BCRP/ABCG2 Transporter Regulates Accumulation of Cadmium in Kidney Cells: Role of the Q141K Variant in Modulating Nephrotoxicity.

Exposure to the environmental pollutant cadmium is ubiquitous, as it is present in cigarette smoke and the food supply. Over time, cadmium enters and accumulates in the kidneys, where it causes tubular injury. The breast cancer resistance protein (BCRP, ATP-Binding Cassette G2 ABCG2) is an efflux transporter that mediates the urinary secretion of pharmaceuticals and toxins. The ABCG2 genetic variant Q141K exhibits altered membrane trafficking that results in reduced efflux of BCRP substrates. Here, we sought to 1) evaluate the in vitro and in vivo ability of BCRP to transport cadmium and protect kidney cells from toxicity and 2) determine whether this protection is impaired by the Q141K variant. Cadmium concentrations, cellular stress, and toxicity were quantified in human embryonic kidney 293 cells expressing an empty vector (EV), BCRP wild-type (WT), or variant (Q141K) gene. Treatment with CdCl2 resulted in greater accumulation of cadmium and apoptosis in EV cells relative to WT cells. Exposure to CdCl2 induced expression of stress-related genes and proteins including MT-1A/MT-2A, NAD(P)H quinone dehydrogenase 1, and heme oxygenase-1 to a higher extent in EV cells compared with WT cells. Notably, the Q141K variant protected against CdCl2-induced activation of stress genes and cytotoxicity, but this protection was to a lesser magnitude than observed with WT BCRP. Lastly, concentrations of cadmium in the kidneys of Bcrp knockout mice were 40% higher than in WT mice, confirming that cadmium is an in vivo substrate of BCRP. In conclusion, BCRP prevents the accumulation of cadmium and protects against toxicity, a response that is impaired by the Q141K variant. SIGNIFICANCE STATEMENT: The breast cancer resistance protein transporter lowers cellular accumulation of the toxic heavy metal cadmium. This protective function is partially attenuated by the Q141K genetic variant in the ABCG2 gene.

Cholesterol efflux pathways, inflammation, and atherosclerosis.

Plasma levels of high-density lipoprotein (HDL) inversely correlate with the incidence of cardiovascular diseases (CVD). The causal relationship between plasma HDL-cholesterol levels and CVD has been called into question by Mendelian randomization studies and the majority of clinical trials not showing any benefit of plasma HDL-cholesterol raising drugs on CVD. Nonetheless, recent Mendelian randomization studies including an increased number of CVD cases compared to earlier studies have confirmed that HDL-cholesterol levels and CVD are causally linked. Moreover, several studies in large population cohorts have shown that the cholesterol efflux capacity of HDL inversely correlates with CVD. Cholesterol efflux pathways exert anti-inflammatory and anti-atherogenic effects by suppressing proliferation of hematopoietic stem and progenitor cells, and inflammation and inflammasome activation in macrophages. Cholesterol efflux pathways also suppress the accumulation of cholesteryl esters in macrophages, i.e. macrophage foam cell formation. Recent single-cell RNASeq studies on atherosclerotic plaques have suggested that macrophage foam cells have lower expression of inflammatory genes than non-foam cells, probably reflecting liver X receptor activation, upregulation of ATP Binding Cassette A1 and G1 cholesterol transporters and suppression of inflammation. However, when these pathways are defective lesional foam cells may become pro-inflammatory.

A Decade's Review of miRNA: A Center of Transcriptional Regulation of Drugmetabolizing Enzymes and Transporters under Hypoxia.

