Readthrough-induced misincorporated amino acid ratios guide mutant-specific therapeutic approaches for two CFTR nonsense mutations.

Cystic fibrosis (CF) is a monogenic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. Premature termination codons (PTCs) represent ∼9% of CF mutations that typically cause severe expression defects of the CFTR anion channel. Despite the prevalence of PTCs as the underlying cause of genetic diseases, understanding the therapeutic susceptibilities of their molecular defects, both at the transcript and protein levels remains partially elucidated. Given that the molecular pathologies depend on the PTC positions in CF, multiple pharmacological interventions are required to suppress the accelerated nonsense-mediated mRNA decay (NMD), to correct the CFTR conformational defect caused by misincorporated amino acids, and to enhance the inefficient stop codon readthrough. The G418-induced readthrough outcome was previously investigated only in reporter models that mimic the impact of the local sequence context on PTC mutations in CFTR. To identify the misincorporated amino acids and their ratios for PTCs in the context of full-length CFTR readthrough, we developed an affinity purification (AP)-tandem mass spectrometry (AP-MS/MS) pipeline. We confirmed the incorporation of Cys, Arg, and Trp residues at the UGA stop codons of G542X, R1162X, and S1196X in CFTR. Notably, we observed that the Cys and Arg incorporation was favored over that of Trp into these CFTR PTCs, suggesting that the transcript sequence beyond the proximity of PTCs and/or other factors can impact the amino acid incorporation and full-length CFTR functional expression. Additionally, establishing the misincorporated amino acid ratios in the readthrough CFTR PTCs aided in maximizing the functional rescue efficiency of PTCs by optimizing CFTR modulator combinations. Collectively, our findings contribute to the understanding of molecular defects underlying various CFTR nonsense mutations and provide a foundation to refine mutation-dependent therapeutic strategies for various CF-causing nonsense mutations.

HERC3 facilitates ERAD of select membrane proteins by recognizing membrane-spanning domains.

Aberrant proteins located in the endoplasmic reticulum (ER) undergo rapid ubiquitination by multiple ubiquitin (Ub) E3 ligases and are retrotranslocated to the cytosol as part of the ER-associated degradation (ERAD). Despite several ERAD branches involving different Ub E3 ligases, the molecular machinery responsible for these ERAD branches in mammalian cells remains not fully understood. Through a series of multiplex knockdown/knockout experiments with real-time kinetic measurements, we demonstrate that HERC3 operates independently of the ER-embedded ubiquitin ligases RNF5 and RNF185 (RNF5/185) to mediate the retrotranslocation and ERAD of misfolded CFTR. While RNF5/185 participates in the ERAD process of both misfolded ABCB1 and CFTR, HERC3 uniquely promotes CFTR ERAD. In vitro assay revealed that HERC3 directly interacts with the exposed membrane-spanning domains (MSDs) of CFTR but not with the MSDs embedded in liposomes. Therefore, HERC3 could play a role in the quality control of MSDs in the cytoplasm and might be crucial for the ERAD pathway of select membrane proteins.

Analysis of AlphaMissense data in different protein groups and structural context.

Single amino acid substitutions can profoundly affect protein folding, dynamics, and function. The ability to discern between benign and pathogenic substitutions is pivotal for therapeutic interventions and research directions. Given the limitations in experimental examination of these variants, AlphaMissense has emerged as a promising predictor of the pathogenicity of missense variants. Since heterogenous performance on different types of proteins can be expected, we assessed the efficacy of AlphaMissense across several protein groups (e.g. soluble, transmembrane, and mitochondrial proteins) and regions (e.g. intramembrane, membrane interacting, and high confidence AlphaFold segments) using ClinVar data for validation. Our comprehensive evaluation showed that AlphaMissense delivers outstanding performance, with MCC scores predominantly between 0.6 and 0.74. We observed low performance on disordered datasets and ClinVar data related to the CFTR ABC protein. However, a superior performance was shown when benchmarked against the high quality CFTR2 database. Our results with CFTR emphasizes AlphaMissense's potential in pinpointing functional hot spots, with its performance likely surpassing benchmarks calculated from ClinVar and ProteinGym datasets.

Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling.

The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR posttranslational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their posttranslational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding.

Folding correctors can restore CFTR posttranslational folding landscape by allosteric domain-domain coupling.

