Light Control of Cell Excitability Using a Photochromic Blocker for Voltage-Gated Ion Channels

Abstract

Rendering proteins light sensitive gives the opportunity to control various cellular functions with great spatial and temporal precision. Native proteins can be photosensitized using photochromic ligands (PCLs), small light sensitive molecules that can be reversibly converted between active and inactive forms with light. We engineered a new PCL for voltage-gated ion channels, QAQ, which consists of an Azobenzene photoswitch flanked by two Quaternary ammoniums. Azobenzenes rapidly photoisomerize between cis and trans configurations upon 380 or 500 nm light irradiation, respectively. Quaternary ammoniums are internal blockers for voltage-gated sodium, potassium and calcium channels. Our studies show that intracellular QAQ blocks indiscriminitely those three types of ion channels in its trans state, while photoisomerization to the cis configuration relieves block.QAQ is a permanently charged, membrane-impermeable molecule that needs hydrophilic pathways to reach its site of action. Loading QAQ into neurons through a patch pipette can be used to control excitability of sub-cellular domains in a single cell. More importantly, QAQ can be loaded into multiple cells at a time, passing through open TRPV1 or P2X7 receptors, two non-selective cation channels that show pore-dilation upon prolonged activation. Experiments in HEK cells and hippocampal neurons show that QAQ can enter cells only upon expression AND activation of TRPV1 or P2X7R. QAQ can also be loaded into cells naturally expressing those channels, like the sensory neurons of the dorsal root ganglia that play a crucial role in pain sensation. QAQ acts like a local anesthetic, silencing sensory neurons by blocking voltage-gated cation channels. In this context, light can then be used to restore nociception. QAQ is therefore a very promising PCL molecule with both scientific and clinical applications.