Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) ion channel are established as the primary causative factor in the devastating lung disease cystic fibrosis (CF). More recently, cigarette smoke exposure has been shown to be associated with dysfunctional airway epithelial ion transport, suggesting a role for CFTR in the pathogenesis of chronic obstructive pulmonary disease (COPD). Here, the identification and characterization of a high throughput screening hit 6 as a potentiator of mutant human F508del and wild-type CFTR channels is reported. The design, synthesis, and biological evaluation of compounds 7-33 to establish structure-activity relationships of the scaffold are described, leading to the identification of clinical development compound icenticaftor (QBW251) 33, which has subsequently progressed to deliver two positive clinical proofs of concept in patients with CF and COPD and is now being further developed as a novel therapeutic approach for COPD patients.

The adult form of the human muscle nicotinicacetylcholine receptor (nAChR) consists of four different subunits forming apentamer around the channel pore, (α1) 2 β1δɛ. The two orthosteric binding sites are located at the α1‐ɛ and α1‐δ interfaces. A library of 2816α1 subunit mutants was created using high throughput mutagenesis by error‐prone PCR and sequences of all mutants were determined using single molecule real timesequencing. Each α1 mutant was transiently transfected with wildtype β1, δ, and ɛ subunits into HEK293 cells, and the channel function examined in an ion flux assay. Mutant hits were selected that responded to the nicotinic agonistepibatidine, but were resistant to the orthosteric blockers α‐bungarotoxin ortubocurarine. Eight α1 subunit mutant hits were discovered, six of which contained mutations at Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were functionally characterized, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations do not impair blocker binding, but rather evoke again‐of‐function phenotype, e . g . increased agonist sensitivity. Immunocytochemical staining showed that the surface expression of mutated α1subunits was comparable to the wildtype protein. Functional analysis of the mutant library containing alterations for each of the 457 amino acids of the human α1 subunit therefore yielded structure‐function insights for the nAChR. Support or Funding Information This work was supported by the Novartis Institutes for BioMedical Research (NIBR).

High throughput random mutagenesis is a powerful tool to identify which residues are important for the function of a protein, and gain insight into its structure-function relation. The human muscle nicotinic acetylcholine receptor was used to test whether this technique previously used for monomeric receptors can be applied to a pentameric ligand-gated ion channel. A mutant library for the α1 subunit of the channel was generated by error-prone PCR, and full length sequences of all 2816 mutants were retrieved using single molecule real time sequencing. Each α1 mutant was co-transfected with wildtype β1, δ, and ε subunits, and the channel function characterized by an ion flux assay. To test whether the strategy could map the structure-function relation of this receptor, we attempted to identify mutations that conferred resistance to competitive antagonists. Mutant hits were defined as receptors that responded to the nicotinic agonist epibatidine, but were not inhibited by either α-bungarotoxin or tubocurarine. Eight α1 subunit mutant hits were identified, six of which contained mutations at position Y233 or V275 in the transmembrane domain. Three single point mutations (Y233N, Y233H, and V275M) were studied further, and found to enhance the potencies of five channel agonists tested. This suggests that the mutations made the channel resistant to the antagonists, not by impairing antagonist binding, but rather by producing a gain-of-function phenotype, e.g. increased agonist sensitivity. Our data show that random high throughput mutagenesis is applicable to multimeric proteins to discover novel functional mutants, and outlines the benefits of using single molecule real time sequencing with regards to quality control of the mutant library as well as downstream mutant data interpretation.

BACKGROUND AND PURPOSE:Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia.EXPERIMENTAL APPROACH:The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC).KEY RESULTS:In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action.CONCLUSIONS AND IMPLICATIONS:NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds.

The calcium-activated chloride channel ANO1 regulates multiple physiological processes. However, little is known about the mechanism of channel gating and regulation of ANO1 activity. Using a high-throughput, random mutagenesis-based variomics screen, we generated and functionally characterized similar to 6000 ANO1 mutants and identified novel mutations that affected channel activity, intracellular trafficking, or localization of ANO1. Mutations such as S741T increased ANO1 calcium sensitivity and rendered ANO1 calcium gating voltage-independent, demonstrating a critical role of the re-entrant loop in coupling calcium and voltage sensitivity of ANO1 and hence in regulating ANO1 activation. Our data present the first unbiased and comprehensive study of the structure-function relationship of ANO1. The novel ANO1 mutants reported have diverse functional characteristics, providing new tools to study ANO1 function in biological systems, paving the path for a better understanding of the function of ANO1 and its role in health and diseases.

