1. Neuroscience

Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands

  1. Aaron L Nichols
  2. Zack Blumenfeld
  3. Chengcheng Fan
  4. Laura Luebbert
  5. Annet EM Blom
  6. Bruce N Cohen
  7. Jonathan S Marvin
  8. Philip M Borden
  9. Charlene H Kim
  10. Anand K Muthusamy
  11. Amol V Shivange
  12. Hailey J Knox
  13. Hugo Rego Campello
  14. Jonathan H Wang
  15. Dennis A Dougherty
  16. Loren L Looger
  17. Timothy Gallagher
  18. Douglas C Rees
  19. Henry A Lester  Is a corresponding author
  1. Division of Biology and Biological Engineering, California Institute of Technology, United States
  2. Keck School of Medicine, University of Southern California, United States
  3. Division of Chemistry and Chemical Engineering, California Institute of Technology, United States
  4. Institute of Biology, Leiden University, Netherlands
  5. Janelia Research Campus, Howard Hughes Medical Institute, United States
  6. School of Chemistry, University of Bristol, United Kingdom
  7. Howard Hughes Medical Institute, California Institute of Technology, United States
https://doi.org/10.7554/eLife.74648
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Cite this article
  1. Aaron L Nichols
  2. Zack Blumenfeld
  3. Chengcheng Fan
  4. Laura Luebbert
  5. Annet EM Blom
  6. Bruce N Cohen
  7. Jonathan S Marvin
  8. Philip M Borden
  9. Charlene H Kim
  10. Anand K Muthusamy
  11. Amol V Shivange
  12. Hailey J Knox
  13. Hugo Rego Campello
  14. Jonathan H Wang
  15. Dennis A Dougherty
  16. Loren L Looger
  17. Timothy Gallagher
  18. Douglas C Rees
  19. Henry A Lester
(2022)
Fluorescence activation mechanism and imaging of drug permeation with new sensors for smoking-cessation ligands
eLife 11:e74648.
https://doi.org/10.7554/eLife.74648
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11 figures, 6 videos, 4 tables and 3 additional files

Figures

Figure 1 with 2 supplements
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Apo and ligand-bound structures of iNicSnFR3adt (dt indicates that His6 and Myc tags have been removed to aid crystallization).

To form an intensity-based drug-sensing fluorescent reporter (iDrugSnFR), a circularly permuted GFP molecule, flanked by two 4-residue linking sequences, is inserted into a PBP at a position (77–78, …

Figure 1—figure supplement 1
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Conformational change of apo (PDB 7S7V) to the liganded, closed form (PDB 7S7T) of iNicSnFR3adt.

The bottom lobe of the PBP is superimposed in the two conformations. With respect to the bottom lobe, the ‘Venus flytrap’ conformational change tilts the top lobe of the PBP but does not change its …

Figure 1—figure supplement 2
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Left: electrostatic surface potential densities for protonated forms of the nicotinic agonists in this study, calculated by SPARTAN at HF/6-31G** theory level.

The display ranges from –10 to 715 kJ/mol. The molecules are shown on the same distance scale. Right: bond-line skeletal structures for the deprotonated forms.

Figure 2
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Nicotinic agonist intensity-based drug-sensing fluorescent reporter (iDrugSnFR) development.

Dose–response relations on intermediate constructs using E. coli lysate were performed with respective drug partners to identify site-saturation mutagenesis (SSM) winners. (A–D) The progenitor …

Figure 3 with 1 supplement
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Dose–response relations of intensity-based drug-sensing fluorescent reporter (iDrugSnFR) protein versus a nicotinic agonist panel.

(A–D) Relevant EC50 values for each iDrugSnFR are listed in Table 2. Dashed lines indicate dose–response relations that did not approach saturation for the concentration ranges tested; therefore, EC5…

Figure 3—figure supplement 1
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Dose–response relations of nicotinic agonist intensity-based drug-sensing fluorescent reporters (iDrugSnFRs) against select endogenous molecules.

