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1.
Nat Commun ; 12(1): 4488, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301944

RESUMO

Opn7b is a non-visual G protein-coupled receptor expressed in zebrafish. Here we find that Opn7b expressed in HEK cells constitutively activates the Gi/o pathway and illumination with blue/green light inactivates G protein-coupled inwardly rectifying potassium channels. This suggests that light acts as an inverse agonist for Opn7b and can be used as an optogenetic tool to inhibit neuronal networks in the dark and interrupt constitutive inhibition in the light. Consistent with this prediction, illumination of recombinant expressed Opn7b in cortical pyramidal cells results in increased neuronal activity. In awake mice, light stimulation of Opn7b expressed in pyramidal cells of somatosensory cortex reliably induces generalized epileptiform activity within a short (<10 s) delay after onset of stimulation. Our study demonstrates a reversed mechanism for G protein-coupled receptor control and Opn7b as a tool for controlling neural circuit properties.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Neurônios/metabolismo , Opsinas/metabolismo , Optogenética/métodos , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Proteínas de Ligação ao GTP/genética , Células HEK293 , Humanos , Camundongos Endogâmicos C57BL , Neurônios/fisiologia , Opsinas/genética , Células Piramidais/metabolismo , Células Piramidais/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/genética , Córtex Somatossensorial/citologia , Córtex Somatossensorial/metabolismo , Sinapses/genética , Sinapses/fisiologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
2.
Chembiochem ; 21(5): 612-617, 2020 03 02.
Artigo em Inglês | MEDLINE | ID: mdl-31468691

RESUMO

Optogenetics uses light-sensitive proteins, so-called optogenetic tools, for highly precise spatiotemporal control of cellular states and signals. The major limitations of such tools include the overlap of excitation spectra, phototoxicity, and lack of sensitivity. The protein characterized in this study, the Japanese lamprey parapinopsin, which we named UVLamP, is a promising optogenetic tool to overcome these limitations. Using a hybrid strategy combining molecular, cellular, electrophysiological, and computational methods we elucidated a structural model of the dark state and probed the optogenetic potential of UVLamP. Interestingly, it is the first described bistable vertebrate opsin that has a charged amino acid interacting with the Schiff base in the dark state, that has no relevance for its photoreaction. UVLamP is a bistable UV-sensitive opsin that allows for precise and sustained optogenetic control of G protein-coupled receptor (GPCR) pathways and can be switched on, but more importantly also off within milliseconds via lowintensity short light pulses. UVLamP exhibits an extremely narrow excitation spectrum in the UV range allowing for sustained activation of the Gi/o pathway with a millisecond UV light pulse. Its sustained pathway activation can be switched off, surprisingly also with a millisecond blue light pulse, minimizing phototoxicity. Thus, UVLamP serves as a minimally invasive, narrow-bandwidth probe for controlling the Gi/o pathway, allowing for combinatorial use with multiple optogenetic tools or sensors. Because UVLamP activated Gi/o signals are generally inhibitory and decrease cellular activity, it has tremendous potential for health-related applications such as relieving pain, blocking seizures, and delaying neurodegeneration.


Assuntos
Proteínas de Peixes/metabolismo , Lampreias/metabolismo , Optogenética/métodos , Receptores Acoplados a Proteínas G/metabolismo , Opsinas de Bastonetes/metabolismo , Animais , Células HEK293 , Humanos , Raios Ultravioleta
3.
Chembiochem ; 20(14): 1766-1771, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-30920724

RESUMO

The primary goal of optogenetics is the light-controlled noninvasive and specific manipulation of various cellular processes. Herein, we present a hybrid strategy for targeted protein engineering combining computational techniques with electrophysiological and UV/visible spectroscopic experiments. We validated our concept for channelrhodopsin-2 and applied it to modify the less-well-studied vertebrate opsin melanopsin. Melanopsin is a promising optogenetic tool that functions as a selective molecular light switch for G protein-coupled receptor pathways. Thus, we constructed a model of the melanopsin Gq protein complex and predicted an absorption maximum shift of the Y211F variant. This variant displays a narrow blue-shifted action spectrum and twofold faster deactivation kinetics compared to wild-type melanopsin on G protein-coupled inward rectifying K+ (GIRK) channels in HEK293 cells. Furthermore, we verified the in vivo activity and optogenetic potential for the variant in mice. Thus, we propose that our developed concept will be generally applicable to designing optogenetic tools.


