this sodium channel with the expected potencies. More recently, DiSBAC

2(3)/CC2-

DMPE was also used for the configuration of a FRET assay in a 1536-well format on a FLIPRTetra platform for primary HTS and the identification of inhibitors of K

ir channels [28].

The established protocol allowed the assay to be fully automated on a robotics platform for screening of a complete small molecule library collection. Potency of a subset of active com-pounds selected for confirmation ranged from 40 to 10,000 nM.

Also in the case of such sophisticated FRET assays, many ‘false positive’ hits are still usu-ally obtained in HTS due to the more indirect nature of the read-out, and no direct voltage control can be obtained. Thus, these fully HTS-compatible FRET assays do not allow the identification of bona fide state-dependent ion channel modulators.

Assays measuring ion fluxRadioactive ion flux assaysThe direct measurement of ion flux through ion channels was originally accomplished by the use of radioactive isotopes of ions that pass through the channel under investigation.

‘Tracer’ ions were either radioactive isotopes of the physiological ion species - as in the case of 22Na+ [29] and 45Ca2+ [30,31] - or ‘xeno-ions’, which are conducted by the channel such as 86Rb+ in the case of K+ and nonselective cat-ion channels [32] and [14C]-guanidinium in the case of Na+ channels [29]. In cell-based assay systems, depending on the nature of the channel, either influx or efflux is measured

Figure 5. The principle of assays measuring ion flux such as the nonradioactive Rb+ efflux assay (for which the direction of ion flux is indicated). For details refer to the text.

Figure 6. The principle of assays measuring Ca2+ flux with Ca2+-sensitive fluorescence dyes such as Fluo 3/4.

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upon channel activation. Whereas influx of radiotracer is measured for sodium, calcium and chloride channels, efflux is measured in the case of potassium channels. In the 1990s such radioactive flux assays were in routine use in major pharmaceutical companies with an interest in ion channels, mainly for HTS of cal-cium, potassium and nonselective cation chan-nels. These assays are robust and less sensitive to disturbances as compared with membrane potential assays, which provide a more indirect measure of channel activity. The major disad-vantage is the need for radioisotopes and the associated safety and environmental hazards.

Ion flux assays using atomic absorption spectroscopy In order to avoid problems associated with the use of radioisotopes, a nonradioactive Rb+ efflux assay was developed for K+ channels and nonselective cation channels (FiguRe 5) , which uses atomic absorption spectroscopy (AAS) for the determination of rubidium [33]. Ion channel-expressing cells are loaded with Rb+ by simply replacing KCl with RbCl in a cell-compatible buffer solution. In a number of wash steps extracellular Rb+ is removed and subsequent channel activation leads to Rb+ efflux, which is quantified as relative Rb+ efflux by AAS determination of rubidium in the cell supernatant and cell lysate. This assay technol-ogy has found widespread application in the pharmaceutical industry for both, drug discov-ery and hERG-related drug-safety screening for the identification of potential QT liabilities that might cause lethal arrhythmias (applica-tions reviewed elsewhere [34,35]). The evaluation of hERG blockade of compounds in this assay was shown to be compromised by a reduced sensitivity as compared with classical electro-physiology, which results in reduced apparent potencies of compounds while their rank order is maintained [36,37]. Thus, the ‘hERG applica-tion’ of this assay might be limited to ‘rank-ordering’ compounds within a chemical series while comparisons between multiple different chemotypes prove difficult [38]. In conjunction with recent innovation in AAS instrumenta-tion including parallelization of measurements (ICR 12000, Aurora Biomed) the nonradio-active Rb+ efflux assay is compatible with the throughput requirements of HTS and has displaced radioactive 86Rb+ flux assays. More recently, the AAS-based ion channel technol-ogy was adapted for measurements of sodium

channels using Li+ influx and lithium quanti-fication by AAS [39]. A more indirect applica-tion of this technology for chloride channels was also described that uses silver nitrate to precipitate chloride anions in the form of a an AgCl complex and determination of free silver by AAS [40].

