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

Summary

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.

Abstract

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.

Introduction

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 ligands^2,3,4. This technique has limited specificity due to diffusion of caged agonists. Genetic specificity is achieved by ectopically expressing light-sensitive opsin channels and pumps^5,6,7, 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 proteins^8,9,10. 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 relationships^12,13,14. 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 cells^15,16,17,18. Uaa incorporation into neuronal proteins has been demonstrated in primary neurons and neural stem cells^19,20. 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 excitable^23,24. 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.

Protocol

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

1. DNA Construction
   1. 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 chosen site with the photoresponsive Uaa incorporated enables optical modulation of Kir2.1 function²¹.
   2. Construct a recombinant DNA encoding Kir2.1 gene with the chosen site mutated to the TAG amber stop codon. Use the standard cloning techniques²⁵ to clone the Kir2.1-TAG DNA into a mammalian expression plasmid.
   3. Clone tRNA/synthetase genes that are specific for the Uaa and incorporate the Uaa in response to the TAG stop codon into another mammalian expression plasmid²¹.
      Note: For optimal Uaa incorporation, different promoters and combinations of gene cassettes (for tRNA, synthetase, and Kir2.1) can be tested in different plasmids.
   4. Obtain high quality plasmid DNAs using miniprep or maxiprep commercial kits according to manufacturer's protocol. Around 1.9 of the 260/280 ratio is optimal for the purified DNA. When necessary, perform an agarose gel electrophoresis to check purity of the prepped DNA²⁵. Supercoiled plasmid DNAs with high purity are best for neuronal transfection.
2. Culture Rat Hippocampal Primary Neurons
   1. Place glass coverslips (circles, 12 mm in diameter) in 24-well plates, one coverslip on each well, and coat each well with 250 µl of 0.5 mg/ml poly-D-lysine (in 100 mM borate buffer: 1.24 g boric acid and 1.90 g sodium tetraborate in 400 ml of deionized distilled H₂O or ddH₂O at pH 8.5) for over 10 hr at room temperature, sealed. Prepare around 10-12 coated coverslips per pup.
   2. On the day of dissection, collect neonatal brains from postnatal rat pups (1-4 postnatal day) after anesthetizing them with isoflurane, and decapitating. Anesthetize the pups in an anesthetization chamber containing isoflurane (2-4%). Wait until they lose consciousness and fail to respond to tactile stimuli and to pinching of the paws.
   3. Using the standard technique²⁶, dissect hippocampi from the brain in warm (~37 °C) saline (10 mM HEPES and 20 mM D-glucose added in Hank's balanced salt solution), and collect hippocampi in a conical tube with 4.5 ml warm saline.
   4. Add 500 µl of 2.5% trypsin to the hippocampi (0.25% final trypsin concentration), and incubate for 10 min in a water bath at 37 °C.
   5. Thoroughly rinse the tissue with saline (3 x 10 ml) and triturate.
   6. Recover the dissociated neurons in 1 ml warm growth media containing Minimum Essential Medium (MEM) supplemented with 5% Fetal Bovine Serum (FBS), 21.2 mM D-glucose, 2 mM L-glutamine, 2% B-27, and 0.1% serum extender.
      Note: Add L-glutamine and B-27 on the day of dissection to prepare fresh growth media.
   7. Count the neurons with a hemocytometer, and plate them onto coverslips in 24-well plates at 1.0-1.5 x 10⁵ cell/well density with 500 µl growth media after filtered through a 40 µm nylon mesh.
   8. Incubate the neuronal culture at 35 °C in a 5% CO₂: 95% air humidified incubator for 2-3 weeks. For the best result, avoid disturbing the culture as much as possible during incubation.
3. Calcium phosphate (Ca-P) Transfection of Primary Neuronal Culture
   1. Make stock solutions and store at 4 °C: 0.5 M BES (N,N-bis[2-hydroxy-ethyl]-2-aminoethanesulfonic acid) buffer (10x); 150 mM Na₂HPO₄ (100x); 2.8 M NaCl (10x); sterile ddH₂O; sterile 1 N NaOH.
   2. On the day of transfection, make fresh 2.5 M CaCl₂ solution in ddH₂O and sterile filter with 0.22 µm filter. Make fresh 2x BES-buffered Saline (BBS) buffer containing 50 mM BES, 1.5 mM Na₂HPO₄, and 280 mM NaCl at pH 7.00 (adjust pH with NaOH) and sterile filter with 0.22 µm filter.
