Permeability of wild-type and mutant cystic fibrosis transmembrane conductance regulator chloride channels to polyatomic anions

Abstract

Permeability of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel to polyatomic anions of known dimensions was studied in stably transfected Chinese hamster ovary cells by using the patch clamp technique. Biionic reversal potentials measured with external polyatomic anions gave the permeability ratio (P/P) sequence NO > Cl > HCO > formate > acetate. The same selectivity sequence but somewhat higher permeability ratios were obtained when anions were tested from the cytoplasmic side. Pyruvate, propanoate, methane sulfonate, ethane sulfonate, and gluconate were not measurably permeant (P/P < 0.06) from either side of the membrane. The relationship between permeability ratios from the outside and ionic diameters suggests a minimum functional pore diameter of approximately 5.3 A. Permeability ratios also followed a lyotropic sequence, suggesting that permeability is dependent on ionic hydration energies. Site-directed mutagenesis of two adjacent threonines in TM6 to smaller, less polar alanines led to a significant (24%) increase in single channel conductance and elevated permeability to several large anions, suggesting that these residues do not strongly bind permeating anions, but may contribute to the narrowest part of the pore.

Figure 4

(A) Proposed topology of CFTR, indicating the 12 transmembrane regions (TM1–12), two nucleotide binding domains (NBD1–2) and the regulatory (R) domain. (B) Amino acid sequence of TM6 according to Riordan et al. (1989). Residues mutated in the present study (T338 and T339) are indicated by asterisks, residues that line the pore according to cysteine scanning mutagenesis (Cheung and Akabas, 1996) are shown by crosses. Not shown are the residues downstream from TM6 (as originally defined), which have also been proposed by Cheung and Akabas (1996) to line the pore (T351, R352, and Q353).

Figure 1

Single CFTR channel currents recorded with different extracellular anions. Traces are shown at 0 mV (left) and at strongly positive potentials when the extracellular anion carried a measurable current. In each case the arrow on the left represents the current level when all channels were in the closed state.

Figure 2

Mean single channel i/V relationships determined for wild-type CFTR with different extracellular anions. Note that control i/Vs in symmetrical Cl⁻ at pH 7.3 are shown in each panel for reference. In A, the control i/V relationship obtained with symmetrical Cl⁻ is also shown at pH 8.4 for comparison with the HCO₃ ⁻ curve, which was obtained immediately after equilibrating the HCO₃ ⁻ solution at this pH with 5% CO₂ (see methods).

Figure 3

Mean single channel i/V relationships determined for wild-type CFTR with different intracellular anions. See Fig. 1 for details.

Figure 5

Conductance properties of single TT338,339AA channels. (A) Representative single channel current records from wild-type and TT338,339AA channels at the membrane potentials indicated. The closed state is indicated “C” on the right. (B) Examples of single channel amplitude histograms for wild-type and TT338,339AA channels at a membrane potential of −60 mV. In these examples, the mean open state current, indicated by the dashed line, was −0.45 pA for wild type and −0.69 pA for TT338,339AA. The apparent difference in open probability suggested by these histograms is an artifact of the short recording periods used. (C) Mean single channel current–voltage relationships for wild-type (○) and TT338,339AA channels (•). (D) Dependence of channel conductance on symmetrical Cl⁻ activity in wild-type (○) and TT338,339AA channels (•). Each point represents the mean ± SEM (where this is larger than the size of the symbol) of data from three to eight patches. The data points have been fit by Eq. 2 (see methods), giving saturating conductances of 11.1 pS for wild-type CFTR and 14.0 pS for TT338,339AA, and a K _m of 45.9 mM for wild type and 52.9 mM for TT338,339AA. Mean conductance was significantly greater in TT338,339AA channels than in wild-type channels at every Cl⁻ concentration studied (P < 0.05, one-tailed t test).

Figure 6

Permeability of TT338,339AA to different extracellular anions. Mean current–voltage relationships were measured under biionic conditions (see methods and Fig. 2). Each point represents the mean ± SEM (where this is larger than the size of the symbol) of data from three to nine patches for Cl⁻, and from three to seven patches for other anions. Although the shape of the curves for both F⁻ and gluconate suggest current reversal might occur at potentials more positive than those studied, we were unable to record outward F⁻ or gluconate currents at large positive potentials where inward Cl⁻ currents become vanishingly small. Currents recorded with extracellular I⁻ showed a similar hysteresis to that seen in wild-type channels (data not shown; see Tabcharani et al., 1997). The reversal potential for I⁻ was therefore estimated before I⁻ block of the channel occurred (see Tabcharani et al., 1997, for details).

Figure 7

Dependence of ion permeability on ion dimensions and hydration energy in wild-type and TT338,339AA channels. (A) Permeability (P_X/P_Cl) as a function of mean ion diameter (estimated as described in the text). The data have been fitted by Eq. 3, giving estimated pore diameters of 5.34 Å for wild type and 5.83 Å for TT338,339AA. A, acetate; E, ethane sulfonate; Fo, formate; G, gluconate; M, methane sulfonate; Pr, propanoate; and Py, pyruvate. (B) Permeability as a function of hydration energy, relative to that of Cl⁻ (ΔG_h). Values for ΔG_h were taken from Halm and Frizzell (1992).