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease.

The human prostacyclin receptor (hIP receptor) is a seven-transmembrane G protein-coupled receptor (GPCR) that plays a critical role in vascular smooth muscle relaxation and platelet aggregation. hIP receptor dysfunction has been implicated in numerous cardiovascular abnormalities, including myocardial infarction, hypertension, thrombosis and atherosclerosis. Genomic sequencing has discovered several genetic variations in the PTGIR gene coding for hIP receptor, however, its structure-function relationship has not been sufficiently explored. Here we set out to investigate the applicability of high throughput random mutagenesis to study the structure-function relationship of hIP receptor. While chemical mutagenesis was not suitable to generate a mutagenesis library with sufficient coverage, our data demonstrate error-prone PCR (epPCR) mediated mutagenesis as a valuable method for the unbiased screening of residues regulating hIP receptor function and expression. Here we describe the generation and functional characterization of an epPCR derived mutagenesis library compromising >4000 mutants of the hIP receptor. We introduce next generation sequencing as a useful tool to validate the quality of mutagenesis libraries by providing information about the coverage, mutation rate and mutational bias. We identified 18 mutants of the hIP receptor that were expressed at the cell surface, but demonstrated impaired receptor function. A total of 38 non-synonymous mutations were identified within the coding region of the hIP receptor, mapping to 36 distinct residues, including several mutations previously reported to affect the signaling of the hIP receptor. Thus, our data demonstrates epPCR mediated random mutagenesis as a valuable and practical method to study the structure-function relationship of GPCRs.

TRPC3, TRPC6 and TRPC7 are Ca2+ permeable non-selective cation channels that have been implicated in cancer, cardiovascular, respiratory and kidney diseases. In this study, CHO-K1 cells stably expressing human M3 muscarinic acetylcholine receptors were infected with human TRPC3, TRPC6 or TRPC7 BacMam viruses and measured on QPatch and IonWorks Quattro automated electrophysiology platforms. In the QPatch HT mode (single cell recording), TRPC currents were rapidly activated by the muscarinic agonist carbachol and then decayed irreversibly with a rank order of TRPC7>TRPC3>TRPC6. In both HT and HTX (10 cells per well) mode there was large variation in current levels between recordings. The lack of a good well-to-well comparison means that QPatch has low suitability for TRPC screening assays. The Quattro in PPC mode (64 cells per well), sufficiently normalised well-to-well variation in carbachol activated TRPC currents to enable assays to be developed for TRPC3 and TRPC6. The time delay on the Quattro between carbachol addition and the first current measurement resulted in TRPC7 currents having almost completely decayed so remaining currents were too small. The TRPC3 and TRPC6 assays were validated with two recently described TRPC inhibitors. Compound 8 [1] inhibited TRPC3 and TRPC6 currents with respective IC50 values of 1.1 ± 0.2 μM and 24 ± 7 nM (Mean ± SD, n=4-5). 2-(amino)-thiazole-4-carboxamide [2] inhibited TRPC3 and TRPC6 currents with respective IC50 values of 1.4 ± 1.1 and 0.9 ± 0.2 μM (Mean ± SD, n=3). These values are in good agreement with the published data. This study demonstrates that TRPC3, TRPC6 and TRPC7 currents can be measured on automated electrophysiology platforms. For TRPC channels, the QPatch is suitable to profile channel biophysics whereas the Quattro is more applicable for compound profiling. [1] Patent WO2011/107474 A1. [2] Patent WO2012/037349 A2.

The canonical transient receptor potential channel subfamily (TRPC3, TRPC6, and TRPC7) contains Ca2+ permeable non-selective cation channels that are widely expressed in a variety of tissues. There is increasing evidence implicating TRPC channels, particularly TRPC3 and 6, in physiological and pathophysiological processes, eliciting interest in these channels as novel drug targets. Electrophysiology remains a benchmark technique for measuring ion channel function and accurately determining the pharmacological effects of compounds. In this report we describe the development of TRPC inhibitor assays on 2 automated planar patch clamp platforms—the IonWorks® Quattro™ and QPatch® systems. To enable activation of TRPC channels by carbachol, Chinese Hamster Ovary-K1 cells stably expressing the muscarinic M3 receptor were transduced with human TRPC3, TRPC6, or TRPC7 using BacMam viruses. TRPC3, 6, and 7 currents could be recorded on both platforms. However, the design of each platform limits which assay parameters can be recorded. Due to its continuous recording capabilities, the QPatch can capture both the activation and decay of the response. However, the transient nature of TRPC channels, the inability to reactivate and the large variation in peak currents limits the ability to develop assays for compound screening. The IonWorks Quattro, due to its discontinuous sampling, did not fully capture the peak of TRPC currents. However, due to the ability of the IonWorks Quattro to record from 64 cells per well, the variation from well to well was sufficiently reduced allowing for the development of medium-throughput screening assays.