Ion flux assays using ion-specific fluorescence dyesMeasurement of ion flux across cell membranes can also be accomplished using ion-specific flu-orescence probes that change their emission characteristics upon ion binding, often associ-ated with an increase in fluorescence intensity. Although such probes exist for a variety of ions including K+, Na+ and Cl- [41–43], their use has generally been limited to measuring Ca2+ flux through calcium channels or nonselective cat-ion channels with conductivity for Ca2+. The reason for this is that sensitive cell-permeable Ca2+ indicators with high binding affinities for this cation (K

d 300–400 nM) exist (e.g., Fura-2

[44] and Fluo-3/4 [Box 2] [42]) and that mea-surements are favored by the low intracellular Ca2+ concentration (100 nM) and concomi-tant steep electrochemical gradient of about four orders of magnitude (FiguRe 6). Typically, adherently-growing cells are loaded with cell-permeable acetoxymethyl ester derivatives of Fluo-3/4 followed by removal of excessive dye by a number of wash steps. The latter can be omitted if a noncell-permeable quenching dye is added or a commercially available premixed assay kit is used. Channel activation results in Ca2+ influx, increased Ca2+ binding to the fluo-rescence dye and, in consequence, increased fluorescence emission, which is measured. In conjunction with specialized fluorescence plate reader instrumentation (e. g. FLIPR, MDS Analytical Technologies), which measures through the bottom of microplates (intracel-lular fluorescence) and allows temporal reso-lution of intracellular Ca2+ peaks, such assays have found widespread routine use in ion chan-nel HTS [45]. The recent development of such an HTS assay for screening of the TRPM2 channel, a member of the transient receptor potential melastatin (TRPM)–related family, also demonstrates its applicability for cells that are growing in suspension [46]. Although this assay technology per se lacks control of trans-membrane potential and thus does not allow bona fide identif ication of state-dependent channel modulators, for certain ion channel

Key TeRm

Atomic absorption spectroscopy (AAS): Traditionally used for the detection of trace elements in environmental, biological and medical samples, AAS utilizes thermal energy to generate free ground-state atoms in a vapor phase that absorb light of a specific wavelength (in the case of rubidium 780 nm). This absorption is proportional to the concentration of the element and can thus be used for its quantitative determination

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Extracellular buffer

Conventional patch clamping

CellA/D

Computer

Amplifier

Planar patch clamping

Population patch clamping

Cell


A/D

Computer


1 hole/well

10 or 64 holes/well

Intracellular buffer

Planar patch plate

Computer

Cells

Amplifier

Amplifier

A/D



applications this might be achieved by gener-ating cell systems that co-express nonvoltage-sensitive potassium channels. This was recently shown for members of the Cav2 subfamiliy of calcium channels that were co-expressed with the inward rectifier Kir

2.3 channel such that

variations of external K+ concentrations can be used to manipulate membrane potential [47]. Clearly, this does not function as a true ‘volt-age clamp’ and differences between the FLIPR assay and classical electrophysiology measure-ments were observed with regards to the degree of state-dependent inhibition of a number of reference compounds. Nevertheless, this assay might be useful for HTS to identify state-dependent Cav2 blockers. A disadvantage of all fluorescent Ca2+ flux assays is the identification of ‘false-positive’ hits as a result of compound effects on cellular processes that change intra-cellular Ca2+ concentrations independent from the channel under investigation (e.g., calcium transporters, calcium release from intracellu-lar stores) and the limited temporal resolution (which is in the range of seconds).

More recently, a fluorescent Tl+ assay was developed for the identification of potassium channel modulators [48]. In this assay Tl+ is used as tracer for K+ and its influx into cells is measured with the thallium-sensitive fluo-rescent dye BTC. Although this assay is ame-nable to HTS, large concentrations (mM) of the highly toxic thallium and Cl--free buffer conditions are necessary in order to generate a robust assay read-out. Using a commercially available thallium assay kit (FluxOR) a pro-tocol for hERG screening was developed that allows screening in a homogeneous 1536-well plate format [49].