BACKGROUND: Hypoxia has a negative effect on the cardiovascular system, nervous system, and metabolism, which contributes to potential changes in drug absorption, distribution, metabolism, and excretion (ADME). However, hypoxia can also alter the expression of microRNA (miRNA), thereby regulating drug-metabolizing enzymes, transporters, and ADME genes, such as hypoxia-inducible factor, inflammatory cytokine, nuclear receptor, etc. Therefore, it is crucial to study the role of miRNA in the regulation of drug-metabolizing enzymes and transporters under hypoxia. METHODS: A systematic review of published studies was carried out to investigate the role of miRNA in the regulation of drug-metabolizing enzymes and transporters under hypoxia. Data and information on expression changes in miRNA, drug-metabolizing enzymes, and transporters under hypoxia were analyzed and summarized. RESULTS: Hypoxia can up or down-regulate the expression of miRNA. The effect of hypoxia on Cytochrome P450 (CYP450) is still a subject of debate. The widespread belief is that hypoxia decreased the activity and expression of CYP1A1, CYP1A2, CYP2E1, and CYP3A1 and increased those of CYP3A6 and CYP2D1 in rats. Hypoxia increased the expression of a multidrug resistance-associated protein, breast cancer resistance protein, peptide transporter, organic cation transporter, and organic anion transporter. miRNA negatively regulated the expression of drugmetabolizing enzymes and transporters. CONCLUSION: The findings of this review indicated that miRNA plays a key role in the expression changes of drugmetabolizing enzymes and transporters under hypoxia.

Opportunities and Challenges in Tunneling Nanotubes Research: How Far from Clinical Application?

Tunneling nanotubes (TNTs) are recognized long membrane nanotubes connecting distance cells. In the last decade, growing evidence has shown that these subcellular structures mediate the specific transfer of cellular materials, pathogens, and electrical signals between cells. As intercellular bridges, they play a unique role in embryonic development, collective cell migration, injured cell recovery, cancer treatment resistance, and pathogen propagation. Although TNTs have been considered as potential drug targets for treatment, there is still a long way to go to translate the research findings into clinical practice. Herein, we emphasize the heterogeneous nature of TNTs by systemically summarizing the current knowledge on their morphology, structure, and biogenesis in different types of cells. Furthermore, we address the communication efficiency and biological outcomes of TNT-dependent transport related to diseases. Finally, we discuss the opportunities and challenges of TNTs as an exciting therapeutic approach by focusing on the development of efficient and safe drugs targeting TNTs.

ER-to-Golgi transport and SEC23-dependent COPII vesicles regulate T cell alloimmunity.

T cell-mediated responses are dependent on their secretion of key effector molecules. However, the critical molecular determinants of the secretion of these proteins are largely undefined. Here, we demonstrate that T cell activation increases trafficking via the ER-to-Golgi pathway. To study the functional role of this pathway, we generated mice with a T cell-specific deletion in SEC23B, a core subunit of coat protein complex II (COPII). We found that SEC23B critically regulated the T cell secretome following activation. SEC23B-deficient T cells exhibited a proliferative defect and reduced effector functions in vitro, as well as in experimental models of allogeneic and xenogeneic hematopoietic cell transplantation in vivo. However, T cells derived from 3 patients with congenital dyserythropoietic anemia II (CDAII), which results from Sec23b mutation, did not exhibit a similar phenotype. Mechanistic studies demonstrated that unlike murine KO T cells, T cells from patients with CDAII harbor increased levels of the closely related paralog, SEC23A. In vivo rescue of murine KO by expression of Sec23a from the Sec23b genomic locus restored T cell functions. Together, our data demonstrate a critical role for the COPII pathway, with evidence for functional overlap in vivo between SEC23 paralogs in the regulation of T cell immunity in both mice and humans.

A Splice Variant in SLC16A8 Gene Leads to Lactate Transport Deficit in Human iPS Cell-Derived Retinal Pigment Epithelial Cells.

Age-related macular degeneration (AMD) is a blinding disease for which most of the patients remain untreatable. Since the disease affects the macula at the center of the retina, a structure specific to the primate lineage, rodent models to study the pathophysiology of AMD and to develop therapies are very limited. Consequently, our understanding relies mostly on genetic studies highlighting risk alleles at many loci. We are studying the possible implication of a metabolic imbalance associated with risk alleles within the SLC16A8 gene that encodes for a retinal pigment epithelium (RPE)-specific lactate transporter MCT3 and its consequences for vision. As a first approach, we report here the deficit in transepithelial lactate transport of a rare SLC16A8 allele identified during a genome-wide association study. We produced induced pluripotent stem cells (iPSCs) from the unique patient in our cohort that carries two copies of this allele. After in vitro differentiation of the iPSCs into RPE cells and their characterization, we demonstrate that the rare allele results in the retention of intron 2 of the SLC16A8 gene leading to the absence of MCT3 protein. We show using a biochemical assay that these cells have a deficit in transepithelial lactate transport.

