Genetic and chemical engineering of ligand-gated ion channels
The atomic structure of each member of the superfamily of ligand-gated ion channels has been recently resolved. This outstanding advance thus encourages the development of new chemical tools to explore in details the functioning of these receptors. P2X receptors are activated by extracellular ATP and are particularly interesting because they are expressed in an almost all tissues in the human body and are involved in many physiological processes as diverse as synaptic transmission, the inflammatory response, recovery from heart failure and ischemic pain perception. Knowledge of the molecular mechanism of activation of these receptors is still quite limited. In particular, the precise stoichiometry of channel activation as well as the steps that coupled agonist binding to channel opening are poorly understood. To answer these questions, we develop a new strategy focused on the photo-regulation of P2X channel activity.
Two disciplines are brought together in this project: biology and organic synthesis. We design new derivatives of ATP carrying a photosensitive function and an irreversible attachment site. These molecules, called photo-switches should be able to open and close the under light when properly tethered to the binding site. We use patch-clamp electrophysiology coupled with LED irradiation system for recording currents from these channels activated by light.
We screened by bioinformatics a series of photo-switches, which carry spacer arms of different sizes, on a three-dimensional model of P2X2 receptor based on a crystal structure of a homologous receptor. We selected a molecule that discriminated between the cis and trans isomers. We then successfully synthesized the spacer arm of this light-switch. In parallel, we have created by molecular biology thirty single cysteine mutations on the P2X2 receptor. We tested the functionality (by patch-clamp electrophysiology) and showed that the majority of these mutants expressed in HEK cells remains functional. Finally, we adapted to the set-up an irradiation source (LED) that will be used to screen for the mutants.
In parallel to this project, we have recently shown the existence of an intermediate state in the P2X2 receptor. By recording single-channel currents activated by zinc on an engineering receptor mutant, we have trapped a non-conductant state that differs from the resting state by its sensitivity to agonists and precedes the open state of the receptor. These results have recently been published.
The new tools developed in this project represents an opportunity to investigate not only the biophysics of these receptors but also their contribution in both physiological and pathological conditions. They should be useful to other scientists to highlight the specific contribution of ATP currents.
- Jiang, R., Taly, A., Lemoine, D., Martz, A., Cunrath, O., Specht, A. & Grutter, T. (2012) Intermediate closed channel state(s) precede(s) activation in the ATP-gated P2X2 receptor. Channels (Austin), in press.
The recent elucidation of the atomic structure of each member of the ligand-gated ion channels super-family requires the development of original chemical tools to investigate in great details the functioning of these receptors. Among them, the ATP-gated P2X receptors are of special interests because they are ubiquitously distributed in the human body and are involved in many physiological processes as diverse as synaptic transmission, the response to inflammation, the rescue of ischemic heart failure and pain perception. Little is known about the molecular mechanism of activation of these recently discovered receptors. In particular, the number of ATP molecules required for channel opening as well as the mechanical steps that link agonist binding to channel gating remain completely unknow. To address these unanswered questions, we propose to develop innovative tools, based on promising preliminary results.
We will develop a strategy focused on the photoregulation of the purinergic P2X receptors. We plan to convert the ATP-gated into a light-gated ion channel, by designing an original ATP analogue that carries a light-sensitive azobenzene photoswitch. As a function of the irradiation wavelength the tethered azobenzene linker should concentrate (or not) the ATP moiety into its binding site. This should in turn cause opening (or closing) of the ion channel. We will synthesize different ATP-derived photoswitch analogues. In parallel, guided by molecular modeling based on the X-ray structure of the P2X receptor and our recent ATP-binding site affinity labeling identification, we will engineer cysteine mutant receptors, both in single and concatenated subunits. These mutations will be introduced, one at a time, near the ATP-binding site to ensure optimal ATP moiety binding of the tethered photoswitch analogues. We will use patch-clamp electrophysiology coupled to ultra-bright LED irradiation to record single-channel currents evoked by the light-gated ion channels. These tools represent an original opportunity to resolve the gating stoichiometry and to understand the initial steps linking binding site occupancy to channel opening. In the future, they should also be suitable for other scientists to reveal the precise contribution of the ATP-gated currents to the relevant physiological actions.
This project requires skills from chemistry and biology. We have thus gathered two teams: the first (partner 1, lab biophysicochimie des récepteurs canaux) is specialized in the structure-function relationships of ligand-gated ion channels, in particular the P2X receptors, and the second (partner 2, lab chimie bioorganique) is specialized on bioorganic chemistry of photo-sensitive compounds acting on proteins. This collaboration is thus ideal for the present proposal.
Monsieur Thomas Grutter (UNIVERSITE DE STRASBOURG) – firstname.lastname@example.org
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
UMR 7199 CNRS-UNISTRA UNIVERSITE DE STRASBOURG
UMR 7199 CNRS-UNISTRA UNIVERSITE DE STRASBOURG
Help of the ANR 376,012 euros
Beginning and duration of the scientific project: - 36 Months