Thiocyanate as a probe of the cystic fibrosis transmembrane conductance regulator chloride channel pore

2001 ◽  
Vol 79 (7) ◽  
pp. 573-579 ◽  
Author(s):  
Paul Linsdell

Immediately following exposure to thiocyanate (SCN–)-containing solutions, the cystic fibrosis conductance regulator Cl– channel exhibits high unitary SCN– conductance and anomalous mole fraction behaviour, suggesting the presence of multiple anion binding sites within the channel pore. However, under steady-state conditions SCN– conductance is very low. Here I show, using patch clamp recording from CFTR-transfected mammalian cell lines, that under steady-state conditions neither SCN– conductance nor SCN– permeability show anomalous mole fraction behaviour. Instead, SCN– conductance, permeability, and block of Cl– permeation can all be reproduced by a rate theory model that assumes only a single intrapore anion binding site. These results suggest that under steady-state conditions the interaction between SCN– and the CFTR channel pore can be understood by a simple model whereby SCN– ions enter the pore more easily than Cl–, and bind within the pore more tightly than Cl–. The implications of these findings for investigating and understanding the mechanism of anion permeation are discussed.Key words: chloride channel, permeation, anion binding, multi-ion pore behaviour, rate theory model.

2016 ◽  
pp. 505-515
Author(s):  
F. QIAN ◽  
L. LIU ◽  
Z. LIU ◽  
C. LU

The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel contains 12 transmembrane (TM) regions that are presumed to form the channel pore. However, there is no direct evidence clearly illustrating the involvement of these transmembrane regions in the actual CFTR pore structure. To obtain insight into the architecture of the CFTR channel pore, we used patch clamp recording techniques and a strategy of co-mutagenesis of two potential pore-forming transmembrane regions (TM1 and TM6) to investigate the collaboration of these two TM regions. We performed a range of specific functional assays comparing the single channel conductance, anion binding, and anion selectivity properties of the co-mutated CFTR variants, and the results indicated that TM1 and TM6 play vital roles in forming the channel pore and, thus, determine the functional properties of the channel. Furthermore, we provided functional evidence that the amino acid threonine (T338) in TM6 has synergic effects with lysine (K95) in TM1. Therefore, we propose that these two residues have functional collaboration in the CFTR channel pore and may collectively form a selective filter.


1995 ◽  
Vol 268 (3) ◽  
pp. C636-C646 ◽  
Author(s):  
J. L. Overholt ◽  
A. Saulino ◽  
M. L. Drumm ◽  
R. D. Harvey

Whole cell epithelial cystic fibrosis transmembrane conductance regulator (CFTR) Cl- currents exhibited a linear current-voltage (I-V) relationship with high symmetrical transmembrane Cl- concentrations. However, when intracellular Cl- (Cli-) was reduced by replacement with glutamate, I-V relationships were outwardly rectifying. Rectification was not affected by reducing extracellular Cl- to eliminate or reverse the gradient, indicating that rectification is not a function of the Cl- gradient. Rectification was affected by Cli- in a concentration-dependent manner, and it was weaker when Cli- was reduced by replacement with sucrose. These characteristics are identical to those of the cardiac isoform of CFTR, and the experimental data could be simulated by an Eyring rate theory model assuming that permeating anions interact at a single binding site within the channel pore. No evidence was found for multiple binding sites. These results indicate that rectification is a function of the concentration and permeability of the anions inside the cell. It is concluded that rectification of CFTR Cl- current is a property of ion channel permeation that would occur under physiological conditions and that permeation of the epithelial and cardiac isoforms of CFTR is identical.


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