Naphthylphthalamic acid associates with and inhibits PIN auxin transporters.

N-1-naphthylphthalamic acid (NPA) is a key inhibitor of directional (polar) transport of the hormone auxin in plants. For decades, it has been a pivotal tool in elucidating the unique polar auxin transport-based processes underlying plant growth and development. Its exact mode of action has long been sought after and is still being debated, with prevailing mechanistic schemes describing only indirect connections between NPA and the main transporters responsible for directional transport, namely PIN auxin exporters. Here we present data supporting a model in which NPA associates with PINs in a more direct manner than hitherto postulated. We show that NPA inhibits PIN activity in a heterologous oocyte system and that expression of NPA-sensitive PINs in plant, yeast, and oocyte membranes leads to specific saturable NPA binding. We thus propose that PINs are a bona fide NPA target. This offers a straightforward molecular basis for NPA inhibition of PIN-dependent auxin transport and a logical parsimonious explanation for the known physiological effects of NPA on plant growth, as well as an alternative hypothesis to interpret past and future results. We also introduce PIN dimerization and describe an effect of NPA on this, suggesting that NPA binding could be exploited to gain insights into structural aspects of PINs related to their transport mechanism.

SLC7A11 as a biomarker and therapeutic target in HPV-positive head and neck Squamous Cell Carcinoma.

Ferroptosis, a regulated form of cell necrosis was previously reported to be induced upon pharmacological targeting of the cystine transporter SLC7A11 in Head and neck Squamous Cell Carcinoma (HNSCC). Whether tumors arising in a context of chronic infection with Human Papillomavirus (HPV) are sensitive to ferroptosis is unknown. Using RNAseq data (both whole-tumor and single-cell sequencing) we report that HPV positive (HPV+ve) tumors have lower expression levels of SLC7A11 compared to HPV negative (HPV-ve) HNSCC. We examined in vitro the effect of erastin, a specific blocker of SLC7A11, applied on two HNSCC cell lines with stable expression of HPV16 E6 and E7 oncoproteins. We report a decrease in total GSH levels and an increased sensitivity to erastin-induced ferroptosis in E6-E7 cells. Cell sensitivity to ferroptosis was specificaly related to a defect in cystine transport since we found no difference in ferroptosis induced by the direct inhibition of GPX4, and N-Acetyl Cystein abolished the difference between WT and E6-E7-expressing cells. Our findings point to SLC7A11 as an HPV-related biomarker of potential therapeutic relevance in HNSCC. Targeting cystine import to promote ferroptosis might be a promising strategy against HPV+ve HNSCC. (188 words).

Human ABCB1 with an ABCB11-like degenerate nucleotide binding site maintains transport activity by avoiding nucleotide occlusion.

Several ABC exporters carry a degenerate nucleotide binding site (NBS) that is unable to hydrolyze ATP at a rate sufficient for sustaining transport activity. A hallmark of a degenerate NBS is the lack of the catalytic glutamate in the Walker B motif in the nucleotide binding domain (NBD). The multidrug resistance transporter ABCB1 (P-glycoprotein) has two canonical NBSs, and mutation of the catalytic glutamate E556 in NBS1 renders ABCB1 transport-incompetent. In contrast, the closely related bile salt export pump ABCB11 (BSEP), which shares 49% sequence identity with ABCB1, naturally contains a methionine in place of the catalytic glutamate. The NBD-NBD interfaces of ABCB1 and ABCB11 differ only in four residues, all within NBS1. Mutation of the catalytic glutamate in ABCB1 results in the occlusion of ATP in NBS1, leading to the arrest of the transport cycle. Here we show that despite the catalytic glutamate mutation (E556M), ABCB1 regains its ATP-dependent transport activity, when three additional diverging residues are also replaced. Molecular dynamics simulations revealed that the rescue of ATPase activity is due to the modified geometry of NBS1, resulting in a weaker interaction with ATP, which allows the quadruple mutant to evade the conformationally locked pre-hydrolytic state to proceed to ATP-driven transport. In summary, we show that ABCB1 can be transformed into an active transporter with only one functional catalytic site by preventing the formation of the ATP-locked pre-hydrolytic state in the non-canonical site.

