Panel a. Wild-type (WT) and variant CFTR proteins were expressed in HEK 293T cells and immunoblotted with anti-CFTR and anti-Aldolase antibodies. Replicate lanes are in small font. Band B, expected size of immature ER core-glycosylated CFTR; band C, mature complex-glycosylated CFTR. Panel b. Whole-cell Cl⁻ currents were measured in WT and variant CFTR-expressing HEK 293T cells, as described in Methods. Panel c. Whole-cell currents of WT-CFTR were measured in HEK 293T cells co-expressed with WNK1 and SPAK using patch pipette contained a low concentration of Cl⁻ (10 mM). A representative trace of reversal potential measurement is shown in the left panel. The permeability ratio P_HCO3/P_Cl was calculated according to the Goldman-Hodgkin-Katz equation. I–V relationships at the indicated points are presented in the accompanying graph. The conductance ratio G_HCO3/G_Cl was calculated by measuring each outward current (i.e., slope between E_rev and E_rev+25 mV). RMP, resting membrane potential. Panel d. Whole-cell currents of R170H-CFTR were measured in HEK 293T cells using the same protocol shown in panel c. Panel e. A summary of the P_HCO3/P_Cl values obtained from WT-CFTR in the standard state (left) compared to WT-CFTR and the nine CFTR^BD variants with WNK1 + SPAK activation (right, underlined). Panel f. A summary of the G_HCO3/G_Cl values in the standard state (left) with WNK1 + SPAK activation (right). Values throughout are means ± SEM. * p<0.05, **p<0.01: difference from WT in cells co-expressed with WNK1 and SPAK.

https://doi.org/10.1371/journal.pgen.1004778.g001