RESUMEN
L-type Ca2+ channels (LTCCs) play a crucial role in excitation-contraction coupling and release of hormones from secretory cells. They are targets of antihypertensive and antiarrhythmic drugs such as diltiazem. Here, we present a photoswitchable diltiazem, FHU-779, which can be used to reversibly block endogenous LTCCs by light. FHU-779 is as potent as diltiazem and can be used to place pancreatic ß-cell function and cardiac activity under optical control.
Asunto(s)
Canales de Calcio Tipo L/metabolismo , Diltiazem/farmacología , Colorantes Fluorescentes/farmacología , Corazón/efectos de los fármacos , Células Secretoras de Insulina/efectos de los fármacos , Imagen Óptica , Canales de Calcio Tipo L/química , Diltiazem/química , Colorantes Fluorescentes/química , Humanos , Células Secretoras de Insulina/metabolismo , Luz , Procesos FotoquímicosRESUMEN
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/metabolismoAsunto(s)
Oxazoles/química , Receptores AMPA/agonistas , Tetrazoles/química , Potenciales de Acción/efectos de los fármacos , Animales , Compuestos Azo/química , Células HEK293 , Humanos , Luz , Ratones , Neuronas/fisiología , Oxazoles/síntesis química , Oxazoles/farmacología , Receptores AMPA/genética , Receptores AMPA/metabolismo , Tetrazoles/síntesis química , Tetrazoles/farmacologíaRESUMEN
AMPA receptors respond to the neurotransmitter glutamate and play a critical role in excitatory neurotransmission. They have been implicated in several psychiatric disorders and have rich pharmacology. Antagonists of AMPA receptors have been explored as drugs and one has even reached the clinic. We now introduce a freely diffusible photoswitchable antagonist that is selective for AMPA receptors and endows them with light-sensitivity. Our photoswitch, ShuBQX-3, is active in its dark-adapted trans-isoform but is significantly less active as its cis-isoform. ShuBQX-3 exhibits a remarkable red-shifting of its photoswitching properties through interactions with the AMPA receptor ligand binding site. Since it can be used to control action potential firing with light, it could emerge as a powerful tool for studying synaptic transmission with high spatial and temporal precision.
RESUMEN
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
Fatty acids (FAs) are not only essential components of cellular energy storage and structure, but play crucial roles in signalling. Here we present a toolkit of photoswitchable FA analogues (FAAzos) that incorporate an azobenzene photoswitch along the FA chain. By modifying the FAAzos to resemble capsaicin, we prepare a series of photolipids targeting the Vanilloid Receptor 1 (TRPV1), a non-selective cation channel known for its role in nociception. Several azo-capsaicin derivatives (AzCAs) emerge as photoswitchable agonists of TRPV1 that are relatively inactive in the dark and become active on irradiation with ultraviolet-A light. This effect can be rapidly reversed by irradiation with blue light and permits the robust optical control of dorsal root ganglion neurons and C-fibre nociceptors with precision timing and kinetics not available with any other technique. More generally, we expect that photolipids will find many applications in controlling biological pathways that rely on protein-lipid interactions.
Asunto(s)
Ácidos Grasos/química , Procesos Fotoquímicos , Fotoquímica/métodos , Canales Catiónicos TRPV/agonistas , Animales , Compuestos Azo/química , Bradiquinina , Capsaicina/análogos & derivados , Ácidos Grasos/metabolismo , Ganglios Espinales/metabolismo , Células HEK293 , Humanos , Técnicas In Vitro , Lidocaína/análogos & derivados , Metabolismo de los Lípidos , Ratones , SerotoninaRESUMEN
Photochromic ligands (PCLs), defined as photoswitchable molecules that are able to endow native receptors with a sensitivity towards light, have become a promising photopharmacological tool for various applications in biology. In general, PCLs consist of a ligand of the target receptor covalently linked to an azobenzene, which can be reversibly switched between two configurations upon light illumination. Gluazo, as a PCL that targets excitatory amino acid receptors, in its dark-adapted trans iso-form was characterized to be a partial agonist of the kainate glutamate receptor GluK2. Application of UV light leads to the formation of the cis form, with remarkedly reduced affinity towards GluK2. The mechanism of the change of ligand affinity induced by the photoisomerization was unresolved. The presented computational study explains how the isomerization of such a PCL affects the structural changes in the target receptor that lead to its activation.
