Dissecting the mechanism of ligand regulation in cyclic nucleotide gated channels at the single molecule level.

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry

Abstract

The ability of a cell to transport ions across the cell membrane is one of the most fundamental biological processes and allows a cell to interact with its environment. Transport of ions is achieved by proteins called ion channels, that provide pathways for ions to cross the otherwise impermeable cell membrane. The opening and closing (gating) of ion channels in response to the binding of other small molecules (ligands) is a critical step in many signal transduction pathways. The cyclic-nucleotide gated (CNG) ion channel governs the signalling mechanisms that permit both sight and smell. Like other members of this family of ion channels, CNG channels are formed by the assembly of four individual subunits, with a portion of each subunit contributing to the channel structure. Each subunit contains a ligand-binding site, and binding to these portions of the protein is thought to cause an structural change that is transduced into opening/closure of the ion channel. This process of ligand binding, transduction, and channel gating is central to understanding the dynamic molecular mechanism of ligand gating in ion channels. A better understanding of ligand regulation in CNG would help us in designing methods to manipulate these important signalling mechaims. We will develop apparatus capable of simultaneous measurements of ligand binding and ion channel gating at the level of individual molecules. By observing ligand binding and transduction at the single-molecule level we will be able to study each step in this signal transduction process; observing both the ligand binding event and the subsequent response of the ion channel. We will use this approach to address the molecular mechanism of ligand regulation in a chimeric cyclic-nucleotide gated potassium channel, KCNG.

Technical Summary

The ability of a cell to facilitate the selective movement of ions and small molecules across the plasma membrane is one of the most fundamental biological processes and allows a cell to interact with its environment. Ion channels are integral membrane proteins that provide ion-selective pathways across the otherwise impermeable cell membrane. The gating of ion channels in response to the binding of chemical ligands is a critical step in many signal transduction pathways. Cyclic-nucleotide gated (CNG) ion channels govern the signalling mechanisms that permit both sight and smell. Like other members of this family of ion channels, CNG channels are formed by the assembly of four individual subunits into a tetramer, with a portion of each subunit contributing to the channel structure. Each subunit contains a ligand-binding site, and binding to these domains is thought to cause an structural change that is transduced into opening/closure of the central pore. This process of ligand binding, transduction, and channel gating is central to understanding the dynamic molecular mechanism of ligand gated ion channels. We will develop apparatus capable of simultaneous measurements of ligand binding and ion channel gating at the single molecule level. This apparatus will combine single-molecule fluorescence measurements of fluorogenic ligands with electrical recording of ion current through the channel. This will enable us to directly relate a single ligand binding event to the electrical response of the channel. By observing ligand binding and transduction in one molecule we will be able to study each step in this signal transduction process. We will then use this approach to address the molecular mechanism of ligand regulation in a chimeric cNTP-gated potassium channel. These measurements will enable us to study in detail the relationship between binding, transduction and gating in a ligand gated ion channel.

Publications

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Heron AJ (2009) Simultaneous measurement of ionic current and fluorescence from single protein pores. in Journal of the American Chemical Society

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Gross LC (2011) Determining membrane capacitance by dynamic control of droplet interface bilayer area. in Langmuir : the ACS journal of surfaces and colloids

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Leptihn S (2011) In vitro reconstitution of eukaryotic ion channels using droplet interface bilayers. in Journal of the American Chemical Society

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Thompson JR (2011) Rapid assembly of a multimeric membrane protein pore. in Biophysical journal

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Harriss LM (2011) Imaging multiple conductance states in an alamethicin pore. in Journal of the American Chemical Society

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Castell OK (2012) Quantification of membrane protein inhibition by optical ion flux in a droplet interface bilayer array. in Angewandte Chemie (International ed. in English)

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Baker MA (2014) Photobleaching reveals heterogeneous stoichiometry for equinatoxin II oligomers. in Chembiochem : a European journal of chemical biology

 
Description New ways of making artificial mimics of the cell membrane and new ways of seeing individual molecules within those membranes.
Exploitation Route Publications, patents.
Sectors Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description ERC Starting Grant
Amount € 1,500,000 (EUR)
Funding ID ERC-2012-StG_20111109 
Organisation European Research Council (ERC) 
Sector Public
Country Belgium
Start  
 
Title Droplet Interface Bilayers 
Description http://www.ncbi.nlm.nih.gov/pubmed/23640169 
Type Of Material Technology assay or reagent 
Year Produced 2008 
Provided To Others? Yes  
Impact 3x patents. Licensing deal. 
 
Title BILAYERS 
Description A method for producing a bilayer of amphipathic molecules comprising providing a hydrated support and providing a hydrophilic body, and bringing the hydrated support and hydrophilic body into contact to form a bilayer of amphipathic molecules. A bilayer produced by the method of the invention, and uses of the bilayer. 
IP Reference WO2009024775 
Protection Patent granted
Year Protection Granted 2009
Licensed Yes
Impact N/A
 
Title BILAYERS 
Description A method for producing a bilayer, the method comprising: (a) providing a hydrated support and a hydrophilic body immersed in a hydrophobic medium; wherein a first monolayer of amphipathic molecules is formed on an interface between the hydrophobic medium and the hydrophilic body and a second monolayer of amphipathic molecules is formed on an interface between the hydrophobic medium and the hydrated support; and (b) bringing the first monolayer into contact with the second monolayer to form a bilayer of amphipathic molecules, wherein at least part of a cell membrane, comprising cell membrane constituents, is provided in or on the hydrated support and/or in the hydrophilic body, and such that constituents of the cell membrane incorporate into the bilayer during or after the bilayer formation.A bilayer produced by the method of the invention, and uses of the bilayer. 
IP Reference WO2011015870 
Protection Patent application published
Year Protection Granted 2011
Licensed Commercial In Confidence
Impact -
 
Description I'm a scientist get me out of here 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact Interaction with ~ 30 primary school classes.

http://imascientist.org.uk
Year(s) Of Engagement Activity 2013
URL http://imascientist.org.uk
 
Description RS MP-Scientist Pairing Scheme 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact RS MP-Scientist Pairing Scheme discussions.

N/A
Year(s) Of Engagement Activity 2006
 
Description Video Podcasting. iTunesU 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact http://www.ox.ac.uk/itunes-u

?
Year(s) Of Engagement Activity 2013
 
Description Wellcome Trust, short story writing. 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach International
Primary Audience Schools
Results and Impact Prepared a short story with a bioethicist, and the writer Jane Rogers ,investigating the ethi- cal implications of membrane-based synthetic biology, sponsored by the Wellcome Trust.

?
Year(s) Of Engagement Activity 2012,2013