Probing ion channel macromolecular interactions using fluorescence resonance energy transfer

Abstract

Ion channels are macromolecular complexes whose functions are exquisitely tuned by interacting proteins. Fluorescence resonance energy transfer (FRET) is a powerful methodology that is adept at quantifying ion channel protein-protein interactions in living cells. For FRET experiments, the interacting partners are tagged with appropriate donor and acceptor fluorescent proteins. If the fluorescently-labeled molecules are in close proximity, then photoexcitation of the donor results in non-radiative energy transfer to the acceptor, and subsequent fluorescence emission of the acceptor. The stoichiometry of ion channel interactions and their relative binding affinities can be deduced by quantifying both the FRET efficiency and the total number of donors and acceptors in a given cell. In this chapter, we discuss general considerations for FRET analysis of biological interactions, various strategies for estimating FRET efficiencies, and detailed protocols for construction of binding curves and determination of stoichiometry. We focus on implementation of FRET assays using a flow cytometer given its amenability for high-throughput data acquisition, enhanced accessibility, and robust analysis. This versatile methodology permits mechanistic dissection of dynamic changes in ion channel interactions.

Keywords: FRET 2-hybrid assay; Flow cytometry; Fluorescence resonance energy transfer; Live-cell binding; Stoichiometry.

Figure 1.

Conceptual framework of FRET 2-hybrid assay for probing the stoichiometry and binding of macromolecular complexes in live cells. (A) Changes in donor and acceptor emission spectra due to FRET. The donor emission is quenched as a result of FRET while the acceptor emission is enhanced. (B) Schematic shows 1:1 interaction of binding partners labeled with donor and acceptor fluorophores. Stochastic expression of the FRET pairs results in variable binding. When the donor and acceptor fluorophores are bound, they undergo FRET. FRET efficiencies can be quantified as either donor-centric E_D (fractional quenching) or acceptor-centric E_A (sensitized emission) methods. Correlating E_A versus the free concentration of the acceptor (A_free) or E_D versus the free concentration of the donor (D_free) yields FRET-binding curves. With 1:1 binding, the saturating maximal FRET efficiency of both curves should equal while the concentration, and relative binding affinity can be measured as D_free or A_free concentration where FRET efficiency is half maximal. (C) For binding interactions with stoichiometry of n_D:n_A, saturating E_A and E_D values are different. The ratio E_A,max / E_D,max yields the stoichiometry n_D/n_A. (D) Schematic shows overall workflow for FRET 2-hybrid experiments. Cells are transfected with FP-tagged proteins and analyzed using a flow cytometer. FRET efficiency and relative binding affinities are obtained following filtering for single cells, background subtraction, and spectral unmixing.

Figure 2.

Filtering single cells and subtracting spectral crosstalk. (A-C) Three-step sequential gating of single cells by forward-scatter (FSC) and side-scatter (SSC) signals. Briefly, SSC-H is plotted again FSC-H and a region of interest R1 is selected (panel A). Second, FSC-H and FSC-A signals from cells within R1 is used to select region of interest R2 (panel B). Finally, SSC-H and SSC-A signals for cells within R2 are utilized to select single cells (R3) that will be subject to further analysis (panel C). (D) Background signals from untransfected or sham-transfected cells are used to estimate autofluorescence in the donor, acceptor, and FRET channels. (E) Left, the spectral crosstalk of the acceptor into the FRET channel. The slope provides estimate for R_A1 parameter. Right, the spectral crosstalk of the donor into the FRET (left axis) and acceptor (right axis) channels. The slopes provide estimates for R_D1 and R_D2 parameters, respectively.

Figure 3.

Obtaining instrument specific calibration coefficients and measuring FRET efficiencies for various cerulean – venus dimers (A) Calculation of donor-centric (E_D) and acceptor-centric (E_D) FRET efficiencies require calculation of two instrument specific parameter g_A / g_D and f_A / f_D corresponding to the ratio of absorption and emissions of the donor and acceptor via FRET cube. Three cube FRET measurements are obtained from cells expressing cerulean-venus dimers and spectral unmixing is used to estimate D_direct, A_direct, and A_FRET. The ratio A_FRET / D_direct is plotted against A_direct/D_direct. The intercept yields f_A / f_D while the slope corresponds to g_D/g_A. (B) Acceptor-centric FRET efficiencies (E_A) obtained from cells expressing Cer-5AA-Ven (light blue), Cer-32AA-Ven (orange), Cer-40AA-Ven (yellow), Cer-50AA-Ven (green), and Cer-traf-Ven (deep blue). (C) Donor-centric FRET efficiencies (E_D) for the same dimers. Format as in panel B. (D) Histogram of single-cell estimates for N_D/N_A for various cerulean-venus dimers. N_D / N_A ~ 1 for all dimers, as expected.

Figure 4.

Application of FRET 2-hybrid analysis to deduce stoichiometry and binding. (A) Cryo-electron microscopy of Myosin Va shows the interaction of 6 CaM molecules with the neck domain that contains 6 tandem IQ domains (PDB code, 2DFS) (J. Liu, Taylor, Krementsova, Trybus, & Taylor, 2006). (B) FRET 2-hybrid analysis of a single IQ domain shows identical saturating values for acceptor-centric (E_A,max) and donor-centric (E_D,max) FRET efficiencies suggesting 1:1 binding. (C) Analysis of the entire neck region containing IQ_1–6 shows that saturating value for acceptor-centric (E_A,max) is ~ 6-fold higher than the donor-centric (E_D,max) FRET efficiencies. This suggest that there are 6 donor molecules (i.e. CaM) per acceptor in the complex. (D) Correlation of estimated stoichiometries for various truncations of myosin Va neck region using FRET with the expected stoichiometry given the number of IQ domains in each truncation. (E) Flow-cytometric FRET 2-hybrid assay confirms robust interaction of Cer-tagged CaM with ven-tagged carboxy-tail segment of Ca_V1.2. As E_A,max = E_D,max, the stoichiometry is 1:1. (F) Flow-cytometric FRET 2-hybrid assay shows PKA-dependent change in binding of the Cerulean tagged Ca_Vβ_2B subunit with Venus tagged Rad. These findings illustrate the ability of FRET to resolve changes in binding due to signaling events. Panels B-D were reproduced with permission from Ben-Johny et al, 2016.