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High-Throughput Fluorescence Assays for Ion Channels and GPCRs

  • Irina VetterEmail author
  • David Carter
  • John Bassett
  • Jennifer R. Deuis
  • Bryan Tay
  • Sina Jami
  • Samuel D. Robinson
Chapter
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1131)

Abstract

Ca2+, Na+ and K+- permeable ion channels as well as GPCRs linked to Ca2+ release are important drug targets. Accordingly, high-throughput fluorescence plate reader assays have contributed substantially to drug discovery efforts and pharmacological characterization of these receptors and ion channels. This chapter describes some of the basic properties of the fluorescent dyes facilitating these assay approaches as well as general methods for establishment and optimisation of fluorescence assays for ion channels and Gq-coupled GPCRs.

Keywords

High-throughput High-content Fluorescence imaging G protein-coupled receptor Voltage-gated ion channel Ligand-gated ion channel Assay development Optimization FLIPR 

Abbreviations

ATP

adenosine triphosphate

Ca2+

calcium ion

CaV and VGCC

Voltage-gated Ca2+ channels

DAG

diacylglycerol

FLIPR

Fluorescent Imaging Plate Reader

GPCR

G-protein coupled receptor

HTS

high throughput screening

IP3

inositol-1,4,5,-triphosphate

LGCC

Ligand-gated Ca2+ channels

NCX

Na+/Ca2+ exchanger

PIP2

phosphatidylinositol 4, 5 bisphosphate

PMCA

Plasma Membrane Ca2+ ATPase

RyR

ryanodine receptors

SERCA

sarco/endoplasmic reticulum Ca2+ ATPase

EGTA

ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid.

APTRA

2-aminophenol-N,N,O-triacetic acid

BAPTA

1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid

Kd

dissociation constant

AM

acetoxymethyl

ER

endoplasmic reticulum

LED

light-emitting diode

CCD

charge-coupled device

EMCCD

Electron Multiplying Charge Coupled Device

ICDD

Intensified CCD

PDL

poly-D-lysine

PLL

poly-L-lysine

PLO

poly-L-ornithine

nAChR

nicotinic acetylcholine receptors

HEPES

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

PAR2

protease-activated receptor 2

RFU

relative fluorescence unit

SBFI

Sodium-binding benzofuran isophthalate

PBFI

Potassium-binding benzofuran isophthalate

DRG

Dorsal Root Ganglion

RGS4

Regulator of G protein signalling 4

NaV

voltage-gated sodium channel

MCU

mitochondrial Ca2+ uniporter

cAMP

cyclic adenosine monophosphate

CANDLES

Cyclic AMP iNdirect Detection by Light Emission from Sensor cells

GTP

guanosine triphosphate

TRP

Transient Receptor Potential

SKCa

small-conductance calcium-activated K+ channel

IKCa

intermediate-conductance calcium-activated K+ channel

BKCa

big-conductance calcium-activated K+ channel

IRK

Inwardly-rectifying K+ channel

TWIK

Tandem of pore domains in a Weakly Inward rectifying K+ channel

TREK

TWIK-related K+ channel

TASK

TWIK-related acid-sensitive K+ channel

TALK

TWIK-related alkaline pH-activated K+ channel

THIK

TWIK-related halothane-inhibited K+ channel

TRESK

TWIK-related spinal cord K+ channel

KV or VGKC

voltage-gated K+ channel

ANG-1 and ANG-2

Asante NaTRIUM Green-1 and -2

Di-4-ANEPPS

Pyridinium, 4-(2-(6(dibutylamino)-2-naphthalenyl)-1-(3-sulfopropyl)-hydroxide

DiBAC4(3)

bis-(1,3-dibutylbarbituric acid) trimethine oxonol

FMP

FLIPR Membrane Potential

FRET

fluorescence resonance energy transfer

PeT

photoinduced electron transfer

CC2-DMPE

N-[6-chloro-7-hydroxycourmarin-3-carbonyl] dimyristroyl phosphatidyl ethanolamine

GEVI

genetically encoded voltage indicators

DPA

dipicrylamine

DiO

oxocyanine

LED

light-emitting diode

sCMOS

scientific complementary metal-oxide-semiconductor

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Copyright information

© Springer Nature Switzerland AG 2020

Authors and Affiliations

  • Irina Vetter
    • 1
    • 2
    Email author
  • David Carter
    • 1
  • John Bassett
    • 2
  • Jennifer R. Deuis
    • 1
  • Bryan Tay
    • 1
  • Sina Jami
    • 1
  • Samuel D. Robinson
    • 1
  1. 1.Institute for Molecular BioscienceThe University of QueenslandSt. LuciaAustralia
  2. 2.School of PharmacyThe University of QueenslandSt. LuciaAustralia

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