Electrophysiological screening technologiesTwo major initiatives have driven the evolu-tion of automated platforms capable of higher-throughput screening for ion channels; one that has successfully recapitulated the gigaohm seal quality typical and expected of manual electro-physiology recording, and the other that has relied on the use of the perforated patch clamp technique and made commercially available as the IonWorks technology [50,51]. A schematic diagram illustrating the transition from the use of conventional glass electrodes for patch clamping to a planar matrix-based approach that can be multiplexed with ease is illustrated in FiguRe 7, and the dynamic evolution of these

planar patch clamp technology platforms is summarized in FiguRe 8 and discussed in the following sections.

IonWorks platformsThe IonWorks platform was the first of the commercially available automated electrophysi-ology platforms to gain widespread utility and validation in the field and is now available in

Figure 7. Schematic of the evolution of the electrophysiological patch clamp technology for ion channel screening. Conventional/manual patch clamping employs complicated micromanipulation of glass microelectrodes to patch onto the cell surface with high resistance (GW) seal to gain electrical access to the intracellular compartment and record low-noise, high-fidelity transmembrane ionic currents. Planar patch clamp technologies replace glass micro electrodes with precisely engineered hole/holes in a glass or silicon wafer or silicone matrix and enable multiplexing and automation and have led to significant increase in throughput.

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2000

2010

Manual patch clamp(GΩ, I/H, DP/D 15–25)

2002IonWorks HT

(MΩ, I/L, DP/D 3000)

2004QPatch 16

(GΩ, I/H, MF, DP/D 1500–3000)

2006Patchliner

(GΩ, I/H, MF, DP/D 1500–3000)

2009Syncropatch 96

(GΩ, MF, DP/D ~10,000)

2010Dynaflow HT

(MΩ, MF, DP/D ~10,000)

2003†Dynaflow

(DP/D 40–100)

2003PatchXpress(GΩ, I/H, DP/D 1500–3000)

2003Port-A-Patch(GΩ, I/H, DP/D 15–25)

2003IonWorks Quattro(MΩ, I/M, DP/D 10,000–12,000)

2006QPatch HT(GΩ, I/H, MF, DP/D 2000–5000)

2006QPatch HT(GΩ, I/H, MF, DP/D 4000–7000)

2009QPatch HTX(GΩ, I/H, MF, DP/D 4000–7000)

2009/2010IonFlux(MΩ, MF, DP/D ~7000)



the original HT mode and the more recently launched Quattro mode [52]. This in itself is a testimony to the rapidly evolving field of ion channel screening automation with both the success and limitations of the HT mode driving the evolution and introduction of the Quattro mode. Using the bottom-up planar patch clamp approach with plates to replace the glass microelectrode used in manual recordings, and antibiotic perfusion to gain electrical access to cells, thus replacing the high fidelity seal at the glass microelectrode tip, the IonWorks HT achieved an unprecedented increase in ion channel screening capability. Yet, the design of one hole per well in the original PatchPlate format of the HT system was recognized as a fundamental limitation relying on a single cell achieving the desired orientation to the hole

and successful electrical access and megaohm seal formation. As a consequence, a high num-ber of well failures per plate was, and is, typi-cal of IonWorks HT recordings with the most optimized cell line yielding somewhere in the region of 60% overall success rate per plate to experiment completion. By reconfiguring the plate design to incorporate 64 holes per plate and averaging recordings across all the success-fully sealed cells in an individual well, the so-called population patch clamp (PPC) mode, this limitation of the first generation IonWorks HT technology has been eliminated. Launched in the form of the Quattro platform the second generation IonWorks platform achieves close to 100% success rate with an optimized cell line.

Various reviews have discussed the util-ity of the IonWorks platform and its impact on ion channel screening [53,54] and as with many of the higher-throughput platforms for ion channel screening the early adoption and implementation was driven largely by the need to screen compounds for activity against the cardiac hERG channel. The IonWorks tech-nology has been demonstrated by a number of investigators to represent a valid approach to hERG screening early in the drug discovery process, where throughput is a major need and consideration [55,56]. One common limitation has been observed for lipophilic compounds with right-shifted (underestimated) compound potencies due to the use of plastic materials for the plates. However, with good assay opti-mization, an acceptable correlation between compound potencies derived from IonWorks assays and manual patch clamp electrophysiol-ogy can be derived for the bulk of compounds tested. Subsequently, ion channel-targeted drug-discovery efforts have also benefited from the availability of the IonWorks platform with robust assays documented for a number of diverse ion channel subtypes including Kv

