Allosteric nature of P2X receptor activation probed by photoaffinity labelling.
OBJECTIVE
In P2X receptors, agonist binding at the interface between neighbouring subunits is efficiently transduced to ion channel gating. However, the relationship between binding and gating is difficult to study because agonists continuously bind and unbind. Here, we covalently incorporated agonists in the binding pocket of P2X receptors and examined how binding site occupancy affects the ability of the channel to gate.


METHODS
We used a strategy for tethering agonists to their ATP-binding pocket, while simultaneously probing ion channel gating using electrophysiology. The agonist 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP), a photoaffinity analogue of ATP, enabled us to trap rat homomeric P2X2 receptor and a P2X2/1 receptor chimera in different agonist-bound states. UV light was used to control the degree of covalent occupancy of the receptors.


RESULTS
Irradiation of the P2X2/1 receptor chimera - BzATP complex resulted in a persistent current that lasted even after extensive washout, consistent with photochemical tethering of the agonist BzATP and trapping of the receptors in an open state. Partial labelling with BzATP primed subsequent agonist binding and modulated gating efficiency for both full and partial agonists.


CONCLUSIONS
Our photolabelling strategy provides new molecular insights into the activation mechanism of the P2X receptor. We show here that priming with full agonist molecules leads to an increase in gating efficiency after subsequent agonist binding.
Department of Biophysical Chemistry, Max-Planck-Institute of Biophysics, Frankfurt am Main, Germany. y.bhargava@wibr.ucl.ac.uk
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Bhargava
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Rettinger
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Mourot
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D015640:Ion Channel Gating / Q000502:physiology*; D009865:Oocytes; D019920:Photoaffinity Labels / Q000494:pharmacology; D011485:Protein Binding; D058911:Purinergic P2X Receptor Agonists / Q000494:pharmacology; D058476:Receptors, Purinergic P2X2 / Q000502:physiology*; D014466:Ultraviolet Rays; D014982:Xenopus laevis
D019920:Photoaffinity Labels; D058911:Purinergic P2X Receptor Agonists; D058476:Receptors, Purinergic P2X2; C033901:3'-O-(4-benzoyl)benzoyladenosine 5'-triphosphate; D000255:Adenosine Triphosphate