This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.
Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and – more importantly – allows investigating numerous cytosolic regulatory mechanisms.
Over 200 unnatural amino acids of various chemical and physical properties have been genetically incorporated into proteins in E. coli, yeast and mammalian cells3. The unnatural amino acid is incorporated in response to a specific stop codon via an orthogonal engineered tRNA/synthetase pair. The genetical approach to modify proteins has provided valuable insights into protein structure and function. Here, we present a protocol for using Voltage-Clamp Fluorometry (VCF) in combination with a fluorescent UAA.
In VCF, the simultaneous observation of functional data and structural rearrangements localized around the fluorescent probe (~5 Å) allows us to obtain dynamic information with millisecond resolution1. The fluorescent probes alter their quenching state upon localized movement of the protein. A movement of only 1-2 Å is sufficient to lead to significant changes in the fluorescence intensity4. After identification of the site of interest in the target protein, the site is mutated by point mutation. Classically, the residue had been mutated to a cysteine whereas now, an amber stop codon (TAG) is introduced for genetic fUAA incorporation. The protein is then in vitro transcribed.
While other expression systems (e.g., mammalian cells) can be used5,6,7, Xenopus oocytes are preferable for structure-function studies because of their larger size, leading to easier manipulation and higher fluorescence intensity (more fluorophores) and, therefore, to-noise ratio. Furthermore, Xenopus oocytes have low background from endogenous proteins2,8, and the dark pigmentation on the animal pole shields against background fluorescence from the cytosol. The Xenopus oocytes are surgically removed and DNA encoding the orthogonal tRNA/tRNA-synthetase pair specific for the fUAA is injected into the nucleus of the oocytes. After a 6-24 h incubation time, the protein RNA is co-injected with the fUAA into the cytosol of the oocytes, followed by a 2-3 days incubation period. In order to prevent any damage to the fUAA (photobleaching), the procedures including Anap have to be carried out under red light to avoid fluorophore excitation.
Oocytes are studied on a cut-open oocyte voltage-clamp setup, which is mounted on an upright fluorescence microscope, and electrical current and fluorescence changes are simultaneously recorded9,10. Alternatively, two-electrode voltage clamp1 or patch-clamp configurations11can be used. Fluorescence is excited by appropriate wavelengths with low RMS noise and emission recorded using a photodiode linked to an amplifier with high amplification.
There are several advantages of using fluorescent unnatural amino acids (fUAAs) in voltage-clamp fluorometry. One is access to the cytosolic side of the membrane proteins; many regulatory processes are located here (e.g., Ca2+– or nucleotide binding sites, fast and closed-state inactivation of voltage-gated ion channels, pore opening, module coupling). All these processes are now accessible for fluorescent labeling.
Another advantage is the small size of the probe leading to less disturbance of the protein. So far, two orthogonal tRNA/tRNA synthetase pairs for fUAAs have been engineered12,13, where 3-(6-acetylnaphthalen-2-ylamino)-2-aminopropanoic acid (Anap) is the only fUAA which has been used in Xenopus oocytes2,8. Anap is an environmentally sensitive fluorophore with a molecular weight of 272.3 g/mol and is only slightly bigger than tryptophan12 (Figures 1A, 1B). Due to its small size, fewer steric effects are likely to be introduced by the fluorophore compared to conventional fluorophores attached via a linker (typically more than 500 g/mol). Moreover, in the case of Anap, the fluorophore is located closer to the protein backbone than those linked to cysteines, and consequently, Anap is probing more localized rearrangements. Finally, removal of endogenous cysteines in conventional VCF in order to ensure site-specific labeling is no longer a requirement in UAA-VCF and therefore (i) leaves the proteins in (almost) their native state and (ii) allows VCF to be applied to study a wider range of proteins in which function may be altered by cysteine substitution.
Figure 1: Anap and Fluorescence Spectra. (A) Chemical structure of Anap. (B) Normalized absorption spectrum and emission spectra for 1 nM Anap, demonstrating the sensitivity of Anap fluorescence to the solvent hydrophobicity. Emission spectra were obtained by exciting at 350 nm. Please click here to view a larger version of this figure.
A disadvantage of using fluorescent UAAs is that a heterogeneous population of proteins may result from stop codon readthrough, translational reinitiation, C-terminal truncated proteins or crosstalk with endogenous aminoacylation if the amount of aminoacylated tRNAs is scarce. Such leak expression should always be checked for in the absence of the fUAA and the tRNA/tRNA synthetase pair. We addressed the issue of translational reinitiation and how to circumvent it for N-terminal insertion sites previously14. However, when the fUAA, tRNA and tRNA synthetase are present in saturated amounts, there only remains a low probability of leak expression.