Mitochondrial pyruvate and fatty acid flux modulate MICU1-dependent control of MCU activity.

The tricarboxylic acid (TCA) cycle converts the end products of glycolysis and fatty acid ß-oxidation into the reducing equivalents NADH and FADH2 Although mitochondrial matrix uptake of Ca2+ enhances ATP production, it remains unclear whether deprivation of mitochondrial TCA substrates alters mitochondrial Ca2+ flux. We investigated the effect of TCA cycle substrates on MCU-mediated mitochondrial matrix uptake of Ca2+, mitochondrial bioenergetics, and autophagic flux. Inhibition of glycolysis, mitochondrial pyruvate transport, or mitochondrial fatty acid transport triggered expression of the MCU gatekeeper MICU1 but not the MCU core subunit. Knockdown of mitochondrial pyruvate carrier (MPC) isoforms or expression of the dominant negative mutant MPC1R97W resulted in increased MICU1 protein abundance and inhibition of MCU-mediated mitochondrial matrix uptake of Ca2+ We also found that genetic ablation of MPC1 in hepatocytes and mouse embryonic fibroblasts resulted in reduced resting matrix Ca2+, likely because of increased MICU1 expression, but resulted in changes in mitochondrial morphology. TCA cycle substrate-dependent MICU1 expression was mediated by the transcription factor early growth response 1 (EGR1). Blocking mitochondrial pyruvate or fatty acid flux was linked to increased autophagy marker abundance. These studies reveal a mechanism that controls the MCU-mediated Ca2+ flux machinery and that depends on TCA cycle substrate availability. This mechanism generates a metabolic homeostatic circuit that protects cells from bioenergetic crisis and mitochondrial Ca2+ overload during periods of nutrient stress.

The Lipid Raft Component Stomatin Interacts with the Na+ Taurocholate Cotransporting Polypeptide (NTCP) and Modulates Bile Salt Uptake.

The sodium taurocholate cotransporting polypeptide (NTCP) is expressed at the basolateral membrane of hepatocytes, where it mediates the uptake of conjugated bile acids and forms the hepatocyte entry receptor for the hepatitis B and D virus. Here, we aimed to identify novel protein-protein interactions that could play a role in the regulation of NTCP. To this end, NTCP was precipitated from HA-tagged hNTCP-expressing HepG2 cells, and chloride channel CLIC-like 1 (CLCC1) and stomatin were identified as interacting proteins by mass spectrometry. Interaction was confirmed by co-immunoprecipitation. NTCP, CLCC1 and stomatin were found at the plasma membrane in lipid rafts, as demonstrated by a combination of immunofluorescence, cell surface biotinylation and isolation of detergent-resistant membranes. Neither CLCC1 overexpression nor its knockdown had an effect on NTCP function. However, both stomatin overexpression and knockdown increased NTCP-mediated taurocholate uptake while NTCP abundance at the plasma membrane was only increased in stomatin depleted cells. These findings identify stomatin as an interactor of NTCP and show that the interaction modulates bile salt transport.

Ubqln4 Facilitates Endoplasmic Reticulum-to-Cytosol Escape of a Nonenveloped Virus during Infection.

