Abstract
Deciphering neuronal networks governing specific brain functions is a longstanding mission in neuroscience, yet global manipulation of protein functions pharmacologically or genetically lacks sufficient specificity to reveal a neuronal protein’s function in a particular neuron or a circuitry. Photostimulation presents a great venue for researchers to control neuronal proteins with high temporal and spatial resolution. Recently, an approach to optically control the function of a neuronal protein directly in neurons has been demonstrated using genetically encoded light-sensitive Unnatural amino acids (Uaas). Here, we describe procedures for genetically incorporating Uaas into target neuronal proteins in neurons in vitro and in embryonic mouse brain. As an example, a photocaged Uaa was incorporated into an inwardly rectifying potassium channel Kir2.1 to render Kir2.1 photo-activatable. This method has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. Uaas with other properties can be similarly incorporated into neuronal proteins in neurons for various applications.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Callaway EM, Katz LC (1993) Photostimulation using caged glutamate reveals functional circuitry in living brain slices. Proc Natl Acad Sci U S A 90:7661–7665
Yoshimura Y, Callaway EM (2005) Fine-scale specificity of cortical networks depends on inhibitory cell type and connectivity. Nat Neurosci 8:1552–1559
Banghart M, Borges K, Isacoff E, Trauner D, Kramer RH (2004) Light-activated ion channels for remote control of neuronal firing. Nat Neurosci 7:1381–1386
Volgraf M, Gorostiza P, Numano R, Kramer RH, Isacoff EY, Trauner D (2006) Allosteric control of an ionotropic glutamate receptor with an optical switch. Nat Chem Biol 2:47–52
Boyden ES, Zhang F, Bamberg E, Nagel G, Deisseroth K (2005) Millisecond-timescale, genetically targeted optical control of neural activity. Nat Neurosci 8:1263–1268
Fenno L, Yizhar O, Deisseroth K (2011) The development and application of optogenetics. Annu Rev Neurosci 34:389–412
Adamantidis A, Arber S, Bains JS, Bamberg E, Bonci A, Buzsaki G, Cardin JA, Costa RM, Dan Y, Goda Y, Graybiel AM, Hausser M, Hegemann P, Huguenard JR, Insel TR, Janak PH, Johnston D, Josselyn SA, Koch C, Kreitzer AC, Luscher C, Malenka RC, Miesenbock G, Nagel G, Roska B, Schnitzer MJ, Shenoy KV, Soltesz I, Sternson SM, Tsien RW, Tsien RY, Turrigiano GG, Tye KM, Wilson RI (2015) Optogenetics: 10 years after ChR2 in neurons--views from the community. Nat Neurosci 18:1202–1212
Wang L, Brock A, Herberich B, Schultz PG (2001) Expanding the genetic code of Escherichia coli. Science 292:498–500
Wang L, Schultz PG (2005) Expanding the genetic code. Angew Chem Int Ed 44:34–66
Wang L (2017) Engineering the genetic code in cells and animals: biological considerations and impacts. Acc Chem Res 50:2767–2775
Liu CC, Schultz PG (2010) Adding new chemistries to the genetic code. Annu Rev Biochem 79:413–444
Wang W, Takimoto JK, Louie GV, Baiga TJ, Noel JP, Lee KF, Slesinger PA, Wang L (2007) Genetically encoding unnatural amino acids for cellular and neuronal studies. Nat Neurosci 10:1063–1072
Shen B, Xiang Z, Miller B, Louie G, Wang W, Noel JP, Gage FH, Wang L (2011) Genetically encoding unnatural amino acids in neural stem cells and optically reporting voltage-sensitive domain changes in differentiated neurons. Stem Cells 29:1231–1240
Kang JY, Kawaguchi D, Coin I, Xiang Z, O’Leary DD, Slesinger PA, Wang L (2013) In vivo expression of a light-activatable potassium channel using unnatural amino acids. Neuron 80:358–370
Beaudoin GM III, Lee SH, Singh D, Yuan Y, Ng YG, Reichardt LF, Arikkath J (2012) Culturing pyramidal neurons from the early postnatal mouse hippocampus and cortex. Nat Protoc 7:1741–1754
Kaech S, Banker G (2006) Culturing hippocampal neurons. Nat Protoc 1:2406–2415
Banker G, Goslin K (1998) Culturing Nerve Cells. MIT Press, Cambridge, MA
Beene DL, Dougherty DA, Lester HA (2003) Unnatural amino acid mutagenesis in mapping ion channel function. Curr Opin Neurobiol 13:264–270
Lemke EA, Summerer D, Geierstanger BH, Brittain SM, Schultz PG (2007) Control of protein phosphorylation with a genetically encoded photocaged amino acid. Nat Chem Biol 3:769–772
Hoppmann C, Lacey VK, Louie GV, Wei J, Noel JP, Wang L (2014) Genetically encoding photoswitchable click amino acids in Escherichia coli and mammalian cells. Angew Chem Int Ed Engl 53:3932–3936
Hoppmann C, Maslennikov I, Choe S, Wang L (2015) In situ formation of an azo bridge on proteins controllable by visible light. J Am Chem Soc 137:11218–11221
Coin I, Katritch V, Sun T, Xiang Z, Siu FY, Beyermann M, Stevens RC, Wang L (2013) Genetically encoded chemical probes in cells reveal the binding path of urocortin-I to CRF class B GPCR. Cell 155:1258–1269
Parrish AR, She X, Xiang Z, Coin I, Shen Z, Briggs SP, Dillin A, Wang L (2012) Expanding the genetic code of Caenorhabditis elegans using bacterial aminoacyl-tRNA synthetase/tRNA pairs. ACS Chem Biol 7:1292–1302
Lu H, Klaassen C (2006) Tissue distribution and thyroid hormone regulation of Pept1 and Pept2 mRNA in rodents. Peptides 27:850–857
Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
Acknowledgment
L.W. acknowledges support from NIH (R01GM118384, RF1MH114079).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Kang, JY., Kawaguchi, D., Wang, L. (2018). Genetically Encoding Unnatural Amino Acids in Neurons In Vitro and in the Embryonic Mouse Brain for Optical Control of Neuronal Proteins. In: Lemke, E. (eds) Noncanonical Amino Acids. Methods in Molecular Biology, vol 1728. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7574-7_17
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7574-7_17
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7573-0
Online ISBN: 978-1-4939-7574-7
eBook Packages: Springer Protocols