Name: optical control of a neuronal protein using a genetically encoded unnatural amino acid in neurons
Uploaded: 2016-03-28T14:00:00
Description: Watch this Scientific Journal Video about Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons at JoVE.com

We thank Dr. S. Szobota for helpful discussion on neuron culture and Ca-P transfection. L.W. acknowledges support from The Salk Innovation Grant, the California Institute for Regenerative Medicine (RN1-00577-1), and the National Institutes of Health (1DP2OD004744).

All procedures in the current study were performed using Institutional Animal Care and Use Committee (IACUC) approved protocols for animal handling at The Salk Institute for Biological Studies, La Jolla, CA.
1. Uaa Incorporation in Kir2.1 and Expression of the Resultant PIRK in the Primary Neuronal Culture
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	<li>DNA Construction
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		<li>Select a target site of Kir2.1 for Uaa incorporation. Exploit prior knowledge and information about structure and function of Kir2.1, so that the cho...

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To achieve effective photo-modulation, the important initial step is to decide where to incorporate the photoresponsive Uaa in the target protein. Structural and functional information of the target protein is very helpful to guide the selection of candidate sites. At the same time, the purpose of light regulation would determine which site is most suitable. After choosing candidate sites, we recommend to test the sites in mammalian cell lines such as the Human Embryonic Kidney (HEK) cells for easier culture and manipula...

Compared to conventional electric stimulation, photostimulation offers greater temporal and spatial resolution with minimum interference to the physiological system of specimens. Since the demonstration of using lasers to stimulate neurons in 19711, many creative ways have been invented to exogenously control neuronal activity with light. Optical release of photocaged agonists has long been used to study physiological response of the neuronal network to ligands2,3,4. This technique has limited specificity due to diffusion of caged agonists. Genetic specificity is achieved by ectopically expressing light-sensitive opsin channels and pumps5,6,7, and it has been successfully applied to modulate selected neuronal networks in diverse model organisms. However, it would be difficult to apply this method to optically control various other neuronal proteins, since grafting photoresponsiveness from the opsin proteins to other proteins would require intense engineering that may alter the natural characteristics of the protein under study. Chemically tethering an exogenous photosensitive ligand to a protein has demonstrated another way to control the functionality of channel proteins8,9,10. The ligand is presented or withdrawn from the binding site of the protein through the photoisomerization of the azobenzene moiety. Tethering chemistry limits the application mainly to the extracellular side of membrane proteins, excluding the intracellular side and intracellular proteins.
Photoresponsive Uaas, after being incorporated into proteins, provide a general strategy to manipulate proteins with light. In early efforts, tRNAs chemically acylated with photocaged Uaas were microinjected into Xenopus oocytes to incorporate the Uaas into membrane receptors and ion channels11, which have advanced the understanding of their structure-function relationships12,13,14. This microinjection approach is mainly limited to large oocytes. Genetic incorporation of a Uaa bypasses the technically challenging tRNA acylation and microinjection by using an orthogonal tRNA/synthetase pair, which incorporates the Uaa through endogenous protein translation in live cells15,16,17,18. Uaa incorporation into neuronal proteins has been demonstrated in primary neurons and neural stem cells19,20. More recently, photoresponsive Uaa has been genetically incorporated into a neuronal protein in the mammalian brain in vivo for the first time21. These advancements make it possible to study neuronal proteins with Uaas in their native cellular environment.
Inwardly rectifying potassium channel Kir2.1 is a strong rectifier that passes K+ currents more readily into than out of the cell, and it is essential in regulating physiological processes including cell excitability, vascular tone, heart rate, renal salt flow and insulin release22. Overexpression of Kir2.1 hyperpolarizes the membrane potential of the target neuron, which becomes less excitable23,24. To optically control Kir2.1 in its native cells, Kang et al. genetically incorporated a photo-responsive Uaa into Kir2.1 expressed in mammalian cells, neurons and embryonic mouse brains21. A brief pulse of light was able to convert the Uaa into a natural amino acid Cys, thus activating the target Kir2.1 protein. When this photo-inducible inwardly rectifying potassium (PIRK) channel protein was expressed in rat hippocampal primary neurons, it suppressed neuronal firing in response to light activation. In addition, the PIRK channel was expressed in the embryonic mouse neocortex, and the light-activated PIRK current in cortical neurons was measured. The successful implementation of the Uaa technology in vivo in the mammalian brain opens the door to optically control neuronal proteins in their native environment, which will enable optical dissection of neuronal processes and mechanisms at the molecular level.
In this protocol we describe procedures for genetic incorporation of Uaas into primary neurons in culture and in the embryonic mouse brain in vivo. The photoresponsive Uaa Cmn and Kir2.1 are used to illustrate the process. Methods to assess successful Uaa incorporation and optical control of neuronal protein activity are provided. This protocol provides a clear guide to genetically encoding Uaas in neurons and in vivo, and to optically regulating neuronal protein function via a photoresponsive Uaa. We expect this protocol to facilitate the adoption of the in vivo Uaa technology for neuroscience and optogenetic biological studies.

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optical control of a neuronal protein using a genetically encoded unnatural amino acid in neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

To genetically incorporate a Uaa into a protein in neurons, the first important step is to design appropriate gene constructs to deliver and express genes efficiently in neurons. There are three genetic components for Uaa incorporation: (1) the target gene with the TAG amber stop codon introduced at the chosen site for Uaa incorporation (2) an orthogonal tRNA to recognize the mutated TAG stop codon, and (3) an orthogonal aminoacyl-tRNA synthetase to charge the Uaa onto the orthogonal tRNA...

Watch this Scientific Journal Video about Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons at JoVE.com

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Research

JoVE Journal

Neuroscience

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Efficient Derivation of Human Neuronal Progenitors and Neurons from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

There is a large unfulfilled need for a clinically-suitable human neuronal cell source for repair or regeneration of the damaged central nervous system ...

Imaging Neuronal Responses in Slice Preparations of Vomeronasal Organ Expressing a Genetically Encoded Calcium Sensor

The vomeronasal organ (VNO) detects chemosensory signals that carry information about the social, sexual and reproductive status of the individuals within ...

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Super-resolution Imaging of Neuronal Dense-core Vesicles

Detection of fluorescence provides the foundation for many widely utilized and rapidly advancing microscopy techniques employed in modern biological and ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Imaging Intracellular Ca2+ Signals in Striatal Astrocytes from Adult Mice Using Genetically-encoded Calcium Indicators

Astrocytes display spontaneous intracellular Ca2+ concentration fluctuations ([Ca2+]i) and in several settings respond to neuronal excitation with ...

Imaging Membrane Potential with Two Types of Genetically Encoded Fluorescent Voltage Sensors

Genetically encoded voltage indicators (GEVIs) have improved to the point where they are beginning to be useful for in vivo recordings. While the ultimate ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...