Scaling the Functional Nanopore (FuN) Screen: Systematic Evaluation of Self-Assembling Membrane Peptides and Extension with a K⁺‑Responsive Fluorescent Protein Sensor

The functional analysis of protein nanopores is typically conducted in planar lipid bilayers or liposomes exploiting high-resolution but low-throughput electrical and optical read-outs. Yet, the reconstitution of protein nanopores in vitro still constitutes an empiric and low-throughput process. Addressing these limitations, nanopores can now be analyzed using the functional nanopore (FuN) screen exploiting genetically encoded fluorescent protein sensors that resolve distinct nanopore-dependent Ca2+ in- and efflux patterns across the inner membrane of Escherichia coli. With a primary proof-of-concept established for the S2168 holin, and thereof based recombinant nanopore assemblies, the question arises to what extent alternative nanopores can be analyzed with the FuN screen and to what extent alternative fluorescent protein sensors can be adapted. Focusing on self-assembling membrane peptides, three sets of 13 different nanopores are assessed for their capacity to form nanopores in the context of the FuN screen. Nanopores tested comprise both natural and computationally designed nanopores. Further, the FuN screen is extended to K+-specific fluorescent protein sensors and now provides a capacity to assess the specificity of a nanopore or ion channel. Finally, a comparison to high-resolution biophysical and electrophysiological studies in planar lipid bilayers provides an experimental benchmark for future studies

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Channelrhodopsin 2 (ChR2) and its variants are the most frequent tools for remote manipulation of electrical properties in cells via light. Ongoing attempts try to enlarge their functional spectrum with respect to ion selectivity, light sensitivity and protein trafficking by mutations, protein engineering and environmental mining of ChR2 variants. A shortcoming in the required functional testing of large numbers of ChR2 variants is the lack of an easy screening system. Baker’s yeast, which was successfully employed for testing ion channels from eukaryotes has not yet been used for screening of ChR2s, because they neither produce the retinal chromophore nor its precursor carotenoids. We found that addition of retinal to the external medium was not sufficient for detecting robust ChR activity in yeast in simple growth assays. This obstacle was overcome by metabolic engineering of a yeast strain, which constitutively produces retinal. In proof of concept experiments we functionally express different ChR variants in these cells and monitor their blue light induced activity in simple growth assays. We find that light activation of ChR augments an influx of Na+ with a consequent inhibition of cell growth. In a K+ uptake deficient yeast strain, growth can be rescued in selective medium by the blue light induced K+ conductance of ChR. This yeast strain can now be used as chassis for screening of new functional ChR variants and mutant libraries in simple yeast growth assays under defined selective conditions.</div

Functional complementation of SHY4 cells by K⁺ channel.

(A) Fluorescence images of SHY4 cells transiently expressing GFP tagged K+ channel KcvPBCV1 (KcvPBCV1::GFP). Scale bar = 5 μm. (B) Serial dilutions of K+ uptake deficient SHY4 cells transformed with KcvPBCV1::GFP plasmid or the corresponding empty vector (ev). Cells spotted on SD-ura plates with either high (100 mM KCl, top) or without K+ supplementation and incubated for 72h. KcvPBCV1 rescues growth of SHY4 cells on SD-ura medium without additional K+ (bottom row). For experimental details see [1]. [1] Gebhardt M, Hoffgaard F, Hamacher K, Kast SM, Moroni A, Thiel G. Membrane anchoring and interaction between transmembrane domains is crucial for K+ channel function. J. Biol. Chem. 2011; 286:11299–11306. (PDF)</p

Engineering of N/K-ChR2.

