ABSTRACT
Photoswitchable blockers of potassium channels can be used to optically control neuronal excitability and hold great promise for vision restoration. Here, we report a series of improved photoswitchable blockers that are furnished with a new pharmacophore based on the local anesthetic bupivacaine. These azobupivacaines (ABs) enable optical control over the delayed rectifier channel Kv2.1. and target the two-pore domain potassium channel TREK-1. For the first time, we have identified a compound that blocks conductance in the dark and potentiates it upon illumination. Using light as a trigger, ABs efficiently and reversibly silence action potential firing of hippocampal neurons in acute mouse brain slices.
Subject(s)
Action Potentials/drug effects , Azo Compounds/pharmacology , Bupivacaine/analogs & derivatives , Light , Neurons/drug effects , Potassium Channel Blockers/pharmacology , Potassium Channels, Tandem Pore Domain/drug effects , Shab Potassium Channels/drug effects , Animals , Azo Compounds/chemical synthesis , HEK293 Cells , Hippocampus/cytology , Hippocampus/drug effects , Hippocampus/metabolism , Humans , Mice , Neurons/metabolism , Optical Phenomena , Potassium Channel Blockers/chemical synthesis , Potassium Channels, Tandem Pore Domain/antagonists & inhibitors , Potassium Channels, Tandem Pore Domain/metabolism , Shab Potassium Channels/antagonists & inhibitors , Shab Potassium Channels/metabolismABSTRACT
Photopharmacological control of neuronal activity using synthetic photochromic ligands, or photoswitches, is a promising approach for restoring visual function in patients suffering from degenerative retinal diseases. Azobenzene photoswitches, such as AAQ and DENAQ, have been shown to restore the responses of retinal ganglion cells to light in mouse models of retinal degeneration but do not recapitulate native retinal signal processing. Here, we describe diethylamino-azo-diethylamino (DAD), a third-generation photoswitch that is capable of restoring retinal ganglion cell light responses to blue or white light. In acute brain slices of murine layer 2/3 cortical neurons, we determined that the photoswitch quickly relaxes to its inactive form in the dark. DAD is not permanently charged, and the uncharged form enables the photoswitch to rapidly and effectively cross biological barriers and thereby access and photosensitize retinal neurons. Intravitreal injection of DAD restored retinal light responses and light-driven behavior to blind mice. Unlike DENAQ, DAD acts upstream of retinal ganglion cells, primarily conferring light sensitivity to bipolar cells. Moreover, DAD was capable of generating ON and OFF visual responses in the blind retina by utilizing intrinsic retinal circuitry, which may be advantageous for restoring visual function.
Subject(s)
Azo Compounds/pharmacology , Blindness/drug therapy , Quaternary Ammonium Compounds/pharmacology , Recovery of Function/drug effects , Retinal Bipolar Cells/metabolism , Vision, Ocular/drug effects , Animals , Blindness/genetics , Blindness/metabolism , Blindness/pathology , Mice , Mice, Knockout , Recovery of Function/genetics , Retinal Bipolar Cells/pathology , Retinal Diseases/drug therapy , Retinal Diseases/genetics , Retinal Diseases/metabolism , Retinal Diseases/pathology , Retinal Neurons/metabolism , Vision, Ocular/geneticsABSTRACT
Nicotinic acetylcholine receptors (nAChRs) are essential for cellular communication in higher organisms. Even though a vast pharmacological toolset to study cholinergic systems has been developed, control of endogenous neuronal nAChRs with high spatiotemporal precision has been lacking. To address this issue, we have generated photoswitchable nAChR agonists and re-evaluated the known photochromic ligand, BisQ. Using electrophysiology, we found that one of our new compounds, AzoCholine, is an excellent photoswitchable agonist for neuronal α7 nAChRs, whereas BisQ was confirmed to be an agonist for the muscle-type nAChR. AzoCholine could be used to modulate cholinergic activity in a brain slice and in dorsal root ganglion neurons. In addition, we demonstrate light-dependent perturbation of behavior in the nematode, Caenorhabditis elegans.
