Development of high-content fluorescence assays to rapidly monitor CFTR-mediated bicarbonate flow - how do CFTR mutations and modulator drugs affect it?

1) Generation and optimization of a fluorescence probe to monitor CFTR-mediated anion flow.

The aim is to create a tool to rapidly quantify CFTR-dependent Cl^- and HCO₃^- flow, in the simplified HEK293 expression system. The new assay will be based on a recently developed high-content assay [1, 2]. Cells will be transformed with a bicistronic plasmid encoding a soluble red pH-sensitive fluorescent protein [3] and an N-terminal fusion of CFTR to the I^--sensitive yellow fluorescent protein, YFP [4]. Cells expressing these proteins will be grown in 96-well plates, and images will be acquired with a confocal screening microscope equipped with automated fluid-addition robotics. CFTR will be pre-activated in HCO₃^- -free vs. Cl^- -free solutions. Once steady-state activation is reached, a second addition will increase extracellular I^-. By monitoring quenching of YFP over time we will compare Cl^-/I^- vs. HCO₃^-/I^- exchange. Fluorescence timelines (on both red and yellow channels) following I^- addition will be fit using a mathematical model describing transmembrane ionic fluxes and intracellular and extracellular pH and buffers. Finally, the average red fluorescence throughout the cell (following cytosolic alkalinisation) will be used as a unit, to quantify how much CFTR is in proximity of the membrane. The assay will allow simultaneous monitoring of multiple readouts (CFTR Cl^- and HCO₃^- conductance, cytosolic pH, CFTR membrane proximity), at the level of individual cells, while automated image analysis will extract information from thousands of cells.

2) Generation and optimization of a cellular anion flux biosensor system

Results from expression and inhibitor studies (carried out in other SRC labs exploiting ex-vivo material), will help identify components likely to be necessary to recapitulate bile duct results: while ducts expressing WT-CFTR had a high HCO₃^- selectivity, CFTR modulator treatment of F508del-CFTR-expressing ducts increased Cl^- but not HCO₃^- secretion [5]. What protein(s) do we need to coexpress to recover this effect in HEK293 cells? By incorporating transport and regulatory proteins, we propose to build a relevant “minimal model” biosensor HEK293 cell-line. In support of the feasibility of this approach, coexpression of CFTR and the protein kinase WNK1, alone, was sufficient to reconstruct a system capable of modulating CFTR anion selectivity [6]. Mechanistic insight will emerge from building the system from its component parts.

3) Characterization of Cl^- vs. HCO₃^- fluxes in mutant CFTR variants, in the absence and presence of CFTR modulator drugs

The minimal model biosensor system will be used to profile how a panel of 62 CF-causing CFTR mutations [2] affect anion fluxes. Further, for each mutant, the effect of 20 hours’ treatment with modulators will be quantified. How do modulators affect Cl^- and HCO₃^- transmembrane flow? Is there any mutant/modulator combination that differentially recovers one or the other? The empirical screens will provide material for generating hypotheses, and pinpoint compounds/residues/conditions requiring more detailed investigations in native systems (to be carried out in other SRC labs).

Intense cross-discipline communication and iterative interactions with other members of the SRC team will be essential and enable a deeper understanding of how CFTR-mediated anion fluxes are modulated.

The PhD studentship is available to start in September 2023. Please contact Dr Paola Vergani ([Email Address Removed]) for informal enquiries.