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Engineering Ion Channels for Selective Neuronal Activation and Silencing

Henry Lester June 2009. Engineering Ion Channels for Selective Neuronal Activation and Silencing. Neuronal Engineering with Cys-loop Receptor Channels. Goal: develop a general technique to selectively and reversibly silence or activate specific sets of neurons in vivo .

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Engineering Ion Channels for Selective Neuronal Activation and Silencing

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  1. Henry Lester June 2009 Engineering Ion Channels for Selective Neuronal Activation and Silencing

  2. Neuronal Engineering with Cys-loop Receptor Channels Goal: develop a general technique to selectively and reversibly silence or activate specific sets of neurons in vivo. • Ideal approach would: • Have on- and off- kinetics on a time scale of minutes • Have simple activation (ie, via drug injected or in animal’s diet) • Avoid nonspecific effects in animal • Maintain target neurons healthy in an “off-state” for a few days without morphological/other changes • Silence or activate “diffuse” molecularly defined sets of neurons, not just spatially defined groups • The chosen channel • Cys-loop receptor (like nicotinic receptors) • Heteropentamer: α2β3 or α3β2 subunits. • This feature allows one to intersect two promoters, to enhance cellular specificity

  3. The “channelohm” is 2% of the human genome, and many other organisms expand the repertoire Voltage (actually, ΔE ~107 V/m) External transmitter Internal transmitter Light Temperature Force/ stretch/ movement Blockers Binding region Switches Resistor Battery = Membrane region 1/r = 0.1 – 100 pS Nernst potential for Na+, K+, Cl-, Ca2+, H+ Colored by subunit (chain) Cytosolic region (incomplete) Invertebrate glutamate-gated Cl- channel . At this resolution, resembles nicotinic acetylcholine receptor

  4. The drugs: “avermectins” • IVM: Lactone originally isolated from Streptomyces avermitilis • AVMs are used as antiparasitics in animals and humans (“River blindness” / Heartgard™) • IVM is probably an allosteric activator of GluCl channels • Also modulates GABA, 5HT3, P2X, and nicotinic channels, at much higher doses (IVM)

  5. IVM-induced silencing in GluCl-expressing cultured rat hippocampal neurons 500 nm IVM 50 nm IVM 5 nm IVM

  6. A B C D Optimized constructs optGluCla,b = “AVMR-Cl” • Binding site: • subunit unmutated; b Tyr182Phe (cation-π site) • suppresses endogenous glutamate sensitivity • M3-M4 intracellular loop: a YFP; b CFP • allows visualization • Coding region: codons adapted for mammalian expression • ~ 10-fold greater expression

  7. AAV-2 constructs injected into mouse striatum; slice experiments Single neurons: correlation between IVM-induced conductance & AP silencing Lerchner et al, 2007 (collaboration with D. J. Anderson at Caltech)

  8. Plans to extend the AVMR system Transfer AVM sensitivity to mammalian glycine receptor  no immune response Tighter AVM binding  increased AVM sensitivity M2 mutations  increased AVM sensitivity • Na+-permeable • selective neuronal activation • Ca2+-permeable • manipulate signal transduction Increased single-channel current  increased AVM sensitivity Optimize ER exit and trafficking → increased surface expression M3-M4 loop

  9. Very slow (several hr) AVM reversibility is puzzling GluCl- heteromer GluCl- homomer No potentiation GluCl- homomer (Etter et al., JBC 1996)

  10. 1mM Glu 1mM IVM Cys-loop 89 loop Location of the AVM binding site is unknown Likely distinct from the glutamate binding site Covalent binding interaction? Within the cavity of the TMD? At the ECD-TMD interface? (where other anesthetics are bind) Yoav Paas, BIU McCammon Lab, UCSD Radioligand binding experiments with [3H]-IVM on C. elegans membrane preps IVM binding sites exhibit high affinity binding (KD = 0.11 nM) IVM does dissociate from its receptor, with a rate constant of 0.005-0.006/min (Cully & Paress, 1991)

  11. The first AVMR-Na GluCl  WT +  WT Muscle nAChR ND98 (10 nM IVM) 0.5 ND98 GluCl  P(-2’)/A(-1’)E +  WT Still too small Still too large (200 nM IVM)

  12. Many AVMRs remain in intracellular compartments, but are chaperoned by IVM (GluClαYFP)GluClβ 24 h incubation (control solution) The intensity ratio, peripheral/whole cell, is 0.86 ± 0.07 in control and 1.51 ± 0.10 in IVM-treated cells (SEM (1 μM IVM) Confocal TIRF

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