Asunto(s)
Luz , Simulación de Dinámica Molecular , Receptores de Ácido Kaínico/química , Receptores de Ácido Kaínico/metabolismo , Enlace de Hidrógeno , Activación del Canal Iónico/efectos de la radiación , Ligandos , Multimerización de Proteína/efectos de la radiación , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Estereoisomerismo , Termodinámica , Receptor de Ácido Kaínico GluK2RESUMEN
N-methyl-D-aspartate receptors (NMDARs) play a central role in synaptic plasticity, learning and memory, and are implicated in various neuronal disorders. We synthesized a diffusible photochromic glutamate analogue, azobenzene-triazole-glutamate (ATG), which is specific for NMDARs and functions as a photoswitchable agonist. ATG is inactive in its dark-adapted trans-isoform, but can be converted into its active cis-isoform using one-photon (near UV) or two-photon (740 nm) excitation. Irradiation with violet light photo-inactivates ATG within milliseconds, allowing agonist removal on the timescale of NMDAR deactivation. ATG is compatible with Ca(2+) imaging and can be used to optically mimic synaptic coincidence detection protocols. Thus, ATG can be used like traditional caged glutamate compounds, but with the added advantages of NMDAR specificity, low antagonism of GABAR-mediated currents, and precise temporal control of agonist delivery.
Asunto(s)
Región CA1 Hipocampal/metabolismo , Corteza Cerebral/metabolismo , Ácido Glutámico/análogos & derivados , Luz , Células Piramidales/metabolismo , Receptores de N-Metil-D-Aspartato/agonistas , Animales , Hipocampo/metabolismo , Ratones , Oocitos , Técnicas de Placa-Clamp , Isoformas de Proteínas , Ratas , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Rayos Ultravioleta , Xenopus laevisRESUMEN
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.
Asunto(s)
Compuestos Azo/farmacología , Red Nerviosa/efectos de los fármacos , Agonistas Nicotínicos/farmacología , Compuestos de Amonio Cuaternario/farmacología , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Animales , Caenorhabditis elegans , Femenino , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Ratas , Ratas Wistar , TransfecciónRESUMEN
Photochromic ligands (PCLs), recently introduced by our group as a tool for researchers in neuroscience, offer the ability to control native receptors with light in a reversible fashion without the need for any genetic manipulation. Here we describe the application of the PCL Azo-Tetrazole-AMPA-3 (ATA-3) to reversibly gate native AMPA-receptors with blue light and thereby control the activity of cortical neurons in brain slices.
Asunto(s)
Receptores AMPA/agonistas , Animales , Encéfalo/citología , Encéfalo/efectos de los fármacos , Células HEK293 , Humanos , Técnicas In Vitro , Activación del Canal Iónico/efectos de la radiación , Ligandos , Potenciales de la Membrana , Ratones Endogámicos C57BL , Oxazoles/química , Oxazoles/farmacología , Técnicas de Placa-Clamp , Procesos Fotoquímicos , Receptores AMPA/fisiología , Tetrazoles/química , Tetrazoles/farmacologíaRESUMEN
In the mammalian central nervous system, slow synaptic excitation involves the activation of metabotropic glutamate receptors (mGluRs). It has been proposed that C1-type transient receptor potential (TRPC1) channels underlie this synaptic excitation, but our analysis of TRPC1-deficient mice does not support this hypothesis. Here, we show unambiguously that it is TRPC3 that is needed for mGluR-dependent synaptic signaling in mouse cerebellar Purkinje cells. TRPC3 is the most abundantly expressed TRPC subunit in Purkinje cells. In mutant mice lacking TRPC3, both slow synaptic potentials and mGluR-mediated inward currents are completely absent, while the synaptically mediated Ca2+ release signals from intracellular stores are unchanged. Importantly, TRPC3 knockout mice exhibit an impaired walking behavior. Taken together, our results establish TRPC3 as a new type of postsynaptic channel that mediates mGluR-dependent synaptic transmission in cerebellar Purkinje cells and is crucial for motor coordination.