The key procedural difference between fUAA-VCF and conventional VCF is the injection and handling of the oocytes; the injection of DNA encoding the tRNA and tRNA synthetase (pAnap) is followed by the introduction of Anap, which is either co-injected with the protein mRNA or alternatively added to the incubation solution as an acetoxymethyl (AM) ester.
Frog manipulations were performed in accordance with the Canadian guidelines and have been approved by the ethics committee (CDEA, protocol #15-042) of University of Montréal.
1. mRNA Preparation for fUAA Incorporation
2. Oocyte Preparation and Injection
Figure 2: Illustration of DNA and RNA Injection into Xenopus Oocytes for Anap Incorporation.
First, pAnap is injected into the nucleus of the Xenopus oocyte (1). After 6-24 h, Anap and channel RNA are coinjected into the vegetal pole (2). Anap will be orthogonally aminoacylated with the tRNA bearing an amber stop anti-codon, by the aminoacyl-tRNA synthetase which is encoded by pAnap. This way, the aminoacylated Anap-tRNAs are recognized by the ribosome at the inserted amber stop codon in the channel RNA, resulting in suppression of the stop codon and insertion of Anap. Please click here to view a larger version of this figure.
3. VCF Setup
Figure 3: VCF setup. (A) Side view of the VCF setup showing the light path inside the microscope. The filter cube contains an excitation filter, dichroic mirror and an emission filter. (B) Selected oocyte chamber dimensions are 3.4 cm for upper chamber radius (1), 5.5 cm for bottom chamber length (2), 1.4 cm for bottom chamber width (3) and 1.7 cm for middle chamber width (4). (C) Front view of the VCF setup. The first ocular on the left is for mounting the oocyte in the cut-open voltage clamp chamber and for permeabilization. Then, the chamber is slid under the microscope at the second ocular to the right. Here, the V1 electrode is inserted into the oocyte using the 4X objective, and fluorescence is recorded using the water-immersion 40X objective. Please click here to view a larger version of this figure.
4. VCF Recording
5. Two-color VCF
Figure 4 shows an example of VCF recordings obtained from an oocyte expressing Shaker channels with fast inactivation removed (IR), L382stop-W434F in presence of pAnap and Anap. The W434F mutation blocks the ionic potassium currents, which makes it possible to measure the transient gating charge displacements (gating currents). The simultaneous recordings of gating currents (upper trace) and Anap fluorescence intensity changes (lower trace) upon depolarization demonstrate the successful incorporation of Anap into position L382. Here, Anap is located on the bottom of each of the four S4 transmembrane helices that move during activation (L382) and thus report on intracellular local rearrangements. A fluorescence change can be caused by solvent relaxation (environmental polarity changes) and/or quenching by other amino acids4. Both mechanisms are a result of relative protein rearrangements.
Figure 5 displays Anap and TMR fluorescence signals using step protocols obtained from the same oocyte. By adding the A359C mutation into the L382stop-W434F background and labelling with TMR-maleimide, it is possible with the described technique to probe real-time movements in different regions in the same protein. In this case, TMR probes the movement of the upper S4 helix (A359C) while Anap probes the movement of the bottom of S4 (L382). From the time course of the fluorescence change one can retrieve dynamical information of the transition monitored by the fluorescence by analyzing the kinetics (Figures 5A, 5B). Moreover, the fluorescence-voltage relationship (FV) is obtained by plotting the steady-state fluorescence intensity against voltage (Figure 5C). The FV reflects the equilibrium between occupancies of monitored states which in the case of ion channels could follow voltage sensor movement, or opening of the central pore. By using fUAAs it is now possible to obtain the FV from the inside of the channel. Figure 5C shows that the upper part of S4 (TMR) has the same voltage dependency as the lower part of S4 (Anap).
The signal-to-noise ratio is mainly dependent on the relative fluorescence change (dF/F) and the total fluorescence. The dF/F defines the signal size whereas the noise is determined by the total fluorescence because of the quantum nature of light (Poisson noise). The dF/F will depend on the state of quenching in the different states of the protein, and the occupancy of the states (e.g., the open probability). The total fluorescence comprises the fluorescence from specific labeling and from unspecific background labeling. The total specific fluorescence is defined by the number of expressed proteins and the quantum yield of the fluorophore. The large surface of the Xenopus oocytes is advantageous because it increases the number of contributing proteins.