Lymphocyte antigen receptor-mediated production of Ins(1,4,5)P3 induces the release of Ca2+ from intracellular stores, resulting in the opening of store-operated Ca2+ (SOC) channels. Mice deficient in inositol(1,4,5)P3-3 kinase B (ITPKb), which converts inositol(1,4,5)P3 (IP3) to inositol(1,3,4,5)P4 (IP4), exhibit a complete block in T cell positive selection. Previous studies demonstrated that IP4 is an inhibitor of SOC channels. To understand the role of ITPKb in mature peripheral lymphocytes, inducible ITPKb-/- mice were generated. Deletion of ITPKb in mature lymphocytes reveals that ITPKb is required for mature T cell function and T-dependent antibody responses. Following antigen receptor activation, the loss of ITPKb leads to enhanced Ca2+ levels and the induction of death effector gene expression resulting in apoptosis. We further demonstrate that IP4 is an inhibitor of open-state Orai1 channels. LMW ITPKb inhibitors were identified using a high-throughput compound screen. Application of ITPKb inhibitors to lymphocytes enhanced Ca2+ responses following antigen receptor stimulation, similar to ITPKb-/- cells. Treatment of mice with ITPKb inhibitors recapitulated the block in T cell development observed in ITPKb-/- mice and inhibited antigen-induced arthritis formation in rats. These data identify ITPKb and IP4 as crucial mediators of lymphocyte development and activation, and suggest that inhibition of ITPKb may provide a novel mechanism to treat autoimmune disease.

BACKGROUND:Alveolar macrophages are one of the first lines of defence against invading pathogens and play a central role in modulating both the innate and acquired immune systems. By responding to endogenous stimuli within the lung, alveolar macrophages contribute towards the regulation of the local inflammatory microenvironment, the initiation of wound healing and the pathogenesis of viral and bacterial infections. Despite the availability of protocols for isolating primary alveolar macrophages from the lung these cells remain recalcitrant to expansion in-vitro and therefore surrogate cell types, such as monocyte derived macrophages and phorbol ester-differentiated cell lines (e.g. U937, THP-1, HL60) are frequently used to model macrophage function.METHODS:The availability of high throughput gene expression technologies for accurate quantification of transcript levels enables the re-evaluation of these surrogate cell types for use as cellular models of the alveolar macrophage. Utilising high-throughput TaqMan arrays and focussing on dynamically regulated families of integral membrane proteins, we explore the similarities and differences in G-protein coupled receptor (GPCR) and ion channel expression in alveolar macrophages and their widely used surrogates.RESULTS:The complete non-sensory GPCR and ion channel transcriptome is described for primary alveolar macrophages and macrophage surrogates. The expression of numerous GPCRs and ion channels whose expression were hitherto not described in human alveolar macrophages are compared across primary macrophages and commonly used macrophage cell models. Several membrane proteins known to have critical roles in regulating macrophage function, including CXCR6, CCR8 and TRPV4, were found to be highly expressed in macrophages but not expressed in PMA-differentiated surrogates.CONCLUSIONS:The data described in this report provides insight into the appropriate choice of cell models for investigating macrophage biology and highlights the importance of confirming experimental data in primary alveolar macrophages.