Assuntos
Opsinas de Bastonetes/química , Opsinas de Bastonetes/efeitos da radiação , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Humanos , Luz , Camundongos , Mutação , Optogenética/métodos , Estudo de Prova de Conceito , Engenharia de Proteínas , Células de Purkinje/metabolismo , Células de Purkinje/efeitos da radiação , Opsinas de Bastonetes/genética , Alinhamento de Sequência , Transdução de Sinais/efeitos da radiação
4.
Commun Biol ; 2: 60, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30793039

RESUMO

The signal specificity of G protein-coupled receptors (GPCRs) including serotonin receptors (5-HT-R) depends on the trafficking and localization of the GPCR within its subcellular signaling domain. Visualizing traffic-dependent GPCR signals in neurons is difficult, but important to understand the contribution of GPCRs to synaptic plasticity. We engineered CaMello (Ca2+-melanopsin-local-sensor) and CaMello-5HT2A for visualization of traffic-dependent Ca2+ signals in 5-HT2A-R domains. These constructs consist of the light-activated Gq/11 coupled melanopsin, mCherry and GCaMP6m for visualization of Ca2+ signals and receptor trafficking, and the 5-HT2A C-terminus for targeting into 5-HT2A-R domains. We show that the specific localization of the GPCR to its receptor domain drastically alters the dynamics and localization of the intracellular Ca2+ signals in different neuronal populations in vitro and in vivo. The CaMello method may be extended to every GPCR coupling to the Gq/11 pathway to help unravel new receptor-specific functions in respect to synaptic plasticity and GPCR localization.


Assuntos
Técnicas Biossensoriais , Cálcio/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Optogenética/métodos , Receptor 5-HT2A de Serotonina/genética , Opsinas de Bastonetes/genética , Animais , Cerebelo/citologia , Cerebelo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Dependovirus/genética , Dependovirus/metabolismo , Eletrodos Implantados , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Neurônios/metabolismo , Transporte Proteico , Ratos , Ratos Long-Evans , Receptor 5-HT2A de Serotonina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Opsinas de Bastonetes/metabolismo , Técnicas Estereotáxicas
5.
Curr Biol ; 26(9): 1206-12, 2016 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-27068418

RESUMO

G-protein-coupled receptors (GPCRs) represent the major protein family for cellular modulation in mammals. Therefore, various strategies have been developed to analyze the function of GPCRs involving pharmaco- and optogenetic approaches [1, 2]. However, a tool that combines precise control of the activation and deactivation of GPCR pathways and/or neuronal firing with limited phototoxicity is still missing. We compared the biophysical properties and optogenetic application of a human and a mouse melanopsin variant (hOpn4L and mOpn4L) on the control of Gi/o and Gq pathways in heterologous expression systems and mouse brain. We found that GPCR pathways can be switched on/off by blue/yellow light. The proteins differ in their kinetics and wavelength dependence to activate and deactivate G protein pathways. Whereas mOpn4L is maximally activated by very short light pulses, leading to sustained G protein activation, G protein responses of hOpn4L need longer light pulses to be activated and decline in amplitude. Based on the different biophysical properties, brief light activation of mOpn4L is sufficient to induce sustained neuronal firing in cerebellar Purkinje cells (PC), whereas brief light activation of hOpn4L induces AP firing, which declines in frequency over time. Most importantly, mOpn4L-induced sustained firing can be switched off by yellow light. Based on the biophysical properties, hOpn4L and mOpn4L represent the first GPCR optogenetic tools, which can be used to switch GPCR pathways/neuronal firing on an off with temporal precision and limited phototoxicity. We suggest to name these tools moMo and huMo for future optogenetic applications.


Assuntos
Encéfalo/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Opsinas de Bastonetes/metabolismo , Animais , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica/fisiologia , Variação Genética , Humanos , Camundongos , Mutação , Células de Purkinje/fisiologia , Opsinas de Bastonetes/genética
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