Anap's quantum yield, i.e. the number of photons emitted per excitation cycle or the "brightness" of the fluorophore, is lower, which leads to lower total fluorescence intensity and thereby lower signal-to-noise ratio. At the same time, Anap labeling leads to low background fluorescence such that the signal-to-background ratio (dF/F) is higher for Anap (Figures 5A, 5B).
An important control experiment is to check expression in absence of Anap, in order to assess the effect of leak expression. It has previously been shown that isolated voltage sensor domains (iVSDs, 1-382) which lack the S4-S5 linker and the pore are functionally expressed19. Therefore, in the absence of Anap, L382stop-W434F channels express as iVSDs as shown in Figure 6. Expression of full-length channels with Anap has little or no iVSD currents, while truncated channels exhibit strong iVSD currents in the absence of Anap. The presence of such C-terminal truncated proteins depends on the position of the inserted stop codon and should always be taken into consideration when working with unnatural amino acids. Control experiments without Anap allow verification whether a heterologous population is present in experiments with Anap.
Figure 4: Simultaneous Gating Currents and Fluorescence Changes from an Cytosolic Protein Surface Obtained with VCF. Gating currents obtained with a P/4 subtraction protocol and simultaneous recording of fluorescence upon depolarization. Anap is incorporated into Shaker at position L382, giving rise to voltage-dependent fluorescence intensities from the intracellular end of the S4 helix. The maximum fluorescence intensity is denoted "F" and the fluorescence change is denoted "dF". The star at the end of the fluorescence change marks the closing of the electrical shutter (the opening of the shutter before the pulse is not shown). Please click here to view a larger version of this figure.
Figure 5: Two-color VCF with Anap and TMR. (A) Anap and (B) TMR fluorescence changes obtained from a Shaker A359C-L382Anap-W434F expressing oocyte labeled with TMR-maleimide. (C) Fluorescence changes from A and B are plotted against the membrane potential (FV). Data shows mean ±SD with n = 5 oocytes. Please click here to view a larger version of this figure.
Figure 6: C-terminal Truncated Shaker Channels are Expressed in Absence of Anap. Isolated voltage sensor domains (iVSD, 1-382) are expressed with zH4IR-L382stop-W434F in the absence of Anap, resulting in iVSD currents at hyperpolarized potentials19. In the presence of Anap however, full length Shaker expression competes with iVSD expression, and as a result, the amount of iVSD is less. Please click here to view a larger version of this figure.
The in vivo aminoacylation of tRNAs which are continuously being transcribed together with the tRNA-synthetase, makes it possible to obtain high expression levels for fluorescence measurements. For efficient fUAA incorporation, it is critical that pAnap is correctly injected into the nucleus. Due to the uncertainty of the exact location of the nucleus, 10-40% of the DNA injections are expected to fail, resulting in non-expressing (or leak-expressing) oocytes. Therefore, it is important to check expression in absence of Anap and pAnap to identify current phenotypes and magnitudes in case of leak channels. That way, it is possible to distinguish oocytes with proper DNA-injections (high expression, Anap dF, full length current phenotype) from those with failed DNA injections (no or low expression, no Anap dF, truncated current phenotype if any). In case of sudden lack of expression with Anap, prepare a new Anap aliquot as Anap is not stable for longer periods in aqueous solution (up to 1 year).
The use of fUAAs with VCF is limited by the choice of fluorophores since each new fUAA requires an orthogonal tRNA/tRNA synthetase pair to be engineered. So far only Anap exists as a functional fUAA to report on protein dynamics in oocytes. High expression levels are required in order to detect Anap fluorescence changes and we estimate a minimal protein density of 2 – 4000 µm–2 depending on the sensitivity of the site. For example, Anap fluorescence changes can be seen at just 0.2 mS of ionic currents for V234Anap (2.4 x 109 channels per clamped area in cut-open voltage clamp), while 5 nC of gating currents (1 x 106 channels channels per clamped area in cut-open voltage clamp) are required for A391Anap-W434F. As Shaker is a tetramer it has four Anap molecules per channel. Consequently, dimers or monomeric proteins likely require higher expression levels. Furthermore, bleaching kinetics of Anap, which depend on the chemical environment as well as light intensity, should be verified in order to ensure that sufficient intensity remains for the entire protocol. We found that kinetics in the 1 second time range can still be recorded with a typical voltage step protocol. The bleaching can be minimized by reducing light intensity (slow processes can be filtered more) or adding triplet-state quenchers such as Trolox (known from single molecule fluorescence studies). If LED excitation is used, one can also setup a protocol to "sample" the fluorescence intensity every 100 ms for 5-10 ms, effectively reducing the excitation time 10-20X.