The folding/misfolding and pharmacological rescue of multidomain ATP-binding cassette (ABC) C-subfamily transporters, essential for organismal health, remain incompletely understood. The ABCC transporters core consists of two nucleotide binding domains (NBD1,2) and transmembrane domains (TMD1,2). Using molecular dynamic simulations, biochemical and hydrogen deuterium exchange approaches, we show that the mutational uncoupling or stabilization of NBD1-TMD1/2 interfaces can compromise or facilitate the CFTR(ABCC7)-, MRP1(ABCC1)-, and ABCC6-transporters posttranslational coupled domain-folding in the endoplasmic reticulum. Allosteric or orthosteric binding of VX-809 and/or VX-445 folding correctors to TMD1/2 can rescue kinetically trapped CFTR post-translational folding intermediates of cystic fibrosis (CF) mutants of NBD1 or TMD1 by global rewiring inter-domain allosteric-networks. We propose that dynamic allosteric domain-domain communications not only regulate ABCC-transporters function but are indispensable to tune the folding landscape of their post-translational intermediates. These allosteric networks can be compromised by CF-mutations, and reinstated by correctors, offering a framework for mechanistic understanding of ABCC-transporters (mis)folding. One-Sentence Summary: Allosteric interdomain communication and its modulation are critical determinants of ABCC-transporters post-translational conformational biogenesis, misfolding, and pharmacological rescue.

Design of Crotoxin-Based Peptides with Potentiator Activity Targeting the ΔF508NBD1 Cystic Fibrosis Transmembrane Conductance Regulator.

We have previously shown that the CBb subunit of crotoxin, a ß-neurotoxin with phospholipase A2 (PLA2) activity, targets the human ΔF508CFTR chloride channel implicated in cystic fibrosis (CF). By direct binding to the nucleotide binding domain 1 (NBD1) of ΔF508CFTR, this neurotoxic PLA2 acts as a potentiator increasing chloride channel current and corrects the trafficking defect of misfolded ΔF508CFTR inside the cell. Here, for a therapeutics development of new anti-cystic fibrosis agents, we use a structure-based in silico approach to design peptides mimicking the CBb-ΔF508NBD1 interface. Combining biophysical and electrophysiological methods, we identify several peptides that interact with the ΔF508NBD1 domain and reveal their effects as potentiators on phosphorylated ΔF508CFTR. Moreover, protein-peptide interactions and electrophysiological studies allowed us to identify key residues of ΔF508NBD1 governing the interactions with the novel potentiators. The designed peptides bind to the same region as CBb phospholipase A2 on ΔF508NBD1 and potentiate chloride channel activity. Certain peptides also show an additive effect towards the clinically approved VX-770 potentiator. The identified CF therapeutics peptides represent a novel class of CFTR potentiators and illustrate a strategy leading to reproducing the effect of specific protein-protein interactions.

Keratin 8 is a scaffolding and regulatory protein of ERAD complexes.

Early recognition and enhanced degradation of misfolded proteins by the endoplasmic reticulum (ER) quality control and ER-associated degradation (ERAD) cause defective protein secretion and membrane targeting, as exemplified for Z-alpha-1-antitrypsin (Z-A1AT), responsible for alpha-1-antitrypsin deficiency (A1ATD) and F508del-CFTR (cystic fibrosis transmembrane conductance regulator) responsible for cystic fibrosis (CF). Prompted by our previous observation that decreasing Keratin 8 (K8) expression increased trafficking of F508del-CFTR to the plasma membrane, we investigated whether K8 impacts trafficking of soluble misfolded Z-A1AT protein. The subsequent goal of this study was to elucidate the mechanism underlying the K8-dependent regulation of protein trafficking, focusing on the ERAD pathway. The results show that diminishing K8 concentration in HeLa cells enhances secretion of both Z-A1AT and wild-type (WT) A1AT with a 13-fold and fourfold increase, respectively. K8 down-regulation triggers ER failure and cellular apoptosis when ER stress is jointly elicited by conditional expression of the µs heavy chains, as previously shown for Hrd1 knock-out. Simultaneous K8 silencing and Hrd1 knock-out did not show any synergistic effect, consistent with K8 acting in the Hrd1-governed ERAD step. Fractionation and co-immunoprecipitation experiments reveal that K8 is recruited to ERAD complexes containing Derlin2, Sel1 and Hrd1 proteins upon expression of Z/WT-A1AT and F508del-CFTR. Treatment of the cells with c407, a small molecule inhibiting K8 interaction, decreases K8 and Derlin2 recruitment to high-order ERAD complexes. This was associated with increased Z-A1AT secretion in both HeLa and Z-homozygous A1ATD patients' respiratory cells. Overall, we provide evidence that K8 acts as an ERAD modulator. It may play a scaffolding protein role for early-stage ERAD complexes, regulating Hrd1-governed retrotranslocation initiation/ubiquitination processes. Targeting K8-containing ERAD complexes is an attractive strategy for the pharmacotherapy of A1ATD.

