On the origin of asymmetric interactions between permeant anions and the cystic fibrosis transmembrane conductance regulator chloride channel pore.
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)4(2-) ion. We suggest that the CFTR pore has at least two anion binding sites at which Au(CN)2(-) and Pt(NO2)4(2-) 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.
Fatehi
Mohammad
M
Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 1X5, Canada.
St Aubin
Chantal N
CN
Linsdell
Paul
P
D000818:Animals; D000838:Anions; D001665:Binding Sites; D002460:Cell Line; D002712:Chlorides / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003485:Cyanates; D003486:Cyanides / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics / Q000502:physiology*; D006046:Gold; D017612:Gold Compounds / Q000378:metabolism; D015640:Ion Channel Gating; D010539:Permeability; D017671:Platinum Compounds / Q000378:metabolism; D017354:Point Mutation
D000838:Anions; C505032:CFTR protein, human; D002712:Chlorides; D003485:Cyanates; D003486:Cyanides; D017612:Gold Compounds; D017671:Platinum Compounds; C043041:gold cyanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006046:Gold