Expression of Drosophila melanogaster P-glycoproteins is associated with ATP channel activity

1996 ◽  
Vol 271 (5) ◽  
pp. C1527-C1538 ◽  
Author(s):  
I. Bosch ◽  
G. R. Jackson ◽  
J. M. Croop ◽  
H. F. Cantiello
Keyword(s):  
Drosophila Melanogaster ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Transmembrane Conductance ◽  
Apparent Permeability ◽  
Transporter Family ◽  
Pathway Expression ◽  
Endogenous Substrate ◽  
Atp Binding Cassettes ◽  
Cystic Fibrosis Transmembrane

Two distinct Drosophila melanogaster P-glycoprotein (Pgp) gene homologues of different chromosomal origin, MDR49 and MDR65, have been previously identified (38). Most Pgps are implicated in the development of the multidrug-resistance phenotype. Despite intense efforts to identify the molecular mechanism(s) associated with Pgp function, the endogenous substrate(s) of these transport molecules is largely unknown. Recent studies from our laboratory indicate that a murine Pgp homologue (E. H. Abraham, A. G. Prat, L. Gerweck, T. Seneveratne, R. J. Arceci, R. Kramer, G. Guidotti, and H. F. Cantiello. Proc. Natl. Acad. Sci. USA 90: 312-316, 1993) and a related protein, the cystic fibrosis transmembrane conductance regulator (CFTR; I. L. Reisin, A. Prat, E. H. Abraham, J. F. Amara, R. J. Gregory, D. A. Ausiello, and H. F. Cantiello. J. Biol. Chem. 269: 20584-20591, 1994), are novel ATP-permeable ion channels. The common feature of these two proteins is the conserved ATP-binding cassettes (ABC); thus molecules structurally linked to the ABC transporter family may be also functionally associated with ATP channel activity. In this study, MDR65 and MDR49 Pgps were functionally expressed in Sf9 cells, and patch-clamp techniques were applied to assess the role of these proteins in the electrodiffusional movement of ATP. In the presence of intracellular ATP and external NaCl, expression of MDR65 was associated with a linear electrodiffusional pathway that was permeable to both ATP and Cl-. Under symmetrical ATP conditions, only voltage depolarization activated a MDR65-mediated ATP-conductive pathway. Expression of MDR49 was also associated with a voltage-activated ATP conductance in symmetrical ATP, but no apparent permeability to either Cl- or ATP was observed under asymmetrical conditions. The different functional properties of MDR65 and MDR49 may be indicative of distinct physiological roles in this organism. The study indicates, however, that the two Drosophila Pgp homologues share strong functional similarities with their mammalian relatives Pgp and CFTR.

scholarly journals Carbon monoxide-releasing molecules inhibit the cystic fibrosis transmembrane conductance regulator Cl− channel

2020 ◽  
Vol 319 (6) ◽  
pp. L997-L1009
Author(s):  
Mayuree Rodrat ◽  
Walailak Jantarajit ◽  
Demi R. S. Ng ◽  
Bartholomew S. J. Harvey ◽  
Jia Liu ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Carbon Monoxide ◽  
Current Flow ◽  
Open Channels ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Flow Through ◽  
Cystic Fibrosis Transmembrane

The gasotransmitter carbon monoxide (CO) regulates fluid and electrolyte movements across epithelial tissues. However, its action on anion channels is incompletely understood. Here, we investigate the direct action of CO on the cystic fibrosis transmembrane conductance regulator (CFTR) by applying CO-releasing molecules (CO-RMs) to the intracellular side of excised inside-out membrane patches from cells heterologously expressing wild-type human CFTR. Addition of increasing concentrations of tricarbonyldichlororuthenium(II) dimer (CORM-2) (1–300 μM) inhibited CFTR channel activity, whereas the control RuCl3 (100 μM) was without effect. CORM-2 predominantly inhibited CFTR by decreasing the frequency of channel openings and, hence, open probability ( Po). But, it also reduced current flow through open channels with very fast kinetics, particularly at elevated concentrations. By contrast, the chemically distinct CO-releasing molecule CORM-3 inhibited CFTR by decreasing Po without altering current flow through open channels. Neither depolarizing the membrane voltage nor raising the ATP concentration on the intracellular side of the membrane affected CFTR inhibition by CORM-2. Interestingly, CFTR inhibition by CORM-2, but not by CFTRinh-172, was prevented by prior enhancement of channel activity by the clinically approved CFTR potentiator ivacaftor. Similarly, when added after CORM-2, ivacaftor completely relieved CFTR inhibition. In conclusion, CORM-2 has complex effects on wild-type human CFTR consistent with allosteric inhibition and open-channel blockade. Inhibition of CFTR by CO-releasing molecules suggests that CO regulates CFTR activity and that the gasotransmitter has tissue-specific effects on epithelial ion transport. The action of ivacaftor on CFTR Cl− channels inhibited by CO potentially expands the drug’s clinical utility.

scholarly journals Ablation of Internalization Signals in the Carboxyl-terminal Tail of the Cystic Fibrosis Transmembrane Conductance Regulator Enhances Cell Surface Expression