Measuring EGFR plasma membrane density, stability, internalization, and recycling in alive adherent cells by cell surface ELISA.

EGFR cell surface density, stability, internalization, and recycling can be measured by cell surface ELISA (cs-ELISA). Performing this experiment on ice impedes receptor internalization; thus the physiological cell surface receptor levels can be measured by cs-ELISA. Cell surface EGFR levels are detected by measuring Amplex Red fluorescence intensity. Although cell surface receptor levels can be measured by flow cytometry, cs-ELISA does not include cell dissociation steps that might affect cell surface receptor levels. For complete details on the use and execution of this protocol, please refer to Kazan et al. (2019).

Nanomechanics combined with HDX reveals allosteric drug binding sites of CFTR NBD1.

Cystic fibrosis (CF) is a frequent genetic disease in Caucasians that is caused by the deletion of F508 (ΔF508) in the nucleotide binding domain 1 (NBD1) of the CF transmembrane conductance regulator (CFTR). The ΔF508 compromises the folding energetics of the NBD1, as well as the folding of three other CFTR domains. Combination of FDA approved corrector molecules can efficiently but incompletely rescue the ΔF508-CFTR folding and stability defect. Thus, new pharmacophores that would reinstate the wild-type-like conformational stability of the ΔF508-NBD1 would be highly beneficial. The most prominent molecule, 5-bromoindole-3-acetic acid (BIA) that can thermally stabilize the NBD1 has low potency and efficacy. To gain insights into the NBD1 (un)folding dynamics and BIA binding site localization, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen-deuterium exchange (HDX) experiments. We found that the NBD1 α-subdomain with three adjacent strands from the ß-subdomain plays an important role in early folding steps, when crucial non-native interactions are formed via residue F508. Our AFM and HDX experiments showed that BIA associates with this α-core region and increases the resistance of the ΔF508-NBD1 against mechanical unfolding, a phenomenon that could be exploited in future developments of folding correctors.

A PI3Kγ mimetic peptide triggers CFTR gating, bronchodilation, and reduced inflammation in obstructive airway diseases.

Cyclic adenosine 3',5'-monophosphate (cAMP)-elevating agents, such as ß2-adrenergic receptor (ß2-AR) agonists and phosphodiesterase (PDE) inhibitors, remain a mainstay in the treatment of obstructive respiratory diseases, conditions characterized by airway constriction, inflammation, and mucus hypersecretion. However, their clinical use is limited by unwanted side effects because of unrestricted cAMP elevation in the airways and in distant organs. Here, we identified the A-kinase anchoring protein phosphoinositide 3-kinase γ (PI3Kγ) as a critical regulator of a discrete cAMP signaling microdomain activated by ß2-ARs in airway structural and inflammatory cells. Displacement of the PI3Kγ-anchored pool of protein kinase A (PKA) by an inhaled, cell-permeable, PI3Kγ mimetic peptide (PI3Kγ MP) inhibited a pool of subcortical PDE4B and PDE4D and safely increased cAMP in the lungs, leading to airway smooth muscle relaxation and reduced neutrophil infiltration in a murine model of asthma. In human bronchial epithelial cells, PI3Kγ MP induced unexpected cAMP and PKA elevations restricted to the vicinity of the cystic fibrosis transmembrane conductance regulator (CFTR), the ion channel controlling mucus hydration that is mutated in cystic fibrosis (CF). PI3Kγ MP promoted the phosphorylation of wild-type CFTR on serine-737, triggering channel gating, and rescued the function of F508del-CFTR, the most prevalent CF mutant, by enhancing the effects of existing CFTR modulators. These results unveil PI3Kγ as the regulator of a ß2-AR/cAMP microdomain central to smooth muscle contraction, immune cell activation, and epithelial fluid secretion in the airways, suggesting the use of a PI3Kγ MP for compartment-restricted, therapeutic cAMP elevation in chronic obstructive respiratory diseases.

Ubr1-induced selective endophagy/autophagy protects against the endosomal and Ca2+-induced proteostasis disease stress.