      Note: Calculate the amount of CaCl₂ solution and BBS buffer depending on the number of coverslips to be transfected. Also look at 1.3.4.
   3. Replace the culture growth media with 500 µl fresh pre-warmed transfection growth media. (Transfection media can be made with MEM plus 21.2 mM D-glucose).
      Note: Do not discard the old media.
   4. Calculate the amount of ddH₂O and prepare to add in order to make the transfection solution (33 µl per each coverslip) containing 1.65 µl CaCl₂, 0.7 µg DNA, and 16.5 µl 2x BBS.
   5. Prepare the transfection solution immediately before adding it to the culture. To prepare, first combine CaCl₂ and ddH₂O while slowly agitating the tube. Continue agitating and slowly add DNA into the solution. Lastly, add 2x BBS buffer drop-wise while agitating.
   6. Immediately add 30 µl of the transfection solution to each coverslip of neuronal culture.
   7. Rock the culture dish a few times to mix the solution and incubate at 35 °C in a 5% CO₂: 95% air humidified incubator for 45 min-1 hr. After neurons are incubated with the transfection solution, very fine Ca-P precipitates would form a layer covering neurons.
   8. Replace the transfection media with 500 µl pre-warmed washing buffer (135 mM NaCl, 20 mM HEPES, 4 mM KCl, 2 mM CaCl₂, 1 mM MgCl₂, 1 mM Na₂HPO₄, 10 mM glucose at pH 7.3, sterile filtered with 0.22 µm filter) and incubate at 35 °C in a 5% CO₂: 95% air humidified incubator for 15-20 min. Ca-P precipitates would disappear after the wash step.
   9. Replace the washing buffer with 500 µl fresh growth media. Again, replace the growth media with the saved original media.
   10. Add the Uaa Cmn (pre-mixed in 50 µl warm growth media) to the culture to reach 1 mM final concentration.
   11. Incubate the transfected culture at 35 °C in a 5% CO₂: 95% air humidified incubator for 12-48 hr before assays.
4. Whole Cell Recording with Light Activation
   1. Prepare extra/intracellular solutions. The intracellular solution contains 135 mM potassium gluconate, 10 mM NaCl, 2 mM MgCl₂, 10 mM HEPES, 1 mM EGTA, 2.56 mM K₂ATP, and 0.3 mM Li₂GTP at pH 7.4. The extracellular recording solution contains 150 mM NaCl, 3 mM KCl, 5 mM MgCl₂, 0.5 mM CaCl₂, 5 mM glucose, and 10 mM HEPES at pH 7.4.
   2. Set up the electrophysiology rig for whole-cell patch clamping: microscope fitted with 4X and 20X objectives, differential interference contrast (DIC), and mCitrine filter (excitation: 495/10 nm, emission: 525/25 nm); manipulator; patch-clamp amplifier; digitizer; data acquisition and analysis software.
      1. Install a light-emitting diode (LED) with emission of 385 nm by the microscope at the rig to deliver light to the focal point from 1 cm away at a 45° angle. Check the power of the LED with a light power meter.
   3. Pull patch pipettes from glass electrodes using a commercial micropipette puller to have 3-6 MΩ pipette resistance. Follow manufacturer's instruction to set up the micropipette puller.
      1. To test pipette resistance, first fill the pipette with the intracellular solution and position it on the electrode holder. Dip the pipette in a 35 mm culture dish filled with the extracellular solution, placed on the microscope platform. Immerse a ground electrode into the dish to complete a circuit.
      2. Turn on the amplifier/digitizer and start a data acquisition software. Monitor pipette resistance with a membrane test protocol.
   4. Take out a coverslip of the neuron culture from the incubator and rinse once in the extracellular solution. Using vacuum grease, hold down the coverslip in the middle of a 35 mm culture dish filled with the fresh extracellular solution.
   5. Place the coverslip/dish on the electrophysiology microscope platform.
   6. Using the standard patch clamping techniques, patch a neuron with mCitrine fluorescence²⁷. Record neuronal activity using current clamp (I-clamp) method. First, adjust the resting potential to around -72 mV by injecting a small current. Then, inject a step current (10-200 pA) to induce continuous firing (5-15 Hz) of action potentials.
   7. Manually, or using the data acquisition software, flash a pulse (a single pulse of 100 msec^-1 sec duration) of the LED light to the neuron while recording, and see if action potentials are affected.
   8. Add 0.5 mM BaCl₂ to the bath and verify if action potentials are recovered.