(A) iDianiSnFR shows no fluorescent response to any of the selected endogenous molecules. (B) iCytSnFR, (C) iCyt_F_SnFR, and (D) iCyt_BrEt_SnFR show no response to any of the selected endogenous …

Figure 4
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Isothermal titration calorimetry traces, fits, and thermodynamic data.

Top row: exemplar heat traces of iCytSnFR, iCyt_F_SnFR, iCyt_BrEt_SnFR, and iDianiSnFR paired with their drug partners obtained by isothermal calorimetry. The heats for iCytSnFR, iCyt_F_SnFR, and …

Figure 5
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Stopped-flow fluorescence kinetic data for (A) iDianiSnFR, (B) iCytSnFR, (C) iCyt_F_SnFR, and (D) iCyt_BrEt_SnFR over 1 s and 100 s.

Fluorescence was activated by mixing with the agonists as noted. Stopped-flow data shows a departure from first-order kinetics for this set of intensity-based drug-sensing fluorescent reporter …

Figure 6
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Decay of the iCytSnFR_PM responses after removal of ACh, cytisine, or varenicline.

(A) The red, blue, and black traces are mean ΔF/F0 values for the ACh (200 µM), cytisine (15 µM), and varenicline (2 µM) responses as a function of time (n = 4–10 areas per ligand). The ΔF/F0 was …

Figure 7 with 3 supplements
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Nicotinic agonist intensity-based drug-sensing fluorescent reporter (iDrugSnFR) dose–response relations in HeLa cells.

(A–D) Each iDrugSnFR detects its drug partner at the plasma membrane (PM) and endoplasmic reticulum (ER) of HeLa cells at the concentrations sampled. BC, buffer control. SEM of data are indicated by …

Figure 7—figure supplement 1
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Traces of fluorescence responses during time-resolved low-concentration dose–response relations for nicotinic agonists in HeLa cells.

BC, buffer control. SEM of data are indicated by semi-transparent shrouds around traces where trace width is exceeded. Cyt (cytisine) in cells expressing iCytSnFR_ER (A) or iCytSnFR_PM (B); 10FC …

Figure 7—figure supplement 2
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Dose–response relations for iCytSnFR and iCyt_F_SnFR against nicotine in HeLa cells.

BC, buffer control. SEM of data are indicated by semi-transparent shrouds around traces where trace width is exceeded. (A) iCytSnFR and (B) iCyt_F_SnFR detect nicotine at both the plasma membrane …

Figure 7—figure supplement 3
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Spinning disk laser scanning confocal inverted microscope images of nicotinic agonist intensity-based drug-sensing fluorescent reporters (iDrugSnFRs) in HeLa cells.

Endoplasmic reticulum (ER)-targeted constructs of iDianiSnFR, iCytSnFR, iCyt_F_SnFR, and iCyt_BrEt_SnFR are shown before (A1–D1) and during (A2–D2) exposure to each drug partner. ER-targeted …

Figure 8 with 1 supplement
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Nicotinic agonist intensity-based drug-sensing fluorescent reporter (iDrugSnFR) dose–response experiments in mouse primary hippocampal neurons transduced with AAV9-hSyn iDrugSnFR.

Cultured primary mouse hippocampal neurons were transduced with endoplasmic reticulum (ER)- or plasma membrane (PM)-targeted constructs. BC, buffer control. SEM of data are indicated by …

Figure 8—figure supplement 1
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Spinning disk laser scanning confocal inverted microscope images of nicotinic agonist intensity-based drug-sensing fluorescent reporters (iDrugSnFRs) in primary mouse hippocampal neurons.

Endoplasmic reticulum (ER)-targeted constructs of iDianiSnFR, iCytSnFR, iCyt_F_SnFR, and iCyt_BrEt_SnFR are shown before (A1–D1) and during (A2–D2) exposure to each drug partner. ER-targeted …

Appendix 2—figure 1
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Rising phase of the iCytSnFR_PM response to cytisine in HEK293T cells.