The cellular defense mechanisms against cumulative endo-lysosomal stress remain incompletely understood. Here, we identify Ubr1 as a protein quality control (QC) E3 ubiquitin-ligase that counteracts proteostasis stresses by facilitating endosomal cargo-selective autophagy for lysosomal degradation. Astrocyte regulatory cluster membrane protein MLC1 mutations cause endosomal compartment stress by fusion and enlargement. Partial lysosomal clearance of mutant endosomal MLC1 is accomplished by the endosomal QC ubiquitin ligases, CHIP and Ubr1 via ESCRT-dependent route. As a consequence of the endosomal stress, a supportive QC mechanism, dependent on both Ubr1 and SQSTM1/p62 activities, targets ubiquitinated and arginylated MLC1 mutants for selective endosomal autophagy (endophagy). This QC pathway is also activated for arginylated Ubr1-SQSTM1/p62 autophagy cargoes during cytosolic Ca2+-assault. Conversely, the loss of Ubr1 and/or arginylation elicited endosomal compartment stress. These findings underscore the critical housekeeping role of Ubr1 and arginylation-dependent endophagy/autophagy during endo-lysosomal proteostasis perturbations and suggest a link of Ubr1 to Ca2+ homeostasis and proteins implicated in various diseases including cancers and brain disorders.

Mechanism of PINK1 activation by autophosphorylation and insights into assembly on the TOM complex.

Mutations in PINK1 cause autosomal-recessive Parkinson's disease. Mitochondrial damage results in PINK1 import arrest on the translocase of the outer mitochondrial membrane (TOM) complex, resulting in the activation of its ubiquitin kinase activity by autophosphorylation and initiation of Parkin-dependent mitochondrial clearance. Herein, we report crystal structures of the entire cytosolic domain of insect PINK1. Our structures reveal a dimeric autophosphorylation complex targeting phosphorylation at the invariant Ser205 (human Ser228). The dimer interface requires insert 2, which is unique to PINK1. The structures also reveal how an N-terminal helix binds to the C-terminal extension and provide insights into stabilization of PINK1 on the core TOM complex.

Endofin is required for HD-PTP and ESCRT-0 interdependent endosomal sorting of ubiquitinated transmembrane cargoes.

Internalized and ubiquitinated signaling receptors are silenced by their intraluminal budding into multivesicular bodies aided by the endosomal sorting complexes required for transport (ESCRT) machinery. HD-PTP, an ESCRT protein, forms complexes with ESCRT-0, -I and -III proteins, and binds to Endofin, a FYVE-domain protein confined to endosomes with poorly understood roles. Using proximity biotinylation, we showed that Endofin forms a complex with ESCRT constituents and Endofin depletion increased integrin α5-and EGF-receptor plasma membrane density and stability by hampering their lysosomal delivery. This coincided with sustained receptor signaling and increased cell migration. Complementation of Endofin- or HD-PTP-depleted cells with wild-type Endofin or HD-PTP, but not with mutants harboring impaired Endofin/HD-PTP association or cytosolic Endofin, restored EGFR lysosomal delivery. Endofin also promoted Hrs indirect interaction with HD-PTP. Jointly, our results indicate that Endofin is required for HD-PTP and ESCRT-0 interdependent sorting of ubiquitinated transmembrane cargoes to ensure efficient receptor desensitization and lysosomal delivery.

Control of membrane protein homeostasis by a chaperone-like glial cell adhesion molecule at multiple subcellular locations.

The significance of crosstalks among constituents of plasma membrane protein clusters/complexes in cellular proteostasis and protein quality control (PQC) remains incompletely understood. Examining the glial (enriched) cell adhesion molecule (CAM), we demonstrate its chaperone-like role in the biosynthetic processing of the megalencephalic leukoencephalopathy with subcortical cyst 1 (MLC1)-heteromeric regulatory membrane protein complex, as well as the function of the GlialCAM/MLC1 signalling complex. We show that in the absence of GlialCAM, newly synthesized MLC1 molecules remain unfolded and are susceptible to polyubiquitination-dependent proteasomal degradation at the endoplasmic reticulum. At the plasma membrane, GlialCAM regulates the diffusional partitioning and endocytic dynamics of cluster members, including the ClC-2 chloride channel and MLC1. Impaired folding and/or expression of GlialCAM or MLC1 in the presence of diseases causing mutations, as well as plasma membrane tethering compromise the functional expression of the cluster, leading to compromised endo-lysosomal organellar identity. In addition, the enlarged endo-lysosomal compartments display accelerated acidification, ubiquitinated cargo-sorting and impaired endosomal recycling. Jointly, these observations indicate an essential and previously unrecognized role for CAM, where GliaCAM functions as a PQC factor for the MLC1 signalling complex biogenesis and possess a permissive role in the membrane dynamic and cargo sorting functions with implications in modulations of receptor signalling.

