RESUMEN
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.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Compuestos Azo/farmacología , Bupivacaína/análogos & derivados , Luz , Neuronas/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio de Dominio Poro en Tándem/efectos de los fármacos , Canales de Potasio Shab/efectos de los fármacos , Animales , Compuestos Azo/síntesis química , Células HEK293 , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Ratones , Neuronas/metabolismo , Fenómenos Ópticos , Bloqueadores de los Canales de Potasio/síntesis química , Canales de Potasio de Dominio Poro en Tándem/antagonistas & inhibidores , Canales de Potasio de Dominio Poro en Tándem/metabolismo , Canales de Potasio Shab/antagonistas & inhibidores , Canales de Potasio Shab/metabolismoRESUMEN
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.
Asunto(s)
Compuestos Azo/farmacología , Ceguera/tratamiento farmacológico , Compuestos de Amonio Cuaternario/farmacología , Recuperación de la Función/efectos de los fármacos , Células Bipolares de la Retina/metabolismo , Visión Ocular/efectos de los fármacos , Animales , Ceguera/genética , Ceguera/metabolismo , Ceguera/patología , Ratones , Ratones Noqueados , Recuperación de la Función/genética , Células Bipolares de la Retina/patología , Enfermedades de la Retina/tratamiento farmacológico , Enfermedades de la Retina/genética , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Neuronas Retinianas/metabolismo , Visión Ocular/genéticaRESUMEN
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.