(A) Example of the biphasic rising phase of a 1 µM cytisine response in an individual area (black trace, mean of four cells). Cytisine was applied for 30 s. The fast and slow time constants of the …

Appendix 2—figure 2
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Concentration dependence of the fast (kfon) and slow rising rate constants (kson) of the cytisine response.

(A) The [Cytisine]-kfon relation was approximately linear between 1 and 15 µM cytisine. Symbols (filled squares) are the mean kfon for the individual cytisine concentrations tested (n = 7–10 areas …

Appendix 2—figure 3
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A three-state kinetic scheme for iCytSnFR.

The diagram contains cartoons of the PBP moiety (blue and red), the linkers (black lines), the Glu78 ‘candle snuffer’ attached to Linker 1 (black), and the cpGFP moiety (gray, dark green, or green). …

Videos

Video 1
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Video morph of PDB 7S7V to 7S7T.

PBP at the left; cpGFP at the right;key side chains in the linkers are shown as spheres. The ligand, varenicline, is shown as sticks.

Video 2
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Video morph of PDB 7S7V to 7S7T.

PBP at the left; cpGFP at the right; key side chains in the linkers are shown as spheres. The ligand, varenicline, is shown as sticks.

Video 3
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iDianiSnFR_ER dose-response relations in HeLa cells.

The dianicline concentrations are shown. The scale bar is shown. The video is 25-fold faster than real time.

Video 4
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iDianiSnFR_PM dose-response relations in HeLa cells.

The dianicline concentrations are shown. The scale bar is shown.The video is 25-fold faster than real time.

Video 5
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iCytSnFR_ER dose-response relations in HeLa cells.

The cytisine concentrations are shown. The scale bar is shown. The video is 25-fold faster than real time.

Video 6
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iCytSnFR_PM dose-response relations in HeLa cells.

The cytisine concentrations are shown. The scale bar is shown. The video is 25-fold faster than real time.

Tables

Table 1
Nicotinic agonist intensity-based drug-sensing fluorescent reporter (iDrugSnFR) naming, dose–response relations, and residues mutated.

Measurements in E. coli lysates (L) or with purified protein (P). ND, not determined. Data for iAChSnFR from Borden et al., 2019; data for iNicSnFR3b from Shivange et al., 2019.


Informal name
Drug of interest		ΔFmax/F0EC50 (µM)S-slope
LPLPLP11434468324360391395
iNicSnFR3bNicotineND10ND19ND0.5EENHSTFG
iDianiSnFRDianicline7.4 ± 0.14.7 ± 0.26.7 ± 0.315 ± 11.10.3DR-SNG-N
iAChSnFRAChND12ND1.3ND9.2IVNHATFG
iCytSnFRCytisine5.0 ± 0.27.3 ± 0.49.4 ± 0.811 ± 10.50.7-Y----W-
iCyt_F_SnFR10-Fluorocytisine7.9 ± 0.12.3 ± 0.11.4 ± 0.041.6 ± 0.35.61.4-NG---W-
iCyt_BrEt_SnFR9-Bromo-10-ethylcytisine4.0 ± 0.033.6 ± 0.045.7 ± 0.14.2 ± 0.20.70.9-QG---W-
Table 2
Intensity-based drug-sensing fluorescent reporter (iDrugSnFR) dose–response relations versus a selected panel of nicotinic agonists.

ND, not determined. *, ** EC50 and ∆Fmax/F0 could not be determined from the data (Figure 3). Therefore, the upper limit to the S-slope is estimated from the data at the foot of the dose–response …