A Precision Medicine Approach to Optimize Modulator Therapy for Rare CFTR Folding Mutants.

Trikafta, a triple-combination drug, consisting of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor) and the gating potentiator VX-770 (ivacaftor) provided unprecedented clinical benefits for patients with the most common cystic fibrosis (CF) mutation, F508del. Trikafta indications were recently expanded to additional 177 mutations in the CF transmembrane conductance regulator (CFTR). To minimize life-long pharmacological and financial burden of drug administration, if possible, we determined the necessary and sufficient modulator combination that can achieve maximal benefit in preclinical setting for selected mutants. To this end, the biochemical and functional rescue of single corrector-responsive rare mutants were investigated in a bronchial epithelial cell line and patient-derived human primary nasal epithelia (HNE), respectively. The plasma membrane density of P67L-, L206W- or S549R-CFTR corrected by VX-661 or other type I correctors was moderately increased by VX-445. Short-circuit current measurements of HNE, however, uncovered that correction comparable to Trikafta was achieved for S549R-CFTR by VX-661 + VX-770 and for P67L- and L206W-CFTR by the VX-661 + VX-445 combination. Thus, introduction of a third modulator may not provide additional benefit for patients with a subset of rare CFTR missense mutations. These results also underscore that HNE, as a precision medicine model, enable the optimization of mutation-specific modulator combinations to maximize their efficacy and minimize life-long drug exposure of CF patients.

Elexacaftor co-potentiates the activity of F508del and gating mutants of CFTR.

Trikafta, the combination of elexacaftor (VX-445), tezacaftor (VX-661) and ivacaftor (VX-770), was approved for therapy of cystic fibrosis (CF) patients with at least one allele of the CFTR mutation F508del. While the corrector function of VX-445 is well established, here we investigated the putative potentiator activity of VX-445 alone and in combination with VX-770. Acute addition of VX-445 increased the VX-770-potentiated F508del- and G551D-CFTR current by ~24% and >70%, respectively, in human bronchial and nasal epithelia. Combinatorial profiling and cluster analysis of G551D- and G1244E-CFTR channel activation with potentiator pairs indicated a distinct VX-445 mechanism of action that is, at least, additive to previously identified potentiator classes, including the VX-770. Since VX-770 only partially normalizes the G551D-CFTR channel function and adult G551D patients still experience progressive loss of lung function, VX-445+VX-770 combination therapy could provide clinical benefit to CF patients with the G551D and other dual potentiator responsive mutants.

Allosteric folding correction of F508del and rare CFTR mutants by elexacaftor-tezacaftor-ivacaftor (Trikafta) combination.

Based on its clinical benefits, Trikafta - the combination of folding correctors VX-661 (tezacaftor), VX-445 (elexacaftor), and the gating potentiator VX-770 (ivacaftor) - was FDA approved for treatment of patients with cystic fibrosis (CF) carrying deletion of phenylalanine at position 508 (F508del) of the CF transmembrane conductance regulator (CFTR) on at least 1 allele. Neither the mechanism of action of VX-445 nor the susceptibility of rare CF folding mutants to Trikafta are known. Here, we show that, in human bronchial epithelial cells, VX-445 synergistically restores F508del-CFTR processing in combination with type I or II correctors that target the nucleotide binding domain 1 (NBD1) membrane spanning domains (MSDs) interface and NBD2, respectively, consistent with a type III corrector mechanism. This inference was supported by the VX-445 binding to and unfolding suppression of the isolated F508del-NBD1 of CFTR. The VX-661 plus VX-445 treatment restored F508del-CFTR chloride channel function in the presence of VX-770 to approximately 62% of WT CFTR in homozygous nasal epithelia. Substantial rescue of rare misprocessing mutations (S13F, R31C, G85E, E92K, V520F, M1101K, and N1303K), confined to MSD1, MSD2, NBD1, and NBD2 of CFTR, was also observed in airway epithelia, suggesting an allosteric correction mechanism and the possible application of Trikafta for patients with rare misfolding mutants of CFTR.

