RESUMEN
Increased levels of the second messenger lipid diacylglycerol (DAG) induce downstream signaling events including the translocation of C1-domain-containing proteins toward the plasma membrane. Here, we introduce three light-sensitive DAGs, termed PhoDAGs, which feature a photoswitchable acyl chain. The PhoDAGs are inactive in the dark and promote the translocation of proteins that feature C1 domains toward the plasma membrane upon a flash of UV-A light. This effect is quickly reversed after the termination of photostimulation or by irradiation with blue light, permitting the generation of oscillation patterns. Both protein kinase C and Munc13 can thus be put under optical control. PhoDAGs control vesicle release in excitable cells, such as mouse pancreatic islets and hippocampal neurons, and modulate synaptic transmission in Caenorhabditis elegans. As such, the PhoDAGs afford an unprecedented degree of spatiotemporal control and are broadly applicable tools to study DAG signaling.
Asunto(s)
Diglicéridos/metabolismo , Diglicéridos/efectos de la radiación , Procesos Fotoquímicos/efectos de la radiación , Proteína Quinasa C/metabolismo , Proteína Quinasa C/efectos de la radiación , Rayos Ultravioleta , Animales , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/efectos de la radiación , Diglicéridos/química , Ratones , Fenómenos Ópticos , Proteína Quinasa C/química , Transducción de Señal/efectos de la radiaciónRESUMEN
Ceramide is a pro-apoptotic sphingolipid with unique physical characteristics. Often viewed as a second messenger, its generation can modulate the structure of lipid rafts. We prepared three photoswitchable ceramides, ACes, which contain an azobenzene photoswitch allowing for optical control over the N-acyl chain. Using combined atomic force and confocal fluorescence microscopy, we demonstrate that the ACes enable reversible switching of lipid domains in raft-mimicking supported lipid bilayers (SLBs). In the trans-configuration, the ACes localize into the liquid-ordered (Lo) phase. Photoisomerization to the cis-form triggers a fluidification of the Lo domains, as liquid-disordered (Ld) "lakes" are formed within the rafts. Photoisomerization back to the trans-state with blue light stimulates a rigidification inside the Ld phase, as the formation of small Lo domains. These changes can be repeated over multiple cycles, enabling a dynamic spatiotemporal control of the lipid raft structure with light.
Asunto(s)
Ceramidas/metabolismo , Luz , Microdominios de Membrana/efectos de la radiación , Fenómenos Ópticos , Ceramidas/química , Isomerismo , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismoRESUMEN
Light is a fascinating phenomenon that ties together physics, chemistry, and biology. It is unmatched in its ability to confer information with temporal and spatial precision and has been used to map objects on the scale of tens of nanometers (10(-8) m) to light years (10(16) m). This information, gathered through super-resolution microscopes or space-based telescopes, is ultimately funneled through the human visual system, which is a miracle in itself. It allows us to see the Andromeda galaxy at night, an object that is 2.5 million light years away and very dim, and ski the next day in bright sunlight at an intensity that is 12 orders of magnitude higher. Human vision is only one of many photoreceptive systems that have evolved on earth and are found in all kingdoms of life. These systems rely on molecular photoswitches, such as retinal or tetrapyrrols, which undergo transient bond isomerizations or bond formations upon irradiation. The set of chromophores that have been employed in Nature for this purpose is surprisingly small. Nevertheless, they control a wide variety of biological functions, which have recently been significantly increased through the rapid development of optogenetics. Optogenetics originated as an effort to control neural function with genetically encoded photoreceptors that use abundant chromophores, in particular retinal. It now covers a variety of cellular functions other than excitability and has revolutionized the control of biological pathways in neuroscience and beyond. Chemistry has provided a large repertoire of synthetic photoswitches with highly tunable properties. Like their natural counterparts, these chromophores can be attached to proteins to effectively put them under optical control. This approach has enabled a new type of synthetic photobiology that has gone under various names to distinguish it from optogenetics. We now call it photopharmacology. Here we trace our involvement in this field, starting with the first light-sensitive potassium channel (SPARK) and concluding with our most recent work on photoswitchable fatty acids. Instead of simply providing a historical account of our efforts, we discuss the design criteria that guided our choice of molecules and receptors. As such, we hope to provide a roadmap to success in photopharmacology and make a case as to why synthetic photoswitches, properly designed and made available through well-planned and efficient syntheses, should have a bright future in biology and medicine.
Asunto(s)
Luz , Fototerapia , Humanos , Canales Iónicos/química , Canales Iónicos/metabolismo , LigandosRESUMEN
Chemical and electrical signaling at the synapse is a dynamic process that is crucial to neurotransmission and pathology. Traditional pharmacotherapy has found countless applications in both academic labs and the clinic; however, diffusible drugs lack spatial and temporal precision when employed in heterogeneous tissues such as the brain. In the field of photopharmacology, chemical attachment of a synthetic photoswitch to a bioactive ligand allows cellular signaling to be controlled with light. Azobenzenes have remained the go-to photoswitch for biological applications due to their tunable photophysical properties, and can be leveraged to achieve reversible optical control of numerous receptors and ion channels. Here, we discuss the most recent advances in photopharmacology which will improve the use of azobenzene-based probes for neuroscience applications.
Asunto(s)
Compuestos Azo , Diseño de Fármacos , Sondas Moleculares , Transmisión Sináptica , Compuestos Azo/metabolismo , Luz , Sondas Moleculares/metabolismo , Transducción de Señal/efectos de la radiación , Transmisión Sináptica/efectos de la radiaciónRESUMEN
Fatty acids activate GPR40 and K+ channels to modulate ß-cell function. Herein, we describe the design and synthesis of FAAzo-10, a light-controllable GPR40 agonist based on Gw-9508. FAAzo-10 is a potent GPR40 agonist in the trans-configuration and can be inactivated on isomerization to cis with UV-A light. Irradiation with blue light reverses this effect, allowing FAAzo-10 activity to be cycled ON and OFF with a high degree of spatiotemporal precision. In dissociated primary mouse ß-cells, FAAzo-10 also inactivates voltage-activated and ATP-sensitive K+ channels, and allows us to control glucose-stimulated Ca2+ oscillations in whole islets with light. As such, FAAzo-10 is a useful tool to study the complex effects, with high specificity, which FA-derivatives such as Gw-9508 exert at multiple targets in mouse ß-cells.
RESUMEN
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.