Drug nameiDianiSnFRiCytSnFRiCyt_F_SnFRiCyt_BrEt_SnFR
ΔFmax/F0EC50 (µM)S-slopeΔFmax/F0EC50 (µM)S-slopeΔFmax/F0EC50 (µM)S-slopeΔFmax/F0EC50 (µM)S-slope
Choline2.0 ± 0.184 ± 20< 0.15.8 ± 0.2240 ± 30< 0.12.6 ± 0.118 ± 10.12.6 ± 0.112 ± 10.2
Acetylcholine7.4 ± 1.0660 ± 80< 0.12.9 ± 0.135 ± 3< 0.14.4 ± 0.3222 ± 50< 0.12.5 ± 0.273 ± 6<0.1
Cytisine--<0.1*7.3 ± 0.411 ± 10.74.4 ± 0.12.6 ± 0.31.74.7 ± 0.13.5 ± 0.21.3
Dianicline4.7 ± 0.215 ± 10.36.5 ± 0.434 ± 40.22.3 ± 0.343 ± 6< 0.14–6>100<0.1**
Nicotine2.2 ± 0.1440 ± 100< 0.16.4 ± 0.214 ± 20.54.7 ± 0.13.8 ± 0.21.24.8 ± 0.15.5 ± 0.20.9
Varenicline2.4 ± 2.01200 ± 500< 0.16.5 ± 0.10.06 ± 0.011107.1 ± 0.20.09 ± 0.02795.3 ± 0.10.06 ± 0.0188
10-FluorocytisineNDNDNDNDNDND2.3 ± 0.11.6 ± 0.31.43.0 ± 0.14.7 ± 0.30.6
9-Bromo-10-ethylcytisineNDNDNDNDNDND3.1 ± 0.131 ± 20.13.6 ± < 0.14.2 ± 0.20.9
Table 3
Affinity, occupancy number, and thermodynamic data calculated from isothermal titration calorimetry.

Data are the mean ± SEM, three runs.

