Fluorescence assay for simultaneous quantification of CFTR ion-channel function and plasma membrane proximity

Abstract

18CFTR mutations cause cystic fibrosis by affecting how many channels reach the membrane, 19 and/or how well they work. This study presents an assay that simultaneously measures CFTR 20 biogenesis and function. A screen of 62 disease-causing mutations provides clues on how 21 approved drug VX-770 works.Abstract 24 Cystic fibrosis (CF) is a life-limiting disease caused by mutations in the CFTR gene, 25 encoding a plasma membrane anion-selective channel. Because CF-causing mutations affect 26 both CFTR permeation/gating and biogenesis, multi-assay approaches have been implemented 27 in drug development, sequentially screening for channel function and membrane density. Here 28 we present the first assay capable of simultaneous assessment of both CFTR characteristics. 29To validate our assay, we investigate F508del-CFTR, the most common disease-30 causing mutant, confirming rescue by incubation at low temperature, treatment with CFTR-31 targeting drugs and introduction of second-site revertant mutation R1070W. In addition, we 32 characterize a panel of 62 CF-causing mutations and profile effects of acute treatment with 33 approved drug VX-770 (ivacaftor). Measurements using the rare mutation panel correlate well 34 with published results, further validating the assay. Furthermore, mapping the potentiation 35 profile on CFTR structures raises mechanistic hypotheses on drug action, suggesting that VX-36 770 might allow an open-channel conformation with an alternative arrangement of domain 37 interfaces around site 1. 38The assay is a powerful tool for investigation of CFTR ion channel biophysics, allowing 39 rapid and accurate inferences on gating/permeation properties and on how these are affected 40 by mutations and compounds. By providing a two-dimensional molecular characterization of 41 mutant CFTR proteins, our assay can better inform development of therapies tailored for 42 individual CFTR variants. Finally, the integrated assay boosts the potential for discovery of 43 dual-acting compounds, simultaneously repairing both biogenesis and function. 44 45 Keywords: protein transport, fluorescence imaging, precision medicine, VX-770.46 3 Non-standard Abbreviations 47 ABC ATP-binding cassette 48 CF Cystic Fibrosis 49 CFTR Cystic Fibrosis Transmembrane Conductance Regulator 50 FmCherry cell average normalized mCherry fluorescence intensity over the entire cell 51 FYFP cell average normalized YFP fluorescence intensity over the entire cell 52 FYFP membrane average normalized YFP fluorescence intensity within the membrane zone 53 GCFTR CFTR conductance 54 Gtrans transient anion conductance 55 IRES internal ribosome entry site 56 NBD nucleotide binding domain 57 PDL poly-D-lysine 58 PO open probability 59  CFTR membrane density, as defined in this paper 60 SSR sum of squared residuals 61 trans time constant of the transient anion conductance 62 TM transmembrane helix 63 VM membrane potential 64 WT wild type 65 YFP yellow fluorescent protein 66 67 Cystic fibrosis (CF) is a common life-limiting genetic disease. Alth...

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“…4). Time course of the anion‐sensitive YFP quenching was measured following an iodide/chloride exchange protocol [101,102]. Unexpectedly, the compounds appear to activate CFTR, increasing cellular anion conductance and depolarizing membrane potential.…”

“…Acute treatment with selective quinoline MsbA inhibitors G592, G593, G247 and G907 [98] results in an increase in cellular anion conductance. Functional analysis of wild‐type YFP‐CFTR expressed in HEK293 cells using an image‐based fluorescence microscopy assay [101,102]. YFP‐CFTR is expressed from a pIRES2‐mCherry‐YFPCFTR plasmid, in which YFP‐CFTR and a soluble mCherry red fluorescent protein are translated from a single bicistronic mRNA.…”

“…Bars represent different pretreatment: vehicle control (DMSO, dark grey bar), 0.3 µ m forskolin (light grey bar), 0.3 µ m forskolin + 10 µ m G592 (blue bar), 0.3 µ m forskolin + 10 µ m G593 (red bar), 0.3 µ m forskolin + 10 µ m G247 (yellow bar) or with 0.3 µ m forskolin + 10 µ m G907 (black bar) ( n ≥ 5, as indicated by solid circles, each representing one measurement obtained on an independent plate). To account for possible differences in transfection efficiency between wells, CFTR conductance is normalized using the mean mCherry fluorescence measured within cells [102]. Data from wells belonging to the same 96‐well plate were paired, and paired t ‐tests were used to determine statistical significance of comparisons (** P < 0.01; **** P < 0.0001).…”

“…The quinoline compounds might affect the dynamics of the helices surrounding the inner vestibule, favouring the yet unknown conformational changes needed to open the extracellular gate, stabilizing this open conformation and/or increasing the anion throughput. It is interesting to note that some evidence suggests that VX‐770‐bound CFTR might also have noncanonical NBD/TMD interfaces [102,108].…”

“…In the meanwhile, an alternative strategy could be to exploit ligand‐based virtual screening tools [156,157], using existing ligands as starting points for a focused exploration of chemical space. Chemical analogues identified in silico could be screened for effects with 2D organoids and other high‐throughput biological assays [102,158,159]. CFTR ligands thought to act at this step, albeit normally speeding up channel opening (e.g.…”