The nonenveloped polyomavirus simian virus 40 (SV40) must penetrate the host endoplasmic reticulum (ER) membrane to enter the cytosol in order to promote infection. How this is accomplished is not entirely clear. Here, we demonstrate that the cytosolic chaperone Ubiquilin4 (Ubqln4) binds directly to the ER membrane J proteins B12 and B14. Strategically localized at the ER-cytosol interface, Ubqln4 captures SV40 emerging from the ER, thereby facilitating escape of the virus from the ER into the cytosol, which leads to infection. Strikingly, Ubqln4 engages the J proteins in a J-domain-independent manner, in contrast to the previously reported Hsc70-Hsp105-SGTA-Bag2 cytosolic complex that also mediates SV40 ER-to-cytosol transport. Our results also reveal that the H domain and STI1 motif (1-2) of Ubqln4 support J protein binding, essential for SV40 infection. Together, these data further clarify the molecular basis by which a nonenveloped virus escapes a host membrane during infectious entry.IMPORTANCE How a nonenveloped virus escapes from a host membrane to promote infection remains enigmatic. In the case of the nonenveloped polyomavirus SV40, penetration of the ER membrane to reach the cytosol is a decisive virus infection step. In this study, we found a new host factor called Ubqln4 that facilitates escape of SV40 from the ER into the cytosol, thereby providing a path for the virus to enter the nucleus to cause infection.

Trans-endocytosis elicited by nectins transfers cytoplasmic cargo, including infectious material, between cells.

Here, we show that cells expressing the adherens junction protein nectin-1 capture nectin-4-containing membranes from the surface of adjacent cells in a trans-endocytosis process. We find that internalized nectin-1-nectin-4 complexes follow the endocytic pathway. The nectin-1 cytoplasmic tail controls transfer: its deletion prevents trans-endocytosis, while its exchange with the nectin-4 tail reverses transfer direction. Nectin-1-expressing cells acquire dye-labeled cytoplasmic proteins synchronously with nectin-4, a process most active during cell adhesion. Some cytoplasmic cargo remains functional after transfer, as demonstrated with encapsidated genomes of measles virus (MeV). This virus uses nectin-4, but not nectin-1, as a receptor. Epithelial cells expressing nectin-4, but not those expressing another MeV receptor in its place, can transfer infection to nectin-1-expressing primary neurons. Thus, this newly discovered process can move cytoplasmic cargo, including infectious material, from epithelial cells to neurons. We name the process nectin-elicited cytoplasm transfer (NECT). NECT-related trans-endocytosis processes may be exploited by pathogens to extend tropism. This article has an associated First Person interview with the first author of the paper.

Identification of Novel Mutation in CNGA3 gene by Whole-Exome Sequencing and In-Silico Analyses for Genotype-Phenotype Assessment with Autosomal Recessive Achromatopsia in Pakistani families.

OBJECTIVE: To identify the underlying genetic anomalies in two consanguineous Pakistani families with autosomal recessive achromatopsia. METHODS: The exploratory study was conducted under the patronage of International Islamic University, Islamabad, Pakistan, and Sungshin Women University, Seoul, South Korea, after two families coded PKCN-02 and PKCN-07 belonging to different ethnic groups were recruited from different areas of Khyber Pakhtunkhawa province of Pakistan in July 2016. The families were originally diagnosed with nystagmus upon medical examination. Exome sequencing was performed to identify the possible causative gene which was found to be cyclic nucleotide-gated channel alpha-3. Sanger sequencing was performed to confirm the mutations. After genetic analysis, clinical analysis was re-evaluated for colour vision using Ishihara 26 plates. Pathogenic potential of these mutations was evaluated using algorithmic mutation prediction tools. In-silico analysis was performed to predict effect of these mutations on protein structure of the gene in question. RESULTS: Exome sequencing revealed a reported missense mutation c .1306C>T (p.R436W) in family PKCN-02 and a novel missense mutation c.1540G>A (p.D514N) in family PKCN-07. After mutational analysis, clinical re-evaluation revealed that both families were segregating autosomal recessive achromatopsia. Further, the topological model of the cyclic nucleotide-gated channel alpha-3 polypeptide describes these missense mutations primarily affecting the C-linker and cyclic guanosine monophosphate-binding sites, respectively. Protein structure modelling of cyclic nucleotide-gated channel alpha-3 protein revealed abnormal structure produced by p.R436W and p.D514N.. CONCLUSIONS: Exome sequencing approach was used to first identify the genetic alteration in families with nystagmus. Two mutations in cyclic nucleotide-gated channel alpha-3gene were uncovered, including one novel mutation. Clinical re-evaluation uncovered that both families had achromatopsia.

