Journal Article

Optical control of trimeric P2X receptors and acid-sensing ion channels

Nunes, João P. M., Browne, Liam E., Sim, Joan A., Chudasama, Vijay, Caddick, Stephen, Bragg, Laricia, North, R. Alan

2014

Overview

P2X receptors are trimeric membrane proteins that function as ion channels gated by extracellular ATP. We have engineered a P2X2 receptor that opens within milliseconds by irradiation at 440 nm, and rapidly closes at 360 nm. This requires bridging receptor subunits via covalent attachment of 4,4'-bis(maleimido)azobenzene to a cysteine residue (P329C) introduced into each second transmembrane domain. The cis – trans isomerization of the azobenzene pushes apart the outer ends of the transmembrane helices and opens the channel in a light-dependent manner. Light-activated channels exhibited similar unitary currents, rectification, calcium permeability, and dye uptake as P2X2 receptors activated by ATP. P2X3 receptors with an equivalent mutation (P320C) were also light sensitive after chemical modification. They showed typical rapid desensitization, and they could coassemble with native P2X2 subunits in pheochromocytoma cells to form light-activated heteromeric P2X2/3 receptors. A similar approach was used to open and close human acid-sensing ion channels (ASICs), which are also trimers but are unrelated in sequence to P2X receptors. The experiments indicate that the opening of the permeation pathway requires similar and substantial movements of the transmembrane helices in both P2X receptors and ASICs, and the method will allow precise optical control of P2X receptors or ASICs in intact tissues.

• Publisher: National Academy of Sciences
• Subject: Adenosine triphosphatase / adenosine triphosphate / Adenosine Triphosphate - chemistry / Agonists / Amino Acid Sequence / Animals / ATP / Azo Compounds - chemistry / Biological Sciences / Calcium / Cells / Complementary DNA / cysteine / Electrophysiology / Gene Expression Regulation, Neoplastic / HEK293 cells / humans / Inurement / Ion Channel Gating - physiology / Ion Channel Gating - radiation effects / Ion channels / Ion Channels - chemistry / Ions / Irradiation / isomerization / Ligands / Light / Microscopy, Confocal / Models, Molecular / Molecular Conformation / Molecular Sequence Data / Mutagenesis, Site-Directed / Mutation / Optical control / PC12 Cells / Permeability / Physiology / Proteins / Rats / Receptors / Receptors, Purinergic P2X2 - chemistry / Receptors, Purinergic P2X2 - physiology / Receptors, Purinergic P2X2 - radiation effects / Receptors, Purinergic P2X3 - chemistry / Receptors, Purinergic P2X3 - physiology / Receptors, Purinergic P2X3 - radiation effects / Sequence Homology, Amino Acid / Tissues