Background The neuronal nicotinic receptors that mediate excitatory transmission in autonomic ganglia are thought to be formed mainly by the α3 and β4 subunits. Expressing this composition in oocytes fails to reproduce the properties of ganglionic receptors, which may also incorporate the α5 and/or β2 subunits. We compared the properties of human α3β4 neuronal nicotinic receptors expressed in Human embryonic kidney cells (HEK293) and in Xenopus oocytes, to examine the effect of the expression system and α∶β subunit ratio. Methodology/Principal Findings Two distinct channel forms were observed: these are likely to correspond to different stoichiometries of the receptor, with two or three copies of the α subunit, as reported for α4β2 channels. This interpretation is supported by the pattern of change in acetylcholine (ACh) sensitivity observed when a hydrophilic Leu to Thr mutation was inserted in position 9′ of the second transmembrane domain, as the effect of mutating the more abundant subunit is greater. Unlike α4β2 channels, for α3β4 receptors the putative two-α form is the predominant one in oocytes (at 1∶1 α∶β cRNA ratio). This two-α form has a slightly higher ACh sensitivity (about 3-fold in oocytes), and displays potentiation by zinc. The putative three-α form is the predominant one in HEK cells transfected with a 1∶1 α∶β DNA ratio or in oocytes at 9∶1 α∶β RNA ratio, and is more sensitive to dimethylphenylpiperazinium (DMPP) than to ACh. In outside-out single-channel recordings, the putative two-α form opened to distinctive long bursts (100 ms or more) with low conductance (26 pS), whereas the three-α form gave rise to short bursts (14 ms) of high conductance (39 pS). Conclusions/Significance Like other neuronal nicotinic receptors, the α3β4 receptor can exist in two different stoichiometries, depending on whether it is expressed in oocytes or in mammalian cell lines and on the ratio of subunits transfected.

Background and purpose: α4 and β2 nicotinic acetylcholine (ACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixed population of (α4)2(β2)3 and (α4)3(β2)2 receptors. In order to express these receptors separately in heterologous systems, we have engineered pentameric concatenated (α4)2(β2)3 and (α4)3(β2)2 receptors.Experimental approach: α4 and β2 subunits were concatenated by synthetic linkers into pentameric constructs to produce either (α4)2(β2)3 or (α4)3(β2)2 receptors. Using two‐electrode voltage‐clamp techniques, we examined the ability of the concatenated constructs to produce functional expression in Xenopus oocytes. Functional constructs were further characterized in respect to agonists, competitive antagonists, Ca2+ permeability, sensitivity to modulation by Zn2+ and sensitivity to up‐regulation by chaperone protein 14‐3‐3.Key results: We found that pentameric concatamers with a subunit arrangement of β2_α4_β2_α4_β2 or β2_α4_β2_α4_α4 were stable and functional in Xenopus oocytes. By comparison, when α4 and β2 were concatenated with a subunit order of β2_β2_α4_β2_α4 or β2_α4_α4_β2_α4, functional expression in Xenopus oocytes was very low, even though the proteins were synthesized and stable. Both β2_α4_β2_α4_β2 and β2_α4_β2_α4_α4 concatamers recapitulated the ACh concentration response curve, the sensitivity to Zn2+ modulation, Ca2+ permeability and the sensitivity to up‐regulation by chaperone protein 14‐3‐3 of the corresponding non‐linked (α4)2(β2)3 and (α4)3(β2)2 receptors respectively. Using these concatamers, we found that most α4β2‐preferring compounds studied, including A85380, 5I‐A85380, cytisine, epibatidine, TC2559 and dihydro‐β‐erythroidine, demonstrate stoichiometry‐specific potencies and efficacies.Conclusions and implications: We concluded that the α4β2 nicotinic ACh receptors produced with β2_α4_β2_α4_β2 or β2_α4_β2_α4_α4 pentameric constructs are valid models of non‐linked (α4)2(β2)3 and (α4)3(β2)2 receptors respectively.

Background and purpose:The epithelial sodium channel (ENaC) is a key regulator of airway mucosal hydration and mucus clearance. Negative regulation of airway ENaC function is predicted to be of clinical benefit in the cystic fibrosis lung. The aim of this study was to develop a small animal model to enable the direct assessment of airway ENaC function in vivo.Experimental approach:Tracheal potential difference (TPD) was utilized as a measure of airway epithelial ion transport in the guinea‐pig. ENaC activity in the trachea was established with a dose–response assessment to a panel of well‐characterized direct and indirect pharmacological modulators of ENaC function, delivered by intra‐tracheal (i.t.) instillation.Key results:The TPD in anaesthetized guinea‐pigs was attenuated by the direct ENaC blockers: amiloride, benzamil and CF552 with ED50 values of 16, 14 and 0.2 μg kg−1 (i.t.), respectively. 5‐(N‐Ethyl‐N‐isopropyl) amiloride, a structurally related compound but devoid of activity on ENaC, was without effect on the TPD. Intra‐tracheal dosing of the Kunitz‐type serine protease inhibitors aprotinin and placental bikunin, which have previously been demonstrated to inhibit proteolytic activation of ENaC, likewise potently attenuated TPD in guinea‐pigs, whereas α1‐antitrypsin and soya bean trypsin inhibitor were without effect.Conclusions and implications:The pharmacological sensitivity of the TPD to amiloride analogues and also to serine protease inhibitors are both consistent with that of ENaC activity in the guinea‐pig trachea. The guinea‐pig TPD therefore represents a suitable in vivo model of human airway epithelial ion transport.British Journal of Pharmacology (2008) 155, 1025–1033; doi:10.1038/bjp.2008.363; published online 22 September 2008