1.5 [51],

Ca-activated K channels [57], KCNQ2/3

channels [58], T-type Ca channels [59] and HCN

1 as well

as HCN4 channels [60]. Despite the inability to

recapitulate the gigaohm seal quality of man-ual recordings, this has not been a significant limitation of the IonWorks platform in practi-cal use, where this caveat has been accepted as a trade-off for increased compound screening throughput, often without compromising data quality and pharmacology.

Recent validated applications of the IonWorks technology have focused on the evaluation of voltage-gated sodium channels (Nav), either in

Figure 8. Chronological evolution of planar patch clamp technologies in the context of ion channel screening.†A laminar perfusion system that increases the throughput of manual patch clamp experiments. GW: Giga Ohm seal resistance; I/(H,M,L): Information (high, medium, low); MF: Microfluidic perfusion channels; DP/D: Data points/day.

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terms of cardiac liability of compounds target-ing the heart Nav

1.5 subtype [61], or the sensory

neuron-specific Nav1.7

channel [39,62] implicated by human genetics as a potential therapeutic target for chronic pain [63,64]. The activation kinetics, current/voltage relationship and phar-macology of hNav

1.5 expressed in CHO cells

measured using the IonWorks HT was found to be in very good agreement with data derived from manual patch clamp experiments as well as to be a reasonable predictor of cardiac effects in more intact preparations such as the canine Purkinje f ibre action potential model [61]. Similarly, state-dependent activity and phar-macology of Nav

1.7 channel blockers was faith-

fully reproduced using the IonWorks Quattro and validated as a high-throughput approach for Nav

1.7-targeted drug discovery [62]. Another

comprehensive comparison of Nav1.7

channel blocker pharmacology between IonWorks HT and three different nonelectrophysiological platforms (ion flux, membrane potential-sen-sitive fluorescence and FRET) demonstrated all assays to be robust for HTS and generally in good correlation with the IonWorks data [39].

Another significant advantage of the avail-ability of high-throughput platforms such as the IonWorks for ion channel screening, and one that may not be so obvious, is the use of the platform itself as a tool to select the high-est quality cell line as a critical reagent in the ion channel screening process [53,65]. As with screening larger numbers of compounds, larger numbers of cell lines can be tested compared with manual recordings and selected based on the best performance on the high-throughput platform. A recent example of this in terms of using IonWorks to optimize for param-eters such as seal rate, current amplitude and expression rates, current/voltage relationships, pharmacology and stability of expression over time has validated this approach for a diverse array of ion channel targets including hERG, Kv

7.1, and Cav

1.2 [66]. Multiple strategies were

employed including the use of current kinetics (Kv

7.1) or amplitudes (Cav

1.2) in order to probe

accessory subunit expression, or in the case of the Ca-activated K channel to reduce variabil-ity in drug response between wells via use of the averaged PPC mode in Quattro. Using this approach at the stage of cell line selection can translate into significant success when moving to the drug screening process.

Despite the robust use of IonWorks HT and Quattro platforms as a valuable drug-discovery

tool for ion channel investigations, limitations of the instrument design to establish gigaohm seals, ability to maintain continuous voltage clamp (due to conflict of access between the robotic recording and perfusion heads) and perfusion capabilities that do not allow for simultaneous ‘add and record’ (the characteris-tics routinely achieved by manual patch clamp recordings) has led to a parallel evolution of systems to overcome these shortcomings.

Gigaohm seal planar array platformsSeveral planar array-based automated electro-physiology systems were developed that incor-porate the precision and accuracy of manual patch clamp experiments. Of these systems, the PatchXpress, QPatch and Patchliner plat-forms gained prominence and acceptance as ion channel drug-discovery platforms in the pharmaceutical industry.

