Molecular determinants of Au(CN)(2)(-) binding and permeability within the cystic fibrosis transmembrane conductance regulator Cl(-) channel pore.
Lyotropic anions with low free energy of hydration show both high permeability and tight binding in the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore. However, the molecular bases of anion selectivity and anion binding within the CFTR pore are not well defined and the relationship between binding and selectivity is unclear. We have studied the effects of point mutations throughout the sixth transmembrane (TM6) region of CFTR on channel block by, and permeability of, the highly lyotropic Au(CN)(2)(-) anion, using patch clamp recording from transiently transfected baby hamster kidney cells. Channel block by 100 microM Au(CN)(2)(-), a measure of intrapore anion binding affinity, was significantly weakened in the CFTR mutants K335A, F337S, T338A and I344A, significantly strengthened in S341A and R352Q and unaltered in K329A. Relative Au(CN)(2)(-) permeability was significantly increased in T338A and S341A, significantly decreased in F337S and unaffected in all other mutants studied. These results are used to define a model of the pore containing multiple anion binding sites but a more localised anion selectivity region. The central part of TM6 (F337-S341) appears to be the main determinant of both anion binding and anion selectivity. However, comparison of the effects of individual mutations on binding and selectivity suggest that these two aspects of the permeation mechanism are not strongly interdependent.
Gong
Xiandi
X
Department of Physiology & Biophysics, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4H7.
Burbridge
Susan M
SM
Cowley
Elizabeth A
EA
Linsdell
Paul
P
D000818:Animals; D002478:Cells, Cultured; D002712:Chlorides / Q000378:metabolism; D006224:Cricetinae; D003485:Cyanates; D003486:Cyanides / Q000378:metabolism* / Q000494:pharmacology*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression / Q000502:physiology; D006046:Gold; D017612:Gold Compounds / Q000378:metabolism* / Q000494:pharmacology*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects* / Q000235:genetics; D007668:Kidney / Q000166:cytology; D008564:Membrane Potentials / Q000187:drug effects; D016296:Mutagenesis / Q000502:physiology; D018408:Patch-Clamp Techniques
C505032:CFTR protein, human; D002712:Chlorides; D003485:Cyanates; D003486:Cyanides; D017612:Gold Compounds; C043041:gold cyanide; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D006046:Gold