Resumen
Binding of cytoplasmic anionic open channel blockers within the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is antagonized by extracellular Cl-. In the present work, patch clamp recording was used to investigate the interaction between extracellular Cl- (and other anions) and cytoplasmic Pt(NO2)42 - ions inside the CFTR channel pore. In constitutively open (E1371Q-CFTR) channels, these different anions bind to two separate sites, located in the outer and inner vestibules of the pore respectively, in a mutually antagonistic fashion. A mutation in the inner vestibule (I344K) that greatly increased Pt(NO2)42 - binding affinity also greatly strengthened antagonistic Cl-:blocker interactions as well as the voltage-dependence of block. Quantitative analysis of ion binding affinity suggested that the I344K mutation strengthened interactions not only with intracellular Pt(NO2)42 - ions but also with extracellular Cl-, and that altered blocker Cl-- and voltage-dependence were due to the introduction of a novel type of antagonistic ion:ion interaction inside the pore that was independent of Cl- binding in the outer vestibule. It is proposed that this mutation alters the arrangement of anion binding sites inside the pore, allowing both Cl- and Pt(NO2)42 - to bind concurrently within the inner vestibule in a strongly mutually antagonistic fashion. However, the I344K mutation does not increase single channel conductance following disruption of Cl- binding in the outer vestibule in R334Q channels. Implications for the arrangement of ion binding sites in the pore, and their functional consequences for blocker binding and for rapid Cl- permeation, are discussed.
Idioma original | English |
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Páginas (desde-hasta) | 1573-1590 |
Número de páginas | 18 |
Publicación | Biochimica et Biophysica Acta - Biomembranes |
Volumen | 1848 |
N.º | 7 |
DOI | |
Estado | Published - jul. 1 2015 |
Nota bibliográfica
Funding Information:I would like to thank Christina Irving and Dr. Yassine El Hiani for their assistance with this work supported by the Canadian Institutes of Health Research and Cystic Fibrosis Canada .
Publisher Copyright:
© 2015 Elsevier B.V.
ASJC Scopus Subject Areas
- Biophysics
- Biochemistry
- Cell Biology