Location of a permeant anion binding site in the cystic fibrosis transmembrane conductance regulator chloride channel pore

Abstract

In the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, lyotropic anions with high permeability also bind relatively tightly within the pore. However, the location of permeant anion binding sites, as well as their relationship to anion permeability, is not known. We have identified lysine residue K95 as a key determinant of permeant anion binding in the CFTR pore. Lyotropic anion binding affinity is related to the number of positively charged amino acids located in the inner vestibule of the pore. However, mutations that change the number of positive charges in this pore region have minimal effects on anion permeability. In contrast, a mutation at the narrow pore region alters permeability with minimal effects on anion binding. Our results suggest that a localized permeant anion binding site exists in the pore; however, anion binding to this site has little influence over anion permeability. Implications of this work for the mechanisms of anion recognition and permeability in CFTR are discussed.

Fig. 1

Block by intracellular Au(CN)₂ ⁻ is weakened in K95Q/E1371Q channels. Example macroscopic I–V relationships for E1371Q (a) and K95Q/E1371Q (b) CFTR channels recorded before (control) and after the addition of Au(CN)₂⁻ to the intracellular (bath) solution at the concentrations stated. c Mean fraction of control current remaining after addition of different concentrations of Au(CN)₂⁻ at a membrane potential of −100 mV for both these channel constructs. Data have been fitted as described in “Materials and methods.” d Mean K _D values obtained from such fits at different membrane potentials. Mean K _D values were significantly different between the two channel constructs at all voltages examined (P < 0.005). Mean of data from 7 to 8 patches

Fig. 2

Block by intracellular SCN⁻ and C(CN)₃⁻ is weakened in K95Q/E1371Q channels. Example macroscopic I–V relationships for E1371Q (a) and K95Q/E1371Q (b) CFTR channels recorded before (control) and after the addition of 10 mM SCN⁻ to the intracellular (bath) solution. c Mean K _D values for SCN⁻ block for these channel constructs, obtained as described for Au(CN)₂⁻ in Fig. 1. Mean K _D values were significantly different between the two channel constructs at all voltages examined (P < 0.05). d Mean K _D values for C(CN)₃⁻ block, obtained from similar experiments (not illustrated). Mean K _D values were significantly different between the two channel constructs at all voltages examined (P < 0.002). Mean of data from 3 to 5 patches

Fig. 3

Strength of Au(CN)₂⁻ block is dependent on the number of positive charges in the pore inner vestibule. a Example macroscopic I–V relationships for K95H/E1371Q CFTR channels recorded using bath solutions at pH 5.5 (left) or pH 9.0 (right, different patch). Currents were recorded before (control) and after the addition of 1 mM Au(CN)₂⁻ to the intracellular (bath) solution. b Mean K _D values for Au(CN)₂⁻ block of K95H/E1371Q at these two different pHs, obtained as described for Au(CN)₂⁻ in Fig. 1. Mean K _D values were significantly different between the two pHs at all voltages examined (P < 0.01). c Relationship between the observed K _D values for Au(CN)₂⁻ block (at −100 mV) and bath solution pH in K95H/E1371Q and E1371Q. d Example macroscopic I–V relationships for I344K/E1371Q (left) and K95Q/I344K/E1371Q (right) CFTR channels recorded before (control) and after the addition of a low concentration (10 μM) of Au(CN)₂⁻ to the intracellular (bath) solution. e Mean K _D values for Au(CN)₂⁻ block for these channel constructs, as well as the additional positive charge mutants V345K/E1371Q and S1141K/E1371Q, obtained as described in Fig. 1. f Relationship between the observed K _D values for Au(CN)₂⁻ block (at −100 mV) and the expected number of fixed positive charges in the pore inner vestibule in different channel constructs. K95H⁰ refers to the unprotonated form of the histidine side chain (as expected at pH 9.0) and K95H⁺ the protonated form (at pH 5.5). Asterisks indicate a significant difference from E1371Q (*P < 0.05, **P < 0.0001). Mean of data from 3 to 8 patches

Fig. 4

Block of F337A/E1371Q channels by intracellular lyotropic permeant anions. a, b Example macroscopic I–V relationships for F337A/E1371Q CFTR channels recorded before (control) and after the addition of Au(CN)₂⁻ (1 mM) or SCN⁻ (10 mM) to the intracellular (bath) solution. c Mean K _D values for Au(CN)₂⁻, SCN⁻, and C(CN)₃⁻ (estimated at −100 mV as described in Figs. 1 and 2) compared in E1371Q, K95Q/E1371Q, and F337A/E1371Q. Asterisks indicate a significant difference from E1371Q (P < 0.01). Mean of data from 3 to 8 patches

Fig. 5

Anion permeability in different CFTR channel variants. a Normalized I–V relationships for the CFTR variants indicated, recorded with Cl⁻-containing extracellular solutions and intracellular solutions containing F⁻, Cl⁻, Br⁻, NO₃ ⁻ or SCN⁻ as indicated. Note that the range of current reversal potentials was greatly reduced in F337A/E1371Q, suggesting a relative loss of permeability selectivity in this mutant. b Mean P _X/P _Cl values calculated from current reversal potential measurements under these conditions as described in “Materials and methods.” Asterisks indicate a significant difference from E1371Q (*P < 0.05; **P < 0.002). c Relationship between P _X/P _Cl and anion free energy of hydration (G _h, taken from Marcus [31] ). Note that the normal lyotropic relationship between relative permeability and G _h is greatly reduced in F337A/E1371Q but retained in K95Q/E1371Q and I344K/E1371Q. Mean of data from 3 to 8 patches

Fig. 6

Molecular bases for CFTR channel lyotropic anion binding and lyotropic anion permeability. a Atomic homology model of CFTR [21] indicating the approximate extent of the membrane spanning domains (MSDs), intracellul ar loops (ICLs) and cytoplasmic nucleotide binding domains (NBDs). Transmembrane helices TM1 and TM6, the sites of mutations used in the present study, are highlighted in red and blue, respectively. b Detailed view of TMs 1 and 6 in this same homology model, indicating the relative location of amino acid side chains proposed to contribute to the lyotropic anion binding site in the pore inner vestibule (“energy well”; K95, I344) and to the lyotropic anion permeability selectivity filter in the pore narrow region (“energy barrier”; F337). c Cartoon model showing the proposed overall architecture of the pore [3] and indicating the approximate locations of these same amino acids within this overall cartoon model. In both a and b, CFTR model structure was based on coordinates provided by Dalton et al. [21] and visualized using PyMol (Schrödinger, LLC, Portland, OR, USA) (color figure online)