(A) Schemes of ChR2-5x and N/K-ChR2. ChR2-5x is a ChR2 variant with 5 point mutations. N/K-ChR2 comprises ChR2-5x scaffold with CR10 (= first 10 amino acids from proton pump rhodopsin CsR) and N17 (= first 17 amino acids from ChR2). The construct contains additional domains in N- and C-terminus namely LR domain (= N-terminal signal peptide Lucy-Rho), T domain (= the plasma membrane trafficking signal from Kir2.1) and E domain (= the endoplasmic reticulum (ER) export signal from Kir2.1). (B) Shift in reversal potential (ΔVr) recorded in Xenopus ooccytes expressing either ChR2-5x or ChR2-XXM upon changing extracellular solution from 1 mM to 120 mM NaCl (ΔVr Na+) or KCl (ΔVr K+). Values represent mean ± SD, n = 3–4. (C) Images of Xenopus oocytes expressing ChR2-5X and N/K-ChR2; scale bar = 1 mm. (D) Representative photocurrent trace from N/K-ChR2 expressing oocyte elicited by illumination with blue light (472 nm, blue bar); oocyte incubated in solution containing (in mM) 110 NaCl, 5 KCl, 2 BaCl2, 1 MgCl2, 5 HEPES/ pH 7.6). Holding potential at -70 mV. (E) Mean photocurrent amplitudes of ChR2-5x and N/K-ChR2 recorded as in C; n = 13–15. P value of statistical significance in B and E from unpaired t-test indicated as *** = P < 0.001.</p

Plasmids used in this study.

Channelrhodopsin 2 (ChR2) and its variants are the most frequent tools for remote manipulation of electrical properties in cells via light. Ongoing attempts try to enlarge their functional spectrum with respect to ion selectivity, light sensitivity and protein trafficking by mutations, protein engineering and environmental mining of ChR2 variants. A shortcoming in the required functional testing of large numbers of ChR2 variants is the lack of an easy screening system. Baker’s yeast, which was successfully employed for testing ion channels from eukaryotes has not yet been used for screening of ChR2s, because they neither produce the retinal chromophore nor its precursor carotenoids. We found that addition of retinal to the external medium was not sufficient for detecting robust ChR activity in yeast in simple growth assays. This obstacle was overcome by metabolic engineering of a yeast strain, which constitutively produces retinal. In proof of concept experiments we functionally express different ChR variants in these cells and monitor their blue light induced activity in simple growth assays. We find that light activation of ChR augments an influx of Na+ with a consequent inhibition of cell growth. In a K+ uptake deficient yeast strain, growth can be rescued in selective medium by the blue light induced K+ conductance of ChR. This yeast strain can now be used as chassis for screening of new functional ChR variants and mutant libraries in simple yeast growth assays under defined selective conditions.</div

Oligonucleotides used in this study.

Channelrhodopsin 2 (ChR2) and its variants are the most frequent tools for remote manipulation of electrical properties in cells via light. Ongoing attempts try to enlarge their functional spectrum with respect to ion selectivity, light sensitivity and protein trafficking by mutations, protein engineering and environmental mining of ChR2 variants. A shortcoming in the required functional testing of large numbers of ChR2 variants is the lack of an easy screening system. Baker’s yeast, which was successfully employed for testing ion channels from eukaryotes has not yet been used for screening of ChR2s, because they neither produce the retinal chromophore nor its precursor carotenoids. We found that addition of retinal to the external medium was not sufficient for detecting robust ChR activity in yeast in simple growth assays. This obstacle was overcome by metabolic engineering of a yeast strain, which constitutively produces retinal. In proof of concept experiments we functionally express different ChR variants in these cells and monitor their blue light induced activity in simple growth assays. We find that light activation of ChR augments an influx of Na+ with a consequent inhibition of cell growth. In a K+ uptake deficient yeast strain, growth can be rescued in selective medium by the blue light induced K+ conductance of ChR. This yeast strain can now be used as chassis for screening of new functional ChR variants and mutant libraries in simple yeast growth assays under defined selective conditions.</div

N/K-ChR2 rescues K⁺ mediated growth of yeast mutant only in high blue light.

