Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Summary

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.

Abstract

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.

Introduction

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 ....

Protocol

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

1. Choose a site of interest in the protein where conformational changes are expected to occur. Select an amino acid in this region to be substituted for the fUAA.
   NOTE: The choice of position is based on the structural rearrangements that are expected. If a high resolution structure exists and a hypothesis of the expected movements, the anap should be placed such that the chemical environment will alter;....

Results

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.......

Discussion

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 abs.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Solutions
Barth's solution
NaCl	Sigma-Aldrich	S7653	90 mM
KCl	Fisher Scientific	BP366-500	3 mM
MgSO4	Sigma-Aldrich	M-9397	0.82 mM
CaCl2	Sigma-Aldrich	C-7902	0.41 mM
Ca(NO3)2	Sigma-Aldrich	C-1396	0.33 mM
HEPES	Sigma-Aldrich	H4034	5 mM
NaOH hydrate	BDH	BDH7225-4	pH 7.6
Penicilin	Invitrogen	15140122	100 U/mL
Streptomycin	Invitrogen	15140122	100 µg/mL
Kanamycin	Invitrogen	15160054	10 mg/100mL
Horse Serum (HS)	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	115 mM
HEPES	Sigma-Aldrich	H4034	10 mM
Calcium hydroxide	Sigma-Aldrich	239232	2 mM
MES hydrate	Sigma-Aldrich	258105	pH 7.2
Internal recording solution
N-methyl-D-glucamine (NMDG)	Alfa Aesar	L14282	115 mM
HEPES	Sigma-Aldrich	H4034	10 mM
Ethylenediamine Tetraacetic Acid (EDTA)	Fisher Scientific	E478-500	2 mM
MES hydrate	Sigma-Aldrich	258105	pH 7.2
Labeling solution
KOH	Fisher Scientific	P250-1	115 mM
HEPES	Sigma-Aldrich	H4034	10 mM
Calcium hydroxide	Sigma-Aldrich	239232	2 mM
MES hydrate	Sigma-Aldrich	258105	pH 7.2
TMR stock solution
Tetramethylrhodamine-5-maleimide (TMR)	Molcular Probes by Life Technologies	T6027	5 mM in DMSO
Anap stock solution
Anap	ABZENA (TCRS)	Custom synthesis TCRS-170	1 mM in nuclease-free water and 1% NaOH 1 N
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