BiosensorKD (μM)nΔH(kcal/mol)-TΔS(kcal/mol)ΔG(kcal/mol)
iCytSnFR13.7 ± 1.10.84 ± 0.05–2.1 ± 0.1–4.6 ± 0.2–6.6 ± 0.1
iCyt_F_SnFR1.8 ± 0.50.83 ± 0.02–5.5 ± 0.1–2.4 ± 0.2–7.9 ± 0.1
iCyt_BrEt_SnFR5.4 ± 0.80.69 ± 0.09–1.12 ± 0.036.1 ± 0.1–7.2 ± 0.1
iDianiSnFR7.6 ± 1.40.92 ± 0.023.2 ± 0.510.1 ± 0.4–7.0 ± 0.2
Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)BL21(DE3)Agilent Technologies, Santa Clara, CA200131Chemically competent
Cell line (Homo sapiens)HeLaATCCCCL-2;RRID:CVCL_0030
Cell line (H. sapiens)HEK293TATCCCRL-3216;RRID:CVCL_0063
Biological sample (Mus musculus)Primary hippocampal neuronsCaltech animal facilitiesRRID:IMSR_JAX:000664Freshly isolated from Mus musculus
Recombinant DNA reagentiAChSnFRLoren LoogerAddgene: 137955Obtainable through Addgene
Recombinant DNA reagentiDianiSnFRThis paperAddgene: 177741
Obtainable through Addgene
Recombinant DNA reagentiCytSnFRThis paperAddgene: 177738Obtainable through Addgene
Recombinant DNA reagentiCyt_F_SnFRThis paperAddgene: 177739Obtainable through Addgene
Recombinant DNA reagentiCyt_BrEt_SnFRThis paperAddgene: 177740Obtainable through Addgene
Recombinant DNA reagentpCMV(MinDis)-iDianiSnFR_PMThis paperAddgene: 177751Obtainable through Addgene
Recombinant DNA reagentpCMV(MinDis)-iDianiSnFR_ERThis paperAddgene: 177750Obtainable through Addgene
Recombinant DNA reagentpCMV(MinDis)-iCytSnFR_PMThis paperAddgene: 177743Obtainable through Addgene
Recombinant DNA reagentpCMV(MinDis)-iCytSnFR_ERThis paperAddgene: 177742Obtainable through Addgene
Recombinant DNA reagentpCMV(MinDis)-iCyt_F_SnFR_PMThis paperAddgene: 177745Obtainable through Addgene
Recombinant DNA reagentpCMV(MinDis)-iCyt_F_SnFR_ERThis paperAddgene: 177744Obtainable through Addgene
Recombinant DNA reagentpCMV(MinDis)-iCyt_BrEt_SnFR_PMThis paperAddgene: 177747Obtainable through Addgene
Recombinant DNA reagentpCMV(MinDis)-iCyt_BrEt_SnFR_ERThis paperAddgene: 177746Obtainable through Addgene
Recombinant DNA reagentpAAV9-hSyn-iDianiSnFR_PMThis paperAddgene: 177759Obtainable through Addgene
Recombinant DNA reagentpAAV9-hSyn-iDianiSnFR_ERThis paperAddgene: 177758Obtainable through Addgene
Recombinant DNA reagentpAAV9-hSyn-iCytSnFR_PMThis paperAddgene: 177753Obtainable through Addgene
Recombinant DNA reagentpAAV9-hSyn-iCytSnFR_ERThis paperAddgene: 177752Obtainable through Addgene
Recombinant DNA reagentpAAV9-hSyn-iCyt_F_SnFR_PMThis paperAddgene: 177755Obtainable through Addgene
Recombinant DNA reagentpAAV9-hSyn-iCyt_F_SnFR_ERThis paperAddgene: 177754Obtainable through Addgene
Recombinant DNA reagentpAAV9-hSyn-iCyt_BrEt_SnFR_PMThis paperAddgene: 177757Obtainable through Addgene
Recombinant DNA reagentpAAV9-hSyn-iCyt_BrEt_SnFR_ERThis paperAddgene: 177756Obtainable through Addgene
Commercial assay or kitPhusion High-Fidelity PCR KitNew England BiolabsE0553L
Commercial assay or kitQ5 Site-Directed Mutagenesis KitNew England BiolabsE0554S
Commercial assay or kitQIAprep Spin Miniprep KitQIAGEN SCR_00853927104
Commercial assay or kitEndoFree Plasmid Maxi KitQIAGEN SCR_00853912362
Commercial assay or kitQIAquick PCR Purification KitQIAGEN SCR_00853928104
Commercial assay or kitQIAquick Gel Extraction KitQIAGEN SCR_00853928704
Commercial assay or kitAAVpro Purification KitTakara Bio Inc.6666
Commercial assay or kitPACT premierMolecular DimensionsMD1-29
Chemical compound, drug10-FluorocytisineTim GallagherRego Campello et al., 2018
Chemical compound, drug9-Bromo-10-ethylcytisineTim GallagherRego Campello et al., 2018
Chemical compound, drugLipofectamine 2000 Transfection ReagentThermo Fisher Scientific11668027
Chemical compound, drugLipofectamine 3000 Transfection ReagentThermo Fisher ScientificL3000015
Software, algorithmSpartan’20Wavefunction, Inc.RRID:SCR_014901
Software, algorithmNanoAnalyzeTA Instrumentshttps://www.tainstruments.com/sw/nano_analyze.html
Software, algorithmOriginPro 2018OriginLabRRID:SCR_014212
Software, algorithmKaleidaGraphSynergyRRID:SCR_014980
Software, algorithmImageJNIHRRID:SCR_003070
Software, algorithmXDS Program PackageMPI for Medical Research, HeidelbergRRID:SCR_015652
Software, algorithmPhenixPhenixRRID:SCR_014224, SCR_016736Adams et al., 2010
Software, algorithmCootMRC Laboratory of Molecular BiologyRRID:SCR_014222Emsley et al., 2010

Additional files

Supplementary file 1

Data collection and refinement statistics of iNicSnFR 3a.

https://cdn.elifesciences.org/articles/74648/elife-74648-supp1-v2.xlsx
Supplementary file 2

Stopped-flow model determined rate constants.

https://cdn.elifesciences.org/articles/74648/elife-74648-supp2-v2.xlsx
Transparent reporting form
https://cdn.elifesciences.org/articles/74648/elife-74648-transrepform1-v2.docx

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