Functionally additive fixed positive and negative charges in the CFTR channel pore control anion binding and conductance

Ion channels use charged amino-acid residues to attract oppositely charged permeant ions into the channel pore. In the cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel, a number of arginine and lysine residues have been shown to be important for Cl⁻ permeation. Among these, two in close proximity in the pore—Lys⁹⁵ and Arg¹³⁴—are indispensable for anion binding and high Cl⁻ conductance, suggesting that high positive charge density is required for pore function. Here we used mutagenesis and functional characterization to show that a nearby pore-lining negatively charged residue (Glu⁹²) plays a functionally additive role with these two positive charges. While neutralization of this negative charge had little effect on anion binding or Cl⁻ conductance, such neutralization was able to reverse the detrimental effects of removing the positive charge at either Lys⁹⁵ or Arg¹³⁴, as well as the similar effects of introducing a negative charge at a neighboring residue (Ser¹¹⁴¹). Furthermore, neutralization of Glu⁹² greatly increased the susceptibility of the channel to blockage by divalent S₂O₃²⁻ anions, mimicking the effect of introducing additional positive charge in this region; this effect was reversed by concurrent neutralization of either Lys⁹⁵ or Arg¹³⁴. Across a panel of mutant channels that introduced or removed fixed charges at these four positions, we found that many pore properties are dependent on the overall charge or charge density. We propose that the CFTR pore uses a combination of positively and negatively charged residues to optimize the anion binding and Cl⁻ conductance properties of the channel.