PatchXpress, the first of these manufactured by Molecular Devices, uses eight dual-channel amplifiers, a 16-channel seal chip that has a proprietary matrix manufactured by Aviva Biosciences, has one hole per well, enables gigaohm seals and allows continuous voltage clamp [67,68]. The electrodes are independent of the seal chip. Sophion instrument’s QPatch is a 16-channel planar system, the first to enable integrated microfluidic channels in the plate to allow fast solution exchanges. A precision hole in the silicon wafer incorporated into the QPlate enables gigaohm seals and embedded recording and ground electrodes in the QPlate to facili-tate efficient contacts with the amplifier and a precise voltage control [69]. The system has since evolved to increase throughput by threefold to a 48-channel system - the QPatch HT/X - that works either in a single-hole mode (HT) [70,71] ideal for compound profiling, or a multihole mode (HTX) [72] ideal for screening. The mul-tihole mode of QPatch HTX, built on a concept analogous to PPC plates of IonWorks Quattro leads to a significant increase in throughput, ensures a success rate close to 100% and reduces biological variability by averaging the response from multiple cells. The QPatch HTX achieves a throughput of approximately 7000 data points a day, sufficient to support the requirements in the lead-optimization phase of drug discovery.

Nanion’s automated platforms, the single channel Port-A-Patch® [73] and the 16-channel Patchliner operate in a single-hole mode and utilize borosilicate glass chips as a patch clamp substrate that allows low-noise high-quality

Key TeRm

Patch clamp technology: Electrophysiological technique that allows the study of single or multiple ion channels expressed recombinantly in cell lines such as HEK or CHO or in a wide variety of excitable primary cells such as neurons, cardiomyocytes and pancreatic beta cells and nonexcitable cells (e.g., T-lymphocytes). It is a refinement of the voltage clamp technique developed by Neher and Sakmann that enabled and enhanced the understanding of bioelectricity. A glass microelectrode (pipette) with an open tip diameter of approximately 1 µm filled with intracellular recording buffer patches onto a tiny fragment of the cell membrane and forms a very high resistance seal upon application of suction, making it possible to measure low noise high-fidelity transmembrane electrical currents. The whole cell configuration of the patch clamp is most commonly employed in the pharmaceutical industry

Planar patch clamp technology: Planar translation of a conventional glass micro electrode where precisely engineered holes are made in a matrix such as borosilicate glass, silicon wafers for example, with an attempt to multiplex and automate patch clamp measurements and increase the throughput of measurements. This technology is gaining prominence in ion channel drug-discovery programs for screening large libraries of compounds

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recordings [55,74–76]. Port-A-Patch, the single channel, manual version of the technology with no integrated liquid handling capability is avail-able as a benchtop instrument. The Patchliner has microfluidic channels that allow rapid per-fusion and exchange of extracellular as well as intracellular solutions. Cellectricon Inc’s mul-tichannel laminar microfluidic perfusion sys-tem Dynaflow, though not an automated elec-trophysiology system in itself, augmented the throughput of manual patch clamping and gave a new impetus to pharmaceutical research efforts on fast ligand-gated ion channels.

As with the IonWorks platforms, all three gigaohm seal platforms have been successfully deployed for use in safety pharmacology studies where the focus is predominantly on investigat-ing the effects of drugs on cardiac ion chan-nels [67,77–81], primarily hERG, Nav

1.5 and Cav

1.2

channels to assess their proarrhythmic liability. They have demonstrated similar electrophysi-ological responses comparable to the conven-tional patch clamp, and revealed a potential for differences in pharmacological responses mediated, at least in part, by the ability of cer-tain compounds to adsorb on the surfaces of the compound plate, recording chamber and the excess cells surrounding the one that is patched [82,83].

