Electrical Biosensors based on GPCR-Kir6.2 functional coupling

1. One channel Current (pA) Amplifier Activator 0 Stimulation Current amplifier i Temps(ms/s/min) n channels Current (pA) Activator 0 Pipette (Ø~1 µm) Patch Time (ms/s/min) www.receptronics.com cDNA GPCR-Kir6.2 In vitro Transcription cRNA GPCR-Kir6.2 mV + 50 Stimulation Ø~10 µm - 50 microinjection µA Ø~1 mm 0 ~48 hr Recording Electrophysiological characterization Ø~1 µm µA Inhibitor Activator Time (s) 0 Analysis +50 mV 0 -50 IBS – Grenoble / LPM Electrical Biosensors based on GPCR-Kir6.2functional coupling Christophe MOREAU, Jean REVILLOUD, Julien DUPUIS & Michel VIVAUDOU Laboratoire des Protéines Membranaires Institut de Biologie Structurale, Grenoble, France www.ibs.fr SulfonylUrea Receptor SUR Ion Channel Kir6.2 • 4)Benefits of ICCRs. • Suitable for electronic microsystems • Fast response to ligand binding (time in seconds) • High ratio signal/noise • Specific and sensitive (according to the GPCR coupled to Kir6.2) • "Proximal" Biosensor (no second messengers, low false-positive) • Label-free and non radioactive assay • Functional in vivo and in vitro • In situ quality control with antagonits • Real time measurements (kinetic analysis) • reusable system (Kd analysis, no inter-sample variability) N EXT N C C Electrical signal Natural KATP channel SUR + Kir6.2 Sensor of the SUR activity 2)The ATP-sensitive potassium channels. The KATP channels are made of the Kir6.2 subunit that tetramerizes to form the pore, the SUR subunit that surrounds the pore and plays a regulatory role. Ion Channel Coupled Receptor The Challenge: create a functional coupling between GPCR and Kir6.2 Photon Ca2+ Odors Pheromones Small endogenous molecules Amino acids – amines Nucletotides – Nucleosides Lipids Peptides Proteins Thyrotropine (TSH) Lutropine (LH) Follitropine (FSH) Interleukine Chemokine GPCR-Kir6.2 1) Ion Channel Coupled Receptor. Using KATP channels as a natural model of an electrical Biosensor, we fused GPCRs to Kir6.2 in order to create the ICCRs. GPCRs are detectors of specific ligands (agonists, antagonists, inverse agonists), and Kir6.2 the sensor producing an electical signal in response to the ligand binding. EXT • ~1200 genes cloned • ~150 orphan GPCRs • Major pharmaceutical target • Large diversity of ligands • Potentially adjustable to new ligands • Specific • High sensibility • Homologous structure GPCR Gb Ga Gg Irina D.Pogozheva 3)G Protein Coupled Receptors. GPCRs play primordial functions in physiology such as neuromediation, inflammation, senses … All GPCRs have 7 transmembrane domains, bind and activate the heterotrimeric G proteins (Gabg) by their cytoplasmic loops. Patch-clamp technique in excised configuration. On left: pipette with a piece of plasma membrane. Current generated by ion channels are recorded by the intra-pipette electrode. This technique gives the opportunity to characterize ion channels off cell. On right: patch-clamp recordings of single channel (upper trace) and several channels (lower trace). µA Heterologous expression of KATP channels in Xenopus oocytes. Coupling of GPCR and Kir6.2 genes by genetic engineering. The related cRNA is produced by in vitro transcription and micro-injected in Xenopus oocytes. After 48 hours at 19°C, ICCR are characterized by electrophysiological techniques. 1st Attempt Two Electrode Voltage Clamp (TEVC). Two electrodes are stuck in oocytes. One for stimulation at 0, -50, 0, +50, 0 mV. The other one for recording. Results are analyzed in current versus time and solution application period is indicated. Hormone s Time 5) Methods. ICCRs are made by genetic engineering, expressed in Xenopus oocytes and characterized by electrophysiological techniques such as the TEVC and the patch-clamp technique in excised configuration. By analysing currents generated by Kir6.2 in presence of GPCR modulators (activators or inhibitors), we identify ICCRs with a functional coupling. Optimized ICCR Hormone • 7)Applications • in vitro diagnostic ("point of care") • Drug or target screening • detection of toxic (Biodefense) • 8)Conclusions • Based on our expertise in structure-function studies of KATP channel, we created a new type of electrical Biosensor by coupling a GPCR to Kir6.2. The direct and functional link between the receptor and the electric sensor give new complementary benefits to current methods of in vitro diagnostic, drug and target screening and detection. Next steps of this work will mainly focus on new ICCRs with different GPCRs. Antagonist Control with antagonist Hormone 6)Characterisation of ICCRs by the TEVC technique. Our first ICCR was well expressed to the plasma membrane, but did not show any functional coupling between the GPCR and Kir6.2. The original ICCR was modified by protein engineering and some new ICCRs showed an increase of current amplitude in response to the hormone binding on the GPCR. The optimized ICCRs are even sensitive to an antagonist that blocks the hormone activation. Those results indicate a fully functional coupling of our optimized ICCRs. www.idealp-pharma.com Ile de Berder, Aug. 25-31, 2007