2002 ◽  
Vol 277 (51) ◽  
pp. 49952-49957 ◽  
Author(s):  
Krisztina Peter ◽  
Karoly Varga ◽  
Zsuzsa Bebok ◽  
Carmel M. McNicholas-Bevensee ◽  
Lisa Schwiebert ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Surface Expression ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Transmembrane Conductance ◽  
Coated Pits ◽  
Double Mutation ◽  
Carboxyl Terminal ◽  
Cystic Fibrosis Transmembrane

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel that undergoes endocytosis through clathrin-coated pits. Previously, we demonstrated that Y1424A is important for CFTR endocytosis (Prince, L. S., Peter, K., Hatton, S. R., Zaliauskiene, L., Cotlin, L. F., Clancy, J. P., Marchase, R. B., and Collawn, J. F. (1999)J. Biol. Chem.274, 3602–3609). Here we show that a second substitution in the carboxyl-terminal tail of CFTR, I1427A, on Y1424A background more than doubles CFTR surface expression as monitored by surface biotinylation. Internalization assays indicate that enhanced surface expression of Y1424A,I1427A CFTR is caused by a 76% inhibition of endocytosis. Patch clamp recording of chloride channel activity revealed that there was a corresponding increase in chloride channel activity of Y1424A,I1427A CFTR, consistent with the elevated surface expression, and no change in CFTR channel properties. Y14124A showed an intermediate phenotype compared with the double mutation, both in terms of surface expression and chloride channel activity. Metabolic pulse-chase experiments demonstrated that the two mutations did not affect maturation efficiency or protein half-life. Taken together, our data show that there is an internalization signal in the COOH terminus of CFTR that consists of Tyr1424-X-X-Ile1427where both the tyrosine and the isoleucine are essential residues. This signal regulates CFTR surface expression but not CFTR biogenesis, degradation, or chloride channel function.

scholarly journals Cholesterol Interaction Directly Enhances Intrinsic Activity of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR)

Cells ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 804 ◽  
Author(s):  
Chin ◽  
Ramjeesingh ◽  
Hung ◽  
Ereño-Oreba ◽  
Cui ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Catalytic Activity ◽  
Intrinsic Activity ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Transmembrane Conductance ◽  
Activity Measurements ◽  
Conduction Pathway ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane

The recent cryo-electron microscopy structures of zebrafish and the human cystic fibrosis transmembrane conductance regulator (CFTR) provided unprecedented insights into putative mechanisms underlying gating of its anion channel activity. Interestingly, despite predictions based on channel activity measurements in biological membranes, the structure of the detergent purified, phosphorylated, and ATP-bound human CFTR protein did not reveal a stably open conduction pathway. This study tested the hypothesis that the functional properties of the detergent solubilized CFTR protein used for structural determinations are different from those exhibited by CFTR purified under conditions that retain associated lipids native to the membrane. It was found that CFTR purified together with phospholipids and cholesterol using amphipol: A8-35, exhibited higher rates of catalytic activity, phosphorylation dependent channel activation and potentiation by the therapeutic compound, ivacaftor, than did CFTR purified in detergent. The catalytic activity of phosphorylated CFTR detergent micelles was rescued by the addition of phospholipids plus cholesterol, but not by phospholipids alone, arguing for a specific role for cholesterol in modulating this function. In summary, these studies highlight the importance of lipid interactions in the intrinsic activities and pharmacological potentiation of CFTR.

scholarly journals Identifying Inhibitors of the Hsp90-Aha1 Protein Complex, a Potential Target to Drug Cystic Fibrosis, by Alpha Technology

2017 ◽  
Vol 22 (7) ◽  
pp. 923-928 ◽  
Author(s):  
Verena Ihrig ◽  
Wolfgang M. J. Obermann
Keyword(s):  
Cystic Fibrosis ◽  
Chloride Channel ◽  
Chloride Ion ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Transmembrane Conductance ◽  
Culture Cells ◽  
Homogeneous Assay ◽  
Chaperone Complex ◽  
Cystic Fibrosis Transmembrane

Deletion of a single phenylalanine residue at position 508 of the protein CFTR (cystic fibrosis transmembrane conductance regulator), a chloride channel in lung epithelium, is the most common cause for cystic fibrosis. As a consequence, folding of the CFTRΔF508 protein and delivery to the cell surface are compromised, resulting in degradation of the polypeptide. Accordingly, decreased surface presence of CFTRΔF508 causes impaired chloride ion conductivity and is associated with mucus accumulation, a hallmark of cystic fibrosis. Molecular chaperones such as Hsp90 and its co-chaperone partner Aha1 are thought to play a key role in targeting folding-deficient CFTRΔF508 for degradation. Thus, pharmacologic manipulation to inhibit Hsp90-Aha1 chaperone complex formation appears beneficial to inhibit proteolysis of CFTRΔF508 and rescue its residual chloride channel activity. Therefore, we have screened a collection of 14,400 druglike chemical compounds for inhibitors of the Hsp90-Aha1 complex by amplified luminescence proximity homogeneous assay (Alpha). We identified two druglike molecules that showed promising results when we tested their ability to restore chloride channel activity in culture cells expressing the mutant CFTRΔF508 protein. The two molecules were most effective in combination with the corrector VX-809 and may therefore serve as a lead compound that can be further developed into a drug to treat cystic fibrosis patients.

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