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The Glycine Residues G551 and G1349 within the ATP-Binding Cassette Signature Motifs Play Critical Roles in the Activation and Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Channels by Phloxine B

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Abstract

The cystic fibrosis transmembrane conductance regulator (CFTR) protein contains a canonical ATP-binding cassette (ABC) signature motif, LSGGQ, in nucleotide binding domain 1 (NBD1) and a degenerate LSHGH in NBD2. Here, we studied the contribution of the conserved residues G551 and G1349 to the pharmacological modulation of CFTR chloride channels by phloxine B using iodide efflux and whole-cell patch clamp experiments performed on the following green fluorescent protein (GFP)-tagged CFTR: wild-type, delF508, G551D, G1349D, and G551D/G1349D double mutant. We found that phloxine B stimulates and inhibits channel activity of wild-type CFTR (Ks = 3.2 ± 1.6 μM, Ki = 38 ± 1.4 μM) and delF508 CFTR (Ks = 3 ± 1.8 μM, Ki = 33 ± 1 μM). However, CFTR channels with the LSGDQ mutated motif (mutation G551D) are activated (Ks = 2 ± 1.13 μM) but not inhibited by phloxine B. Conversely, CFTR channels with the LSHDH mutated motif (mutation G1349D) are inhibited (Ki = 40 ± 1.01 μM) but not activated by phloxine B. Finally, the double mutant G551D/G1349D CFTR failed to respond not only to phloxine B stimulation but also to phloxine B inhibition, confirming the importance of both amino acid locations. Similar results were obtained with genistein, and kinetic parameters were determined to compare the pharmacological effects of both agents. These data show that G551 and G1349 control the inhibition and activation of CFTR by these agents, suggesting functional nonequivalence of the signature motifs of NBD in the ABC transporter CFTR.

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Acknowledgement

We thank Dr. V. Thoreau, Dr. F. Bilan, Dr. K. H. Karlson and Dr. B. A. Stanton for the pS65T/EGFP-C1/WT-CFTR construct; Marlène Baudis for the delF508-CFTR construct; and N. Bizard for assistance with cell cultures. This work was performed as part of the thesis of P. M. at Ingénierie chimique, Biologique et Géologique (ICBG) thesis school at Poitiers University, supported by a thesis fellowship from the Region Poitou-Charentes. This work was also supported by specific grants from CF-Pronet and Vaincre La Mucoviscidose and an institutional grant from the CNRS and Région Poitou-Charentes.

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Authors and Affiliations

  1. Institut de Physiologie et Biologie Cellulaires, CNRS UMR6187, Université de Poitiers, 40 avenue du recteur Pineau, 86022, Poitiers, France

    Patricia Melin, Caroline Norez, Isabelle Callebaut & Frédéric Becq

  2. Département de biologie structurale, IMPMC, CNRS UMR7590, Université Paris 6 et 7, case 115, 4 place Jussieu, 75252, Paris cedex 05, France

    Frédéric Becq

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  1. Patricia Melin
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  2. Caroline Norez
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  3. Isabelle Callebaut
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  4. Frédéric Becq
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Correspondence to Frédéric Becq.

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Melin, P., Norez, C., Callebaut, I. et al. The Glycine Residues G551 and G1349 within the ATP-Binding Cassette Signature Motifs Play Critical Roles in the Activation and Inhibition of Cystic Fibrosis Transmembrane Conductance Regulator Channels by Phloxine B. J Membrane Biol 208, 203–212 (2006). https://doi.org/10.1007/s00232-005-7001-0

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