Involvement of ARHGEF10, GEF for RhoA, in Rab6/Rab8-mediating membrane traffic.

Small GTPases play crucial roles in the maintenance of a homeostatic environment and appropriate movements of the cell. In these processes, the direct or indirect interaction between distinct small GTPases could be required for regulating mutual signaling pathways. In our recent study, ARHGEF10, known as a guanine nucleotide exchange factor (GEF) for RhoA, was indicated to interact with Rab6A and Rab8A, which are known to function in the exocytotic pathway, and colocalized with these Rabs at exocytotic vesicles. Moreover, it was suggested that ARHGEF10 is involved in the regulation of Rab6A and Rab8A localization and invasion of breast carcinoma cells, in which Rab8 also acts via regulation of membrane trafficking. These results may reveal the existence of a novel small GTPase cascade which connects the signaling of these Rabs with RhoA during membrane trafficking. In this mini-review, we consider the possible functions of ARHGEF10 and RhoA in the Rab6- and Rab8-mediated membrane trafficking pathway.

A common pathomechanism in GMAP-210- and LBR-related diseases.

Biallelic loss-of-function mutations in TRIP11, encoding the golgin GMAP-210, cause the lethal human chondrodysplasia achondrogenesis 1A (ACG1A). We now find that a homozygous splice-site mutation of the lamin B receptor (LBR) gene results in the same phenotype. Intrigued by the genetic heterogeneity, we compared GMAP-210- and LBR-deficient primary cells to unravel how particular mutations in LBR cause a phenocopy of ACG1A. We could exclude a regulatory interaction between LBR and GMAP-210 in patients' cells. However, we discovered a common disruption of Golgi apparatus architecture that was accompanied by decreased secretory trafficking in both cases. Deficiency of Golgi-dependent glycan processing indicated a similar downstream effect of the disease-causing mutations upon Golgi function. Unexpectedly, our results thus point to a common pathogenic mechanism in GMAP-210- and LBR-related diseases attributable to defective secretory trafficking at the Golgi apparatus.

Excessive tubulin polyglutamylation causes neurodegeneration and perturbs neuronal transport.

Posttranslational modifications of tubulin are emerging regulators of microtubule functions. We have shown earlier that upregulated polyglutamylation is linked to rapid degeneration of Purkinje cells in mice with a mutation in the deglutamylating enzyme CCP1. How polyglutamylation leads to degeneration, whether it affects multiple neuron types, or which physiological processes it regulates in healthy neurons has remained unknown. Here, we demonstrate that excessive polyglutamylation induces neurodegeneration in a cell-autonomous manner and can occur in many parts of the central nervous system. Degeneration of selected neurons in CCP1-deficient mice can be fully rescued by simultaneous knockout of the counteracting polyglutamylase TTLL1. Excessive polyglutamylation reduces the efficiency of neuronal transport in cultured hippocampal neurons, suggesting that impaired cargo transport plays an important role in the observed degenerative phenotypes. We thus establish polyglutamylation as a cell-autonomous mechanism for neurodegeneration that might be therapeutically accessible through manipulation of the enzymes that control this posttranslational modification.

cTAGE5/MEA6 plays a critical role in neuronal cellular components trafficking and brain development.

Normal neural development is essential for the formation of neuronal networks and brain function. Cutaneous T cell lymphoma-associated antigen 5 (cTAGE5)/meningioma expressed antigen 6 (MEA6) plays a critical role in the secretion of proteins. However, its roles in the transport of nonsecretory cellular components and in brain development remain unknown. Here, we show that cTAGE5/MEA6 is important for brain development and function. Conditional knockout of cTAGE5/MEA6 in the brain leads to severe defects in neural development, including deficits in dendrite outgrowth and branching, spine formation and maintenance, astrocyte activation, and abnormal behaviors. We reveal that loss of cTAGE5/MEA6 affects the interaction between the coat protein complex II (COPII) components, SAR1 and SEC23, leading to persistent activation of SAR1 and defects in COPII vesicle formation and transport from the endoplasmic reticulum to the Golgi, as well as disturbed trafficking of membrane components in neurons. These defects affect not only the transport of materials required for the development of dendrites and spines but also the signaling pathways required for neuronal development. Because mutations in cTAGE5/MEA6 have been found in patients with Fahr's disease, our study potentially also provides insight into the pathogenesis of this disorder.