Voltage-clamp fluorometry has been a powerful technique to study structure function relations of membrane proteins, and it has been combined with a variety of fluorescence techniques including labeled ligands11, Lanthanide-based and Förster Resonance Energy Transfer (LRET/FRET)20,21 and transition metal FRET22. However, limitations on the labeling techniques hindered the widespread application. Genetically-encoded fluorescent proteins, while invaluable in many other applications, are too large to offer a general alternative. They have been successfully employed in the C-terminus23 and the ligand binding domain of BKCa channels24, but cannot be introduced into "hotspots" of the protein. The introduction of fluorescent unnatural amino acids into the field opened the technique in two ways2: (i) many more proteins can be studied since no longer endogenous cysteines have to be removed; (ii) cytosolic and buried sites can be labeled allowing a wider range of regulatory mechanisms to be investigated.
Future engineering of orthogonal tRNA/synthetase pairs for new fUAAs would increase the applicability of VCF even further. Also, multiple incorporations of different fUAAs could be made possible using a four-base or five-base codon strategy25,26. Due to the high efficiency by which Xenopus oocytes are capable of incorporating UAAs and the widespread application of VCF, we expect fUAA-VCF to become a key-technique in the field of protein structure and function.
The authors have nothing to disclose.
pAnap was a kind gift from Dr. Peter Schultz (Scripps Research Institute). This work was funded by the Canadian Institutes for Health Research Grants MOP-102689 and MOP-136894 (to R.B.) and Canadian Foundation for Innovation Grant 950-225005.
Solutions | |||
Barth's solution | |||
NaCl | Sigma-Aldrich | S7653 | 90mM |
KCl | Fisher Scientific | BP366-500 | 3mM |
MgSO4 | Sigma-Aldrich | M-9397 | 0.82mM |
CaCl2 | Sigma-Aldrich | C-7902 | 0.41mM |
Ca(NO3)2 | Sigma-Aldrich | C-1396 | 0.33mM |
HEPES | Sigma-Aldrich | H4034 | 5mM |
NaOH hydrate | BDH | BDH7225-4 | pH 7.6 |
Penicilin | Invitrogen | 15140122 | 100U/mL |
Streptomycin | Invitrogen | 15140122 | 100µg/mL |
Kanamycin | Invitrogen | 15160054 | 10mg/100mL |
Horse serum | Invitrogen | 16050122 | 5% |
SOS Standard Oocyte Solution | |||
NaCl | Sigma-Aldrich | 746398 | 102 mM |
KCl | Sigma-Aldrich | 746436 | 3 mM |
MgCl2 | Sigma-Aldrich | M9272 | 1 mM |
HEPES | Sigma-Aldrich | H4034 | 5 mM |
External recording solution | |||
N-methyl-D-glucamine (NMDG) | Alfa Aesar | L14282 | 115mM |
HEPES | Sigma-Aldrich | H4034 | 10mM |
Calcium hydroxide | Sigma-Aldrich | 239232 | 2mM |
MES hydrate | Sigma-Aldrich | 258105 | pH 7.2 |
Internal recording solution | |||
N-methyl-D-glucamine (NMDG) | Alfa Aesar | L14282 | 115mM |
HEPES | Sigma-Aldrich | H4034 | 10mM |
Ethylenediamine Tetraacetic Acid (EDTA) | Fisher Scientific | E478-500 | 2mM |
MES hydrate | Sigma-Aldrich | 258105 | pH 7.2 |
Labeling solution | |||
KOH | Fisher Scientific | P250-1 | 115mM |
HEPES | Sigma-Aldrich | H4034 | 10mM |
Calcium hydroxide | Sigma-Aldrich | 239232 | 2mM |
MES hydrate | Sigma-Aldrich | 258105 | pH 7.2 |
TMR stock solution | |||
Tetramethylrhodamine-5-maleimide (TMR) | Molcular Probes by Life Technologies | T6027 | 5mM in DMSO |
Anap stock solution | |||
Anap | ABZENA (TCRS) | Custom synthesis TCRS-170 | 1mM in nuclease-free water and 1% NaOH 1N |
Name | Company | Catalog Number | Comments |
Material/Equipment | |||
pAnap | Addgene | 48696 | |
High Performance Oocyte Clamp | Dagan Corporation | CA-1B | |
Gpatch Acquisition software | Department of Anesthesiology, University of California, Los Angeles | ||
Analysis software | Department of Anesthesiology, University of California, Los Angeles | ||
Recording Chamber | Custom machined | ||
Photo diode detection system | Dagan Corporation | PhotoMax-200/PIN | |
Electrical shutter driver | UNIBLITZ | VCM-D1 |