In addition, these systems have been used successfully for measuring state-dependent modulation of voltage-gated ion channels such as sodium [62,79,84] and calcium channels. While all three systems offer excellent continuous voltage control, they differ to a certain extent in the flexibility available to users to design protocols and quantitatively extract biophysi-cal parameters such as voltage to half-maximal activation and steady-state inactivation, chan-nel activation, inactivation, and deactivation kinetics that are useful in structure–activity relationship investigations in the hit-to-lead and lead-optimization stages of drug discovery. The software and the data handling modules are constantly evolving and improving for all the systems. These systems have been routinely incorporated in the pharmaceutical industry for screening cascades for a variety of voltage-gated channels including Nav

1.7 [62], Nav

1.8,

KCNQ2/3

, KCNQ4 [80], Cav

2.2, Cav

2.1, Kv

1.3,

Kv1.5

, and ICRAC

[85].The rapid solution exchange times (50–100

ms) and perfusion capability of these systems have also made them amenable for ligand-gated ion channel investigations. The QPatch system

with an 8-channel pipettor and dual robotic arms has been successfully implemented for fast ligand-gated ion channel investigations such as the a

7nAChR [86,87], a

4b

2 nAChR

and GABA receptor channels. In a similar manner the Patchliner has been successfully implemented for investigation of GABA and glycine channels. Primary cells are at times needed for electrophysiological experiments to gain a mechanistic understanding of the mode of drug action. Primary human T-lymphocytes have been successfully used in QPatch and Patchliner for recording Kv

1.3 currents and

ICRAC

. Patchliner has additionally been used for recording currents from acutely dissociated primary neuronal cells.

The success of gigaohm seal automated systems has created a need and demand for newer systems with greater throughput while maintaining the highest quality and precision of recording. Towards this goal, three systems that are evolving are the SynchroPatch96 from Nanion, Dynaf low HT from Cellectricon and Ionf lux from Fluxion. SyncroPatch 96 (a 96-well system) uses the same borosilicate patch matrix as the Patchliner and supports gigaohm seal recordings in the whole-cell con-figuration, recording from 96 cells in parallel. It offers an approximately 60% success rate for completed experiments that include obtaining whole-cell recordings with an average record-ing stability of 20 minutes. In the b-testing phase the SyncroPatch 96 has performed well with a number of different cell lines (e.g., CHO, HEK, Jurkat and LTK) and ion chan-nels (e.g., Kv

1.3, Nav

1.5, TRPV

1 and GABA). It

allows a throughput of about 5000 data points per day, which is comparable to Sophion’s QPatch HTX. In early 2010, Celletricon Inc. launched Dynaflow HT, a 96-well automated patch clamp system that employs a 96-channel digital amplifier. Its microfluidic chip technol-ogy coupled to a complex perfusion algorithm achieves an extremely rapid and continuous perfusion (~20 ms exchange time), a feature that closely mimics and, to an extent, improves on the perfusion capabilities achieved on a conventional set-up and facilitates investiga-tions of voltage-gated ion channels as well as fast ligand-gated ion channels with higher throughput. Immediately prior to the start of each experiment the Dynaflow HT plates are exposed to oxygen plasma and filled with intracellular buffer. This ensures bubble-free priming of the microfluidics and renders the

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chip surface more hydrophilic and enhances seal stability. The chip matrix, however, is based on loose patch technology and achieves resistances in the range of 50–150 MW. The 16-channel pipettor with dual robotic arm offers an advantage. The plate design allows for 16 separate experiments per plate, with up to six cells measured individually in each experiment. The manufacturer suggests a daily throughput of this system to be 10,000 data points, but validation in the field will be required to corroborate this information.

Fluxion’s IonFlux , a benchtop instrument, is another system that has recently evolved. It uses either a 96-well plate with eight experi-mental zones or a 384-well plate format with 32 experimental zones and has a 16 or 64-channel amplifier corresponding to the plate format. Recordings are from a parallel ensemble of 20 cells and each zone provides 16 recordings (two recordings from eight compounds). The rapid microfluidic technology integrated into the plate automates compound application and eliminates the need for pipetting and wash-ing. It achieves complete solution exchange in approximately 50 ms thus facilitating use with fast ligand-gated channels. According to the manufacturer’s specification the 384-well sys-tem provides a throughput of 1000 data points an hour, again requiring full validation of this expectation in the field.

Clearly, the continued evolution of existing and new platforms for automated ion chan-nel screening by electrophysiology speaks to the increasing demand for these systems both for ion channel safety profiling and ion channel-targeted drug discovery.