Selective Binding of HSC70 and its Co-Chaperones to Structural Hotspots on CFTR.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel cause cystic fibrosis. Chaperones, including HSC70, DNAJA1 and DNAJA2, play key roles in both the folding and degradation of wild-type and mutant CFTR at multiple cellular locations. DNAJA1 and HSC70 promote the folding of newly synthesized CFTR at the endoplasmic reticulum (ER), but are required for the rapid turnover of misfolded channel at the plasma membrane (PM). DNAJA2 and HSC70 are also involved in the ER-associated degradation (ERAD) of misfolded CFTR, while they assist the refolding of destabilized channel at the PM. These outcomes may depend on the binding of chaperones to specific sites within CFTR, which would be exposed in non-native states. A CFTR peptide library was used to identify binding sites for HSC70, DNAJA1 and DNAJA2, validated by competition and functional assays. Each chaperone had a distinct binding pattern, and sites were distributed between the surfaces of the CFTR cytosolic domains, and domain interfaces known to be important for channel assembly. The accessibility of sites to chaperones will depend on the degree of CFTR folding or unfolding. Different folded states may be recognized by unique combinations of HSC70, DNAJA1 and DNAJA2, leading to divergent biological effects.

High-throughput phenotyping of heteromeric human ether-à-go-go-related gene potassium channel variants can discriminate pathogenic from rare benign variants.

BACKGROUND: KCNH2 encodes the human ether-à-go-go-related gene potassium channel, which passes the rapid delayed rectifier potassium current. Loss-of-function variants in KCNH2 cause long QT syndrome type 2, which is associated with a markedly increased risk of cardiac arrhythmias. The majority of rare KCNH2 variants, however, are likely to be benign. OBJECTIVE: The purpose of this study was to develop a high-throughput assay for discriminating pathogenic from benign KCNH2 variants. METHODS: Nonsynonymous homozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 cell lines were phenotyped using an automated patch-clamp platform and a cell surface enzyme-linked immunosorbent assay. Functional phenotyping of heterozygous KCNH2 variants stably expressed in Flp-In human embryonic kidney 293 cell lines using a bicistronic vector was performed using an automated patch-clamp platform. RESULTS: In homozygous KCNH2 variant cell lines, discrepancies between current density and cell surface expression levels measured using an enzyme-linked immunosorbent assay can be explained by changes in gating properties of the variant channels. For the 30 heterozygous KCNH2 variant cell lines studied, the assay correctly predicted the ClinVar ascribed classification for 17/17 pathogenic/likely pathogenic/benign variants. Of the 13 pore-domain variants studied, 11 had a dominant-negative expression defect while the remaining 2 had enhanced inactivation gating, resulting in a dominant-negative phenotype. CONCLUSION: High-throughput electrophysiological phenotyping of heterozygous KCNH2 variants can accurately distinguish between dominant-negative, haploinsufficient loss-of-function, and benign variants. This assay will help with future classification of KCNH2 variants.

Mutation-specific dual potentiators maximize rescue of CFTR gating mutants.

BACKGROUND: The potentiator ivacaftor (VX-770) has been approved for therapy of 38 cystic fibrosis (CF) mutations (∼10% of the patient population) associated with a gating defect of the CF transmembrane conductance regulator (CFTR). Despite the success of VX-770 treatment of patients carrying at least one allele of the most common gating mutation G551D-CFTR, some lung function decline and P. aeruginosa colonization persist. This study aims at identifying potentiator combinations that can considerably enhance the limited channel activity of a panel of CFTR gating mutants over monotherapy. METHODS: The functional response of 13 CFTR mutants to single potentiators or systematic potentiator combinations was determined in the human bronchial epithelial cell line CFBE41o- and a subset of them was confirmed in primary human nasal epithelia (HNE). RESULTS: In six out of thirteen CFTR missense mutants the fractional plasma membrane (PM) activity, a surrogate measure of CFTR channel gating, reached only ∼10-50% of WT channel activity upon VX-770 treatment, indicating incomplete gating correction. Combinatorial potentiator profiling and cluster analysis of mutant responses to 24 diverse investigational potentiators identified several compound pairs that improved the gating activity of R352Q-, S549R-, S549N-, G551D-, and G1244E-CFTR to ∼70-120% of the WT. Similarly, the potentiator combinations were able to confer WT-like function to G551D-CFTR in patient-derived human nasal epithelia. CONCLUSION: This study suggests that half of CF patients with missense mutations approved for VX-770 administration, could benefit from the development of dual potentiator therapy.