Inherited defects in glycine receptors lead to hyperekplexia, or startle disease. A mutant mouse, spasmodic, that has a startle phenotype, has a point mutation (A52S) in the glycine receptor α1 subunit. This mutation reduces the sensitivity of the receptor to glycine, but the mechanism by which this occurs is not known. We investigated the properties of A52S recombinant receptors by cell‐attached patch‐clamp recording of single‐channel currents elicited by 30–10000 μm glycine. We used heteromeric receptors, which resemble those found at adult inhibitory synapses. Activation mechanisms were fitted directly to single channel data using the HJCFIT method, which includes an exact correction for missed events. In common with wild‐type receptors, only mechanisms with three binding sites and extra shut states could describe the observations. The most physically plausible of these, the ‘flip’ mechanism, suggests that preopening isomerization to the flipped conformation that follows binding is less favoured in mutant than in wild‐type receptors, and, especially, that the flipped conformation has a 100‐fold lower affinity for glycine than in wild‐type receptors. In contrast, the efficacy of the gating reaction was similar to that of wild‐type heteromeric receptors. The reduction in affinity for the flipped conformation accounts for the reduction in apparent cooperativity seen in the mutant receptor (without having to postulate interaction between the binding sites) and it accounts for the increased EC50 for responses to glycine that is seen in mutant receptors. This mechanism also predicts accurately the faster decay of synaptic currents that is observed in spasmodic mice.

The beta3 neuronal nicotinic subunit is localized in dopaminergic areas of the central nervous system, in which many other neuronal nicotinic subunits are expressed. So far, beta3 has only been shown to form functional receptors when expressed together with the alpha3 and beta4 subunits. We have systematically tested in Xenopus laevis oocytes the effects of coexpressing human beta3 with every pairwise functional combination of neuronal nicotinic subunits likely to be relevant to the central nervous system. Expression of alpha7 homomers or alpha/beta pairs (alpha2, alpha3, alpha4, or alpha6 together with beta2 or beta4) produced robust nicotinic currents for all combinations, save alpha6beta2 and alpha6beta4. Coexpression of wild-type beta3 led to a nearly complete loss of function (measured as maximum current response to acetylcholine) for alpha7 and for all functional alpha/beta pairs except for alpha3beta4. This effect was also seen in hippocampal neurons in culture, which lost their robust alpha7-like responses when transfected with beta3. The level of surface expression of nicotinic binding sites (alpha3beta4, alpha4beta2, and alpha7) in tsA201 cells was only marginally affected by beta3 expression. Furthermore, the dominant-negative effect of beta3 was abolished by a valine-serine mutation in the 9' position of the second transmembrane domain of beta3, a mutation believed to facilitate channel gating. Our results show that incorporation of beta3 into neuronal nicotinic receptors other than alpha3beta4 has a powerful dominant-negative effect, probably due to impairment in gating. This raises the possibility of a novel regulatory role for the beta3 subunit on neuronal nicotinic signaling in the central nervous system.

Much of our understanding of ligand-gated ion channels comes from heterologous expression studies. However, this technique cannot produce receptors with a predetermined subunit composition for channels formed by several different subunits and cannot insert a single mutation copy if the subunit of interest is present in several copies in the channel. Here, we describe a novel approach that overcomes these problems by expressing pentameric constructs, in which the code of the five subunits is linked (i.e., β4_β4_α3_β4_α3). This is the first time that a concatemer of the complete pentameric receptor has been expressed for channels in the cysteine-loop superfamily. The presence of the linker did not change the agonist or antagonist sensitivity of α3β4 nicotinic receptors. We show evidence that the expressed receptors were made up of α3 and β4 subunits in one pentameric fusion protein as designed in the construct. This approach can be applied to any nicotinic superfamily receptor to produce channels with a defined subunit arrangement and to introduce specific mutations at any desired location of the pentameric fusion protein.