(A) Growth of SHY4 cells transformed with either empty vector (ev, open symbols)) or N/K-ChR2 (closed symbols) in SDAP medium with 0.1 (black), 1 (blue), 10 (green) or 100 (red) mM KCl. Growth was monitored as in Fig 5B. When cells were illuminated by 500 nm light from the plate-reader, they grew over a period of 48h only in medium with ≥10 mM KCl but without appreciable difference between presence/absence of N/K-ChR2. Data are mean ± SD of 3 independent experiments. (B) Relative growth of SHY4 cells expressing N/K-ChR2 in dark (black bar) or illuminated with 40 μW/mm2 of light (465 nm: blue bar) in SD-ura medium ± 400 mM NaCl on a background of low (7 mM) or medium high (17 mM) KCl. Growth expressed as OD value (rel. OD600) of N/K-ChR2 expressing cell (grey bar) relative to the respective control with empty vector (open bars). Growth is substantially reduced by NaCl only in cells expressing N/K-ChR2 upon light exposure. (C) Growth is increased in blue light grown cells expressing N/K-ChR2 only in low K+ medium e.g. when K+ influx is the limiting growth factor, but not in medium high K+ (17 mM). (D) In low K+ (7 mM), growth of cells expressing N/K-ChR2 is increased in cells exposed to blue light (blue bar), but not in cells exposed to red light (650 nm, red bar) or kept in the dark (black bar). Data in (B) and (C) represent growth after 48 h, data in (D) represent growth after 24 h. All data are mean ± SD of ≥3 independent experiments. P value of statistical significance from t-test indicated as * = P<0.05, ** = P<0.01 and *** = P<0.001.</p

Expression and functional characterization of N/K-ChR2 in SHY4 cells.

(A) Fluorescence (left) and transmission light (center) images of SHY4 cells constitutively expressing N/K-ChR2-eYFP. Overlay (right) confirms localization of fluorescent tagged channel (in blue) with endo-membranes and the plasma membrane. The cell indicated by arrow exhibits fluorescence nearly exclusively in plasma membrane. Scale bar = 5 μm. (B) Growth of SHY4 cells transfected either with empty vector (open symbols) or channelrhodopsin-2 variant (N/K-ChR2, closed symbols) in SD-ura + 10 mM K+ medium with 0 (black) or 400 mM NaCl (blue). Growth measured in 30 sec intervals as change in OD500. While the presence of N/K-ChR2 has little impact on growth in absence of NaCl it reduces growth in the presence of salt stress. Inset: data in 400 mM NaCl replotted for cells ± N/K-ChR2. After ≥12 h cells without N/K-ChR2 grow (P(C) Maximum growth rate (μmax) from data in B for cells with empty vector (open bars) and with N/K-ChR2 (filled bars) in medium with 0 or 400 mM NaCl. Data are mean ± SD of 3 independent experiments. P values were calculated by student t-test.</p

Illumination bleaches retinal in medium.

(A) Agar plates containing 1 mM retinal directly after preparation (left, ctrl) and 24 h after keeping plate in the dark (center, 24 h dark) or in the light (right, 24h light, right). Illumination bleaches retinal-containing agar. (B) Yellow color of agar plate above background was obtained from deconvolution of images and plotted as relative value of plates after 24 h incubation in light or dark relative to pre-incubation (ctrl). Mean ± SD of independent replicates.</p

ChR2 is properly expressed in yeast strain BY4741 but shows no sign of function.

(A) Confocal images of BY4741 yeast strain in SGal-ura overnight cultures. Transmission images (left) and corresponding fluorescent images (right) of eYFP-tagged ChR2. Inset: Magnification and overlay of transmission and fluorescent images from cell indicated in right panel showing that fluorescence is visible in position of the plasma membrane. (B) Droplet test of BY4741 transformed with the pGREG576 plasmid bearing the ORF for the capsaicin receptor TRPV1 from Rattus norvegicus. In SD-ura, without induction of transcription, the presence of either the channel activator capsaicin, the permeant Na+ or both had very little effect on growth (left panel). With induction of transcription by galactose (right panel), channel activation by capsaicin alone had little effect on growth. However, the additional presence of 500 mM NaCl resulted in a strong growth inhibition. (C) Luminometric assay reveals capsaicin triggered calcium influx in BY4741 yeast cells expressing TRPV1, but not in control cells harbouring the empty pGREG576 plasmid. Arrow indicates injection of 10 μM capsaicin. Curves are the means of 6 recordings for the TRPV1 strain and 2 recordings for the empty vector control. (D). Droplet test of BY4741 transformants without (-) and with (+) 500 mM NaCl salt stress. Cells grown in absence (-) and presence (+) of 10 μM retinal for three days in dark and light. Cells harboring the empty pGREG506 plasmid (ev) and cells expressing either ChR2 (ChR2) or its D156C mutant (D156C) where spotted as serial dilutions from an OD600 = 1 on SGal-ura agar plates at 10 μL per spot.</p