Regulation of conductance by the number of fixed positive charges in the intracellular vestibule of the CFTR chloride channel pore

Abstract

Rapid chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel is dependent on the presence of fixed positive charges in the permeation pathway. Here, we use site-directed mutagenesis and patch clamp recording to show that the functional role played by one such positive charge (K95) in the inner vestibule of the pore can be “transplanted” to a residue in a different transmembrane (TM) region (S1141). Thus, the mutant channel K95S/S1141K showed Cl− conductance and open-channel blocker interactions similar to those of wild-type CFTR, thereby “rescuing” the effects of the charge-neutralizing K95S mutation. Furthermore, the function of K95C/S1141C, but not K95C or S1141C, was inhibited by the oxidizing agent copper(II)-o-phenanthroline, and this inhibition was reversed by the reducing agent dithiothreitol, suggesting disulfide bond formation between these two introduced cysteine side chains. These results suggest that the amino acid side chains of K95 (in TM1) and S1141 (in TM12) are functionally interchangeable and located closely together in the inner vestibule of the pore. This allowed us to investigate the functional effects of increasing the number of fixed positive charges in this vestibule from one (in wild type) to two (in the S1141K mutant). The S1141K mutant had similar Cl− conductance as wild type, but increased susceptibility to channel block by cytoplasmic anions including adenosine triphosphate, pyrophosphate, 5-nitro-2-(3-phenylpropylamino)benzoic acid, and Pt(NO2)42− in inside-out membrane patches. Furthermore, in cell-attached patch recordings, apparent voltage-dependent channel block by cytosolic anions was strengthened by the S1141K mutation. Thus, the Cl− channel function of CFTR is maximal with a single fixed positive charge in this part of the inner vestibule of the pore, and increasing the number of such charges to two causes a net decrease in overall Cl− transport through a combination of failure to increase Cl− conductance and increased susceptibility to channel block by cytosolic substances.

Relative Movements of Transmembrane Regions at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Pore during Channel Gating

“…At the end of the experiment, remaining currents were confirmed as being carried by CFTR by their sensitivity to the specific CFTR inhibitor GlyH-101. As described previously (9,18), channels bearing the E1371Q mutation were constitutively active, and whole cell currents carried by such channels were not further increased in amplitude by application of cAMP mixture, although they were sensitive to GlyH-101. CuPhe was prepared freshly before each experiment by mixing stock solutions of CuSO 4 (200 mM in distilled water) with 1,10-phenanthroline (200 mM in ethanol) in a 1:4 molar ratio.…”

“…The functional consequences of both CuPhe-induced cross-linking and Cd 2ϩ coordination have previously been used to infer distances between pairs of cysteine side chains of Ͻ10 Å in other channel types (31)(32)(33)(34)(35). Furthermore, both CuPhe-induced cysteine cross-linking (18,19) and Cd 2ϩ inhibition of single cysteine mutants (23) have been investigated in the CFTR channel pore using electrophysiological recording.…”

“…and function of these parts of the protein have been studied in isolation, and there is very little direct functional information relating to the three-dimensional arrangement of different TMs (18,19).…”

The CFTR Ion Channel: Gating, Regulation, and Anion Permeation

“…This vestibule appears to be lined by positively charged residues given that blocker efficacy is reduced by mutating several basic residues in the cytosolic regions of the TMs (e.g., K95, R303, and R352) (Linsdell 2005;St. Aubin and Linsdell 2006;Zhou et al 2010). As noted above, the interpretation of such results is complicated by possible effects of mutations on pore structure.…”

“…In this regard, K95 substitutions have multiple, complicated effects on permeation. K95 has been argued more recently to attract anions into the pore from the cytosol by residing near the entrance to the pore (inferred from strong voltage-dependent rectification of macroscopic currents for certain K95 mutants) (Linsdell 2006;Zhou et al 2010). Some K95 mutations (e.g., K95S) also markedly decrease single channel conductance at both depolarizing and hyperpolarizing voltages (Zhou et al 2010), which implies an effect on pore structure separate from a charge-attracting role for K95.…”