Endogenous CAL limits ΔF508-CFTR half-life in polarized human airway epithelial cells and represents a potential target for CFTR ‘stabilizers.’ (a) ΔF508-CFTR exhibits three functional defects: (1) a failure to fold properly in the ER, leading to ER associated degradation (ERAD) (“folding”); (2) reduced open probability (“P_o”) of ΔF508-CFTR channels that are found in the apical membrane; and (3) accelerated breakdown (“stability”). Aberrant flux is highlighted by red arrows. (b) Classes of therapeutic agents (blue) are being developed to address the folding defect (‘correctors’) and the gating defect (‘potentiators’), but ‘stabilizers’ that specifically address the half-life deficiency have not yet been identified.

iCAL36 is a competitive inhibitor. (a) HSQC spectra of ¹⁵N-CALP were determined in the absence (red) and in the presence (blue) of 800 μM iCAL36. Crosspeak perturbations are labeled by residue. (b, c) Surface representations of the CAL PDZ domain (PDB entry 2DC2) show the overlapping interfaces (red) associated with binding of the iCAL36 decamer ANSRWPTSII (b) or with the CFTR C-terminal octamer EEVQDTRL (c)[18]. The binding surface of the iCAL36₁₀ peptide extends beyond that of CFTR₈ (b, arrow).

Pull-down binding assays validate the specificity profiles of PDZ inhibitors for endogenous full-length targets. (a) BT-CFTR, (b) BT-SSR5, and (c) BT-iCAL36 (iCAL) peptides were conjugated to streptavidin beads. Control experiments were performed with unconjugated beads (Beads). Following incubation with CFBE-ΔF cell extracts, bound proteins were eluted with the corresponding unlabeled peptide or SCR, and the indicated volumes were immunoblotted (IB) with CAL-, NHERF1 (N1)-, NHERF2 (N2)-, and NHERF3 (N3)-specific antibodies. Dilutions of cell extracts were blotted on the same membrane as positive controls. Arrowhead indicates the position of the CAL band in (a). Representative blots are shown, with M_r standards at right (n = 3).

CAL inhibitors enhance the functional stability of ΔF508-CFTR at the apical membrane. (a, b) Following treatment with F*-iCAL36 or F*-SCR, polarized CFBE-ΔF cells were treated with CHX for 0 h or 2 h, and chloride efflux (I_SC) was measured. (b) The residual ΔI_SC values after 2 h CHX exposure are plotted as a fraction of the corresponding 0 h starting value. CAL-specific inhibition increased residual ΔISC from 60% to 75% of baseline (n = 9). (c) Cell-surface retention of ΔF508-CFTR is enhanced by CAL knockdown. CFBE-ΔF cells were treated with CAL-specific (siCAL) or control (siNEG) siRNAs. Post-maturational stability of ΔF508-CFTR was determined by incubating CFBE-ΔF cells in CHX and measuring the cell-surface abundance of ΔF508-CFTR at t = 2 h as a fraction of the amount present at t = 0 h. siCAL-treated cells exhibited 71% CAL knockdown and retain 40% of ΔF508-CFTR, compared to 15% retention seen in siNEG-treated cells (n = 12 for siNEG and n = 8 for siCAL). Values shown are mean ± SEM.

CAL selectivity improves the efficacy of ΔF508-CFTR rescue. Polarized monolayers were treated with a scrambled control peptide, or else with the CFTR, SSR5, or iCAL36 peptides in the presence of BioPORTER. Following channel activation, the change in short-circuit currents (ΔI_SC) was monitored upon application of the CFTR-specific inhibitor CFTR_inh172[21]. The differential free energy of binding (ΔΔG) for the CAL PDZ domain vs. the NHERF1 PDZ1 domain is shown below the graph for the unlabeled decamer corresponding to each inhibitor peptide[17]. Rescue (ΔI_SC) increases with selectivity (ΔΔG). p-values are shown for pairwise comparisons (n ≥ 5). Values shown are mean ± SEM.

iCAL36 and corr-4a represent complementary rescue strategies. CFBE-ΔF monolayers were treated with DMSO or with corr-4a[8] for 24 h, and were subsequently treated with BioPORTER and either F*-SCR or F*-iCAL36. Chloride efflux values (ΔI_SC) were determined, showing enhancements for both iCAL36 (11%) and corr-4a (15%), with nearly additive effects upon co-application (25%). p-values are shown for pairwise comparisons (n ≥ 9). Values shown are mean ± SEM.