HALIDE PERMEATION IN WILD-TYPE AND MUTANT CYSTIC-FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR CHLORIDE CHANNELS

Permeation of cystic fibrosis transmembrane conductance regulator (CFT R) Cl- channels by halide ions was studied in stably transfected Chine se hamster ovary cells by using the patch clamp technique. In cell-att ached patches with a high Cl- pipette solution, the CFTR channel displ ayed outwardly rectifying currents and had a conductance near-the memb rane potential of 6.0 pS at 22 degrees C or 8.7 pS at 37 degrees C. Th e current-voltage relationship became linear when patches were excised into symmetrical, N-tris(hydroxymethyl)methyl-2-aminomethane sulfonat e (TES)-buffered solutions. Under these conditions, conductance increa sed from 7.0 PS at 22 degrees C to 10.9 pS at 37 degrees C. The conduc tance at 22 degrees C was similar to 1.0 pS higher when TES and HEPES were omitted from the solution, suggesting weak, voltage-independent b lock by pH buffers. The relationship between conductance and Cl- activ ity was hyperbolic and well fitted by a Michaelis-Menten-type) functio n having a K-m of similar to 38 mM and maximum conductance of 10 pS at 22 degrees C. Dilution potentials measured with NaCl gradients indica ted high anion selectivity (P-Na/P-Cl = 0.003-0.028). Biionic reversal potentials measured immediately after exposure of the cytoplasmic sid e to various test anions indicated P, (1.8) > P-Br (1.3) > P-Cl (1.0) > P-F (0.17), consistent with a ''weak field strength'' selectivity si te. The same sequence was obtained for external halides, although inwa rd F- flow was not observed. Iodide currents were protocol dependent a nd became blocked after 1-2 min. This coincided with a large shift in the (extrapolated) reversal potential to values indicating a greatly r educed I-/Cl- permeability ratio (P-I/P-Cl < 0.4). The switch to lo iv I- permeability was enhanced at potentials that favored Cl- entry int o the pore and was not observed in tile R347D mutant, which is thought to lack an anion binding site involved in multi-ion pore behavior. In teractions between Cl- and I- ions may influence I- permeation and be responsible for the wide range of P-I/P-Cl ratios that have been repor ted for the CFTR channel. The low P-I/P-Cl ratio usually reported for CFTR only occurred after entry into an altered permeability state and thus may not be comparable with permeability ratios for other anions, which are obtained in the absence of iodide. We propose that CFTR disp lays a ''weak field strength'' anion selectivity sequence.