Evidence for the critical role of transmembrane helices 1 and 7 in substrate transport by human P-glycoprotein (ABCB1).

P-glycoprotein (P-gp) is an ABC transporter that exports many amphipathic or hydrophobic compounds, including chemically and functionally dissimilar anticancer drugs, from cells. To understand the role of transmembrane helices (TMH) 1 and 7 in drug-binding and transport, we selected six residues from both TMH1 (V53, I59, I60, L65, M68 and F72) and TMH7 (V713, I719, I720, Q725, F728 and F732); and substituted them with alanine by gene synthesis to generate a variant termed "TMH1,7 mutant P-gp". The expression and function of TMH1,7 mutant P-gp with twelve mutations was characterized using the BacMam baculovirus-HeLa cell expression system. The expression and conformation of TMH1,7 mutant P-gp was not altered by the introduction of the twelve mutations, as confirmed by using the human P-gp-specific antibodies UIC2, MRK16 and 4E3. We tested 25 fluorescently-labeled substrates and found that only three substrates, NBD-cyclosporine A, Rhod-2-AM and X-Rhod-1-AM were transported by the TMH1,7 mutant. The basal ATPase activity of TMH1,7 mutant P-gp was lower (40-50%) compared to wild-type (WT) P-gp, despite similar level of expression. Although most of the substrates modulate ATPase activity of P-gp, the activity of TMH1,7 mutant transporter was not significantly modulated by any of the tested substrates. Docking of selected substrates in homology models showed comparable docking scores for the TMH1,7 mutant and WT P-gp, although the binding conformations were different. Both the ATPase assay and in silico docking analyses suggest that the interactions with residues in the drug-binding pocket are altered as a consequence of the mutations. We demonstrate that it is possible to generate a variant of P-gp with a loss of broad substrate specificity and propose that TMH1 and TMH7 play a critical role in the drug efflux function of this multidrug transporter.

Ivacaftor-induced sweat chloride reductions correlate with increases in airway surface liquid pH in cystic fibrosis.

BACKGROUND: Disruption of cystic fibrosis transmembrane conductance regulator (CFTR) anion channel function causes cystic fibrosis (CF), and lung disease produces most of the mortality. Loss of CFTR-mediated HCO3- secretion reduces the pH of airway surface liquid (ASL) in vitro and in neonatal humans and pigs in vivo. However, we previously found that, in older children and adults, ASL pH does not differ between CF and non-CF. Here, we tested whether the pH of CF ASL increases with time after birth. Finding that it did suggested that adaptations by CF airways increase ASL pH. This conjecture predicted that increasing CFTR activity in CF airways would further increase ASL pH and also that increasing CFTR activity would correlate with increases in ASL pH. METHODS: To test for longitudinal changes, we measured ASL pH in newborns and then at 3-month intervals. We also studied people with CF (bearing G551D or R117H mutations), in whom we could acutely stimulate CFTR activity with ivacaftor. To gauge changes in CFTR activity, we measured changes in sweat Cl- concentration immediately before and 48 hours after starting ivacaftor. RESULTS: Compared with that in the newborn period, ASL pH increased by 6 months of age. In people with CF bearing G551D or R117H mutations, ivacaftor did not change the average ASL pH; however reductions in sweat Cl- concentration correlated with elevations of ASL pH. Reductions in sweat Cl- concentration also correlated with improvements in pulmonary function. CONCLUSIONS: Our results suggest that CFTR-independent mechanisms increase ASL pH in people with CF. We speculate that CF airway disease, which begins soon after birth, is responsible for the adaptation. FUNDING: Vertex Inc., the NIH (P30DK089507, 1K08HL135433, HL091842, HL136813, K24HL102246), the Cystic Fibrosis Foundation (SINGH17A0 and SINGH15R0), and the Burroughs Wellcome Fund.