Future perspectiveWith increased recognition of the impor-tance of ion channels in disease processes, the last ten years have witnessed tremendous progress in functional ion channel screening technologies and their implementation for ion channel drug discovery. In addition to non-electrophysiological technologies, which are now fully compatible with the needs of HTS, the ‘gold standard’ electrophysiology has also been developed into an automated plate-based screening technology. It can be expected that in the near future such electrophysiological screening technologies will advance further to become fully compatible with HTS and thus the method-of-choice for primary HTS of large diversity compound libraries. As full voltage

control can be exerted with this technology, it is highly likely that new state-dependent ion channel modulators will be identified that interact with the open, closed or inactivated states of ion channels. Such state-specific ion channel modulators will largely reduce poten-tial side effects as only the ‘disease-state’ of an ion channel can be targeted. Clearly, only those compounds can be identified that are present in large compound collections in which the chemical diversity is limited even if millions of compounds are present. To further increase the probability of identifying new ion channel-targeted drugs, structure-based rational drug design will play a major role with more and more ion channel structures being investigated by crystallography and NMR. Another impor-tant advancement will come from the contin-ued investigation of the pathophysiology of major diseases. This will most likely lead to the identification of ion channels that have so far not been associated with diseases, thus lead-ing to new validated drug targets. The indepth characterization of such new disease-relevant targets will also lead to the elucidation of mul-tiprotein complexes of which ion channels are part and their tissue expression patterns. This will have a consequence for the development of screening assays, which ideally need to mimic faithfully the disease-relevant ion channel species in its native context. Thus, primary cells and induced pluripotent stem cells will be used more and more for screening, in addi-tion to recombinant cell systems. The current functional screening technologies have been advanced to a point where understanding of the biology and pathophysiological relevance of ion channels in a disease context, and the design of enhanced ion channel-targeted chemical libraries become the rate-limiting factors for meaningful drug discovery. In any case, it can be expected that in the next five to ten years many more drugs that target the ‘ion channe-lome’ will enter clinical development and reach the market than is presently the case.

Financial & competing interests disclosureThe authors have no relevant affiliations or financial involve-ment with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, con-sultancies, honoraria, stock ownership or options, expert t estimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.

Key TeRms

States of ion channels: Ion channels are highly dynamic macromolecules, which exist in multiple states such as open, closed and inactivated. The interconversions between these states are currently not well understood

Rational drug design: Rational drug design is a strategy by which drug molecules are developed based on knowledge of the target protein, in particular its 3D structure and/or ligands that bind to it

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Executive summary

Ion channel diseases and drugs

In addition to the approximately 50 inherited ion channel disorders (channelopathies) known in man, many more disease conditions exist in which ion channels are critically involved.

Currently, ion channel drugs constitute approximately 10% of drugs on the market and around 15% of the top 100 best selling drugs.

To date, only a small fraction (~15) of the 400 ion channels has been explored in terms of drug discovery; thus, there is an enormous potential for novel ion channel therapeutics.

Ion channel screening

Whereas high-throughput screening assays are well established for target classes such as G protein-coupled receptors and enzymes, ion channel drug discovery is less developed due to the technical difficulties in developing such assays.

Advances in the development of functional ion channel screening assays over the last decade are currently enabling a more systematic exploitation of this important target class.

Since activation of ion channels results in the movement (flux) of charged molecular species across a cell membrane - which is associated with a concomitant change in membrane potential - these two features are exploited by functional screening assays.

Nonelectrophysiological screening technologies

Assays measuring changes in membrane potential employ membrane potential-sensitive dyes (‘voltage sensors’) and measure either fluorescence or, in combination with a suitable donor molecule, fluorescence resonance energy transfer.

Assays measuring ion flux employ tracer ions such as rubidium and lithium, and use atomic absorption spectroscopy for their determination. Ca2+ flux through calcium channels or nonselective cation channels with conductivity for Ca2+ is typically measured with calcium-sensitive fluorescence dyes.

Electrophysiological screening technologies

Patch clamp recordings represent the gold standard method for studying ion channel function in mammalian cells but throughput is extremely limited.

Planar array-based recording methods using parallel recordings in plate or chip format, in combination with instrumentation supporting full automation, have dramatically increased the throughput and multiple validated assays on these systems have been successfully implemented.

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