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Transporters, Receptors, and Ion ChannelsGroup leader : Michel Vivaudou Group
Molecular mechanisms and engineering of ion channels and associated proteinsWe study the molecular mechanisms of the function and the pharmacology of ion channels and other membrane proteins that may associate with them, such as ABC proteins and G-protein coupled receptors. Our work makes extensive use of ion-channel specific electrophysiological techniques (patch clamp, two-microelectrode voltage-clamp) as well as all manners of protein engineering techniques. Our favorite model is the ATP-sensitive potassium channel (KATP) that is formed by assembly of a K+ channel (Kir6.2) and an ABC protein (SUR). This channel, which has a key role in insulin secretion as well as in regulation of the activity of cardiac and neuronal cells, is a major pharmaceutical target. We study the physiological and pharmacological modulation of this channel, the structure-function relations of SUR and its homologues from the ABC family, MRP1, P-gp, CFTR and YCF1, and the interactions between membrane proteins within natural complexes (SUR and Kir6.2) as well as within artificial complexes that are being built to serve as biosensors.
Legend : Secondary structure and molecular models of the 2 subunits of KATP channel. (A) Kir6.2 ( 400 amino acids) and SUR ( 1600 amino acids) are the constitutive subunits of the KATP channel. Four subunits of the inwardly-rectifying K+ channel, Kir6.1 or Kir6.2, associate with four ATP-binding cassette proteins, SUR, to form a functional KATP channel octamer. Kir6.x has two transmembrane helices M1 and M2, and a large cytoplasmic domain harboring an inhibitory binding site for ATP formed by the proximal C-terminal of one subunit and N-terminal of its neighbor. SUR possesses 3 transmembrane domains (TMD0,1&2), and 2 cytoplasmic nucleotide binding domains (NBD1&2) incorporating the Walker A, Walker B and Linker L consensus sequences. NBD1&2 probably function as a dimer with nucleotide sites made up of Walker A&B of one NBD and linker L of the other. Cytoplasmic loops L0 and L1 are indicated. (B&C) Homology model of Kir6.2 tetramer based on the KirBac1.1 structure (center) and model of Sav1866 together with a cartoon representation of TMD0&L0 in yellow. Panel B is a side view with 2 Sav1866 and TMD0 and a Kir6.2 tetramer. Panel C is viewed from the extracellular side of the full complex. The relative position of the subunits is arbitrary. Alpha-helices are in red and beta-sheets are in blue. ATP in the Kir6.2 and NBD sites is in green.
Legende: KATP channels and insulin secretion in pancreatic b-cells.(A) In pancreatic b-cells, glucose is shuttled in the cytosol tby the glucose transporter GLUT2 and metabolized by glucokinase (GK) and mitochondria to produce ATP from ADP. Cytosolic ATP raises and ADP decreases.These changes cause closure of KATP channels by the cumulative effects of increased inhibition of Kir6.2 by ATP and decreased activation of SUR1 by MgADP. The ensuing depolarization elicits opening of voltage-dependent Ca2+ channels, Ca2+ entry, and Ca2+-initiated insulin granules exocytosis. Co-secreted Zn2+ could reactivate KATP channels in a negative feedback loop. KATP channel blockers like sulfonylureas mimick high glucose and upregulate secretion while openers like diazoxide downregulate secretion. (B) In cartoon form, the b-cell produces insulin as a function of glucose. Output is increased by high glucose, channel blockers, or channel-disabling hyperinsulinemic mutations. It is decreased by low glucose, openers, or channel-activating diabetic mutations. Mechanism of action of KATP channel modulatorsThe KATP channel is modulated via SUR by the nucleotides ATP and ADP and by a large number of molecules, blockers with antidiabetic properties and openers with antihypertensive and antiischemic properties. Using mutagenesis and chimeragenesis, we are searching for the structural elements of SUR that are responsible for the binding of these agents and for the coupling to the channel subunit Kir6.2.
Légend : Fundamental role of 2 residues of the last transmembrane helix of SUR in mediating KATP channel activation by openers. KATP channels were reconstituted in Xenopus oocytes by coexpression of the Kir6.2 subunit with wild-type or modified SUR subunits and functionnally assayed in excised inside-out patch-clamp recordings. The opener response of these channels reflects the binding of the opener to the SUR subunit. SR47063, a cromakalim analogue, was used as a test opener at a saturating concentration. Progressively smaller regions of SUR2A were transferred in SUR1 until single residues were found to be sufficient to confer to SUR1 the capacity to respond chemically distinct openers. Pharmacology of ABC proteinsStudy of the structure-activity relations of ABC protein ligands through synthesis and screening of new compounds and study of their specificity using tests with different ABC transporters. In this family of great therapeutic interest, we are focusing on several highly-homologous proteins:
Legend : Topology and function of ABC family members having sequence homologies with SUR New approaches toward the study of transporters and receptorsDesign of hybrid proteins incorporating ion channels and GPCR receptors or ABC transporters to permit characterization with electrophysiological techniques and integration with microelectronics. This project is part of the European project Receptronics (www.receptronics.org).
Legend : The 3 levels of development of biosensing devices for diagnostics and screening as envisioned in the European Project Receptronics (Label-free Biomolecular Detectors: at the Convergence of Bioengineered Receptors and Microelectronics) TechniquesKeywords Ion channel – K+ channel - K-ATP channel – ABC transporter – YCF1 – SUR – MRP1 – P-glycoprotein - biosensor Techniques Construction of mutants, chimeras, fusion proteins – Heterologous expression in Xenopus oocytes – Patch-clamp – Two-microelectrode voltage clamp – Measurement of surface expression of membrane proteins (chemiluminescence, confocal microscopy) – Western blot – Measurement of ATPase activity by fluorescence – Bioinformatics and molecular modelling |