2. Uaa Incorporation in Kir2.1 and Expression of the Resultant PIRK in the Mouse Embryonic Brain In Vivo

1. DNA Construction
   1. Design the plasmid DNA similarly as in step 1.1. For in vivo expression, use a strong promoter such as CAG (chicken beta-actin promoter with CMV enhancer).
   2. Purify DNA with an endotoxin-free maxiprep commercial kit according to the manufacturer's protocol. Perform phenol-chloroform extraction followed by ethanol precipitation²⁵ to acquire extremely high quality DNA (condensed to 2-5 µg/µl).
2. In Utero Electroporation to the Mouse Embryonic Neocortex
   Note: The basic technique for in utero electroporation has been described previously²⁸.
   1. Anesthetize a timed pregnant mouse at the embryonic day 14.5 (E14.5) with sodium pentobarbital (intraperitoneal injection, 50 µg per gram body weight) or isoflurane (inhalation, 2-4%). Monitor anesthetic depth by checking loss of consciousness and no response to tactile stimuli and to pinching of the paws.
   2. Make a small incision at the abdominal midline. Gently expose the uterine horns with forceps and fingertips.
   3. Inject about 1 µl DNA solution (2-5 µg/µl of each plasmid, depending on the construct) into the lateral ventricle of each littermate with a glass pipette inserted through the uterine wall. In most cases, about 60-80% of total embryos are injected.
   4. Electroporate the embryos with an electroporator (33-35 V, 50 msec duration, 950 msec interval, 4-8 pulses).
   5. Return the uterine horns to the abdominal cavity gently with forceps and fingertips. Suture the muscle wall and then the skin with surgical suture to allow the embryos to continue development.
3. Uaa Microinjection
   1. Make a small incision at the abdominal midline again at E16.5, and gently expose the uterine horns with forceps and fingertips.
   2. Inject about 2-5 µl Cmn-Ala (500 mM) to the electroporated side or both sides of the lateral ventricle with a glass pipette inserted through the uterine wall. To increase Cmn bioavailability, use the dipeptide Cmn-Ala (Cmn-alanine) to deliver Cmn in vivo²¹.
   3. Again, return the uterine horns to the abdominal cavity gently with forceps and fingertips. Suture the muscle wall and then the skin with surgical suture to allow the embryos to continue development.
4. Obtain Acute Brain Slices
   1. Make 1 L artificial cerebrospinal fluid (ACSF) containing 119 mM NaCl, 2.5 mM KCl, 1.3 mM MgCl₂, 2.5 mM CaCl₂, 1 mM NaH₂PO₄, 26.2 mM NaHCO₃, and 11 mM glucose at pH 7.3.
   2. Take 200 ml ACSF and fast-freeze at -80 °C for 20-30 min. Bubble the rest of ACSF with 5% CO₂: 95% O₂ gas at room temperature.
   3. Prepare and sterilize surgery tools to harvest brains from the embryos. Also prepare a bucket of ice.
   4. Make ~100 ml 4% low melting point agarose solution in a flask by heating in a microwave. Cool down solution for about 5 min before it starts to solidify.
   5. Euthanize the mouse 12-24 hr after Cmn-Ala injection by CO₂ overdose. Make large incisions at the abdominal area, and dissect out the electroporated/microinjected embryos from the uterus with fine scissors and forceps.
   6. Harvest brains from the embryos with fine scissors and forceps.
   7. Place each brain on a 10 cm culture dish placed on the ice bucket. Divide two hemispheres using a sharp blade, and place each hemisphere on the dish with the midsagittal plane touching the bottom of the dish. Quickly pour over the agarose solution over brains (around 500 µl per hemisphere).
