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1.
J Biol Chem ; 291(52): 26899-26912, 2016 Dec 23.
Artículo en Inglés | MEDLINE | ID: mdl-27875296

RESUMEN

Temperature sensors are crucial for animals to optimize living conditions. The temperature response of the ion channel transient receptor potential A1 (TRPA1) is intriguing; some orthologs have been reported to be activated by cold and others by heat, but the molecular mechanisms responsible for its activation remain elusive. Single-channel electrophysiological recordings of heterologously expressed and purified Anopheles gambiae TRPA1 (AgTRPA1), with and without the N-terminal ankyrin repeat domain, demonstrate that both proteins are functional because they responded to the electrophilic compounds allyl isothiocyanate and cinnamaldehyde as well as heat. The proteins' similar intrinsic fluorescence properties and corresponding quenching when activated by allyl isothiocyanate or heat suggest lipid bilayer-independent conformational changes outside the N-terminal domain. The results show that AgTRPA1 is an inherent thermo- and chemoreceptor, and analogous to what has been reported for the human TRPA1 ortholog, the N-terminal domain may tune the response but is not required for the activation by these stimuli.


Asunto(s)
Repetición de Anquirina , Culicidae/metabolismo , Activación del Canal Iónico/fisiología , Canales de Potencial de Receptor Transitorio/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Frío , Cristalografía por Rayos X , Calor , Humanos , Unión Proteica , Conformación Proteica , Homología de Secuencia de Aminoácido , Canales de Potencial de Receptor Transitorio/aislamiento & purificación
2.
Methods Mol Biol ; 1987: 39-50, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31028672

RESUMEN

Cryo electron microscopy (cryo-EM) is a powerful technique that can be used to elucidate the structural architecture of a protein molecule in a physiologically relevant environment. In this method, purified protein is frozen in its aqueous buffer in a thin layer of vitreous ice in which the biological macromolecules are embedded in various orientations. Images of this frozen sample are collected with an electron microscope, and the data is processed using different software algorithms resulting in high-resolution structures of the protein. Proteins in the presence of various ligands or other macromolecular complexes can also be studied by this method. Here, we present a protocol for the purification and vitrification of TRP channels for single particle cryo-EM.


Asunto(s)
Microscopía por Crioelectrón/métodos , Canales de Potencial de Receptor Transitorio/aislamiento & purificación , Canales de Potencial de Receptor Transitorio/ultraestructura , Algoritmos , Programas Informáticos , Vitrificación
3.
Cells ; 8(2)2019 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-30754715

RESUMEN

(1) Background: Human transient receptor potential (TRP) channels constitute a large family of ion-conducting membrane proteins that allow the sensation of environmental cues. As the dysfunction of TRP channels contributes to the pathogenesis of many widespread diseases, including cardiac disorders, these proteins also represent important pharmacological targets. TRP channels are typically produced using expensive and laborious mammalian or insect cell-based systems. (2) Methods: We demonstrate an alternative platform exploiting the yeast Saccharomyces cerevisiae capable of delivering high yields of functional human TRP channels. We produce 11 full-length human TRP members originating from four different subfamilies, purify a selected subset of these to a high homogeneity and confirm retained functionality using TRPM8 as a model target. (3) Results: Our findings demonstrate the potential of the described production system for future functional, structural and pharmacological studies of human TRP channels.


Asunto(s)
Proteínas Recombinantes/biosíntesis , Saccharomyces cerevisiae/metabolismo , Canales de Potencial de Receptor Transitorio/biosíntesis , Canales de Potencial de Receptor Transitorio/aislamiento & purificación , Animales , Detergentes/farmacología , Humanos , Filogenia , Solubilidad , Canales de Potencial de Receptor Transitorio/química
4.
Neuroscience ; 396: 66-72, 2019 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-30458219

RESUMEN

Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.


Asunto(s)
Activación del Canal Iónico/efectos de la radiación , Luz , Microvellosidades/metabolismo , Microvellosidades/efectos de la radiación , Células Fotorreceptoras de Invertebrados/citología , Canales de Potencial de Receptor Transitorio/metabolismo , Canales de Potencial de Receptor Transitorio/efectos de la radiación , Animales , Diacilglicerol Quinasa/biosíntesis , Diglicéridos/farmacología , Proteínas de Drosophila/genética , Proteínas de Drosophila/aislamiento & purificación , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/efectos de la radiación , Drosophila melanogaster , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Potenciales de la Membrana/efectos de los fármacos , Proteína Quinasa C/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Sulfonamidas/farmacología , Canales de Potencial de Receptor Transitorio/aislamiento & purificación , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/genética
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