Resumen
Single channel and macroscopic current recording was used to investigate block of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl - channel pore by the permeant anion Au(CN)2-. Block was 1-2 orders of magnitude stronger when Au(CN)2- was added to the intracellular versus the extracellular solution, depending on membrane potential. A point mutation within the pore, T-338A, strongly decreased the asymmetry of block, by weakening block by intracellular Au(CN) 2- and at the same time strengthening block by external Au(CN)2-. Block of T-338A, but not wild-type, was strongest at the current reversal potential and weakened by either depolarization or hyperpolarization. In contrast to these effects, the T-338A mutation had no impact on block by the impermeant Pt(NO2) 42- ion. We suggest that the CFTR pore has at least two anion binding sites at which Au(CN)2- and Pt(NO 2)42- block Cl- permeation. The T-338A mutation decreases a barrier for Au(CN)2- movement between different sites, leading to significant changes in its blocking action. Our finding that apparent blocker binding affinity can be altered by mutagenesis of a residue which does not contribute to a blocker binding site has important implications for interpreting the effects of mutagenesis on channel blocker effects.
Idioma original | English |
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Páginas (desde-hasta) | 1241-1253 |
Número de páginas | 13 |
Publicación | Biophysical Journal |
Volumen | 92 |
N.º | 4 |
DOI | |
Estado | Published - feb. 2007 |
Nota bibliográfica
Funding Information:Our results suggest that T-338 contributes to a barrier to anion movement inside the pore. For Cl − permeation, the consequence of lowering this barrier is an increase in unitary conductance. For anions that interact more strongly with binding sites inside the pore, such as Au ( CN ) 2 − , the existence of a barrier divides the pore into intracellularly and extracellularly accessible compartments, resulting in strongly side-dependent interactions between anions and the channel. Lowering of the barrier, as occurs in the T-338A mutant, results in a channel that interacts much more symmetrically with high affinity permeant anions. We thank Dr. Osvaldo Alvarez for providing the AJUSTE computer program used for rate theory modeling, and Elizabeth VandenBerg for technical assistance. This work was supported by the Canadian Institutes of Health Research.
ASJC Scopus Subject Areas
- Biophysics
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't