   8. Using a sharp blade, square cut the agarose around a brain to make a brain-embedded agarose block.
   9. Using tissue adhesive, glue down the midsagittal plane surface of the agarose block on a mount for vibratome. Fill the vibratome chamber with pre-chilled ACSF and cut 200 µm sagittal brain slices.
   10. Incubate acute slices in ACSF supplemented with 3 mM myo-inositol, 0.4 mM ascorbic acid, and 2 mM sodium pyruvate at 33 °C for 42 min while bubbling with 5% CO₂: 95% O₂ gas.
   11. After 42 min, turn off the heater and continue to incubate the slices at room temp with bubbling. Start whole-cell patch recording at least 15 min after turning off the heater.
5. Whole Cell Recording with Light Activation
   1. Prepare the intracellular solution containing 130 mM potassium gluconate, 4 mM MgCl₂, 5 mM HEPES, 1.1 mM EGTA, 3.4 mM Na₂ATP, 10 mM sodium creatine phosphate, and 0.1 mM Na₃GTP at pH 7.3 adjusted with KOH.
   2. Pull patch pipettes from glass electrodes using a commercial micropipette puller to have 3-6 MΩ pipette resistance.
   3. Set up the slice electrophysiology rig for whole-cell patch clamping and superfuse the recording chamber with ACSF at 2 ml/min rate with a perfusion pump. Adjust the chamber temperature to around 33 °C.
      Note: The slice electrophysiology rig is equipped with a perfusion chamber, a water immersion objective and a temperature controller. Also, GFP filter (excitation: 480/30 nm, emission: 535/40 nm) and mCherry filter (excitation: 580/20 nm emission: 675/130 nm) are required.
      1. Install an LED with emission of 385 nm by the microscope at the rig to deliver light to the focal point from 1 cm away at a 45° angle. Check the power of the LED with a light power meter.
   4. Carefully pick up a brain slice with a glass pipette and place in the perfusion chamber. Hold down the slice with a harp.
   5. Patch a neuron with mCherry/GFP fluorescence from the neocortical region²⁷. Record PIRK activity using voltage clamp (V-clamp) method. First, hold the membrane potential at -60 mV. Then, record currents at fixed negative membrane potentials (-100 mV) or voltage ramps (-100 mV to +40 mV). Specifically, monitor Kir2.1 specific inward currents at -100 mV.
   6. Manually, or using the data acquisition software, flash a pulse (a single pulse of 100 msec^-1 sec duration) of LED light to the neuron while recording, and see if PIRK proteins are activated. Once PIRK is activated, inward currents at -100 mV would increase significantly.
   7. Add 0.5 mM BaCl₂ to the bath and verify if PIRK is inactivated again.

Results

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...

Discussion

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cover Glasses, Circles, 12 mm, Thickness 0.13-0.17 mm	Carolina Biologicals	633029
Corning BioCoat Poly-D-Lysine	Corning Discovery Labware	354210
D-(+)-Glucose solution	Sigma-Aldrich	G8769
Iris Spatula-curved	Fine Science Tool	10092-12
Dissecting Knife - Fine Angled Tip	Fine Science Tool	10056-12
GlutaMAX-I Supplement	Life Technologies	35050-061
MITO+ Serum Extender	BD Biosciences	355006
Falcon 40 µm Cell Strainer	Corning Life Sciences	352340
BES (N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, N,N-Bis(2-hydroxyethyl)taurine)	Sigma-Aldrich	B6420
LED LIGHT SOURCE – Black LED 385	Prizmatix Ltd.
Agarose, Low Melting Point, Analytical Grade	Promega	V2111