Cysteine-independent inhibition of the CFTR chloride channel by the cysteine-reactive reagent sodium (2-sulphonatoethyl) methanethiosulphonate

Abstract

Background and purpose: Methanethiosulphonate (MTS) reagents are used extensively to modify covalently cysteine side chains in ion channel structure-function studies. We have investigated the interaction between a widely used negatively charged MTS reagent, (2-sulphonatoethyl) methanethiosulphonate (MTSES), and the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel.

Experimental approach: Patch clamp recordings were used to study a 'cys-less' variant of human CFTR, in which all 18 endogenous cysteine residues have been removed by mutagenesis, expressed in mammalian cell lines. Use of excised inside-out membrane patches allowed MTS reagents to be applied to the cytoplasmic face of active channels.

Key results: Intracellular application of MTSES, but not the positively charged MTSET, inhibited the function of cys-less CFTR. Inhibition was voltage dependent, with a K(d) of 1.97 mmol x L(-1) at -80 mV increasing to 36 mmol x L(-1) at +80 mV. Inhibition was completely reversed on washout of MTSES, inconsistent with covalent modification of the channel protein. At the single channel level, MTSES caused a concentration-dependent reduction in unitary current amplitude. This inhibition was strengthened when extracellular Cl(-) concentration was decreased.

Conclusions and implications: Our results indicate that MTSES inhibits the function of CFTR in a manner that is independent of its ability to modify cysteine residues covalently. Instead, we suggest that MTSES functions as an open channel blocker that enters the CFTR channel pore from its cytoplasmic end to physically occlude Cl(-) permeation. Given the very widespread use of MTS reagents in functional studies, our findings offer a broadly applicable caveat to the interpretation of results obtained from such studies.

Figure 2

Effect of (2-sulphonatoethyl) methanethiosulphonate (MTSES) on cystic fibrosis transmembrane conductance regulator (CFTR) single channel currents. (A) An example of the single channel currents recorded at membrane potentials of +50 mV and −50 mV as indicated. In each case, the closed state of the channel is indicated by the line on the far left. Currents were recorded in the absence of MTSES (control) or with 2 or 6 mmol·L⁻¹ MTSES present in the intracellular solution as indicated. (B) Mean unitary current–voltage relationships recorded in the absence of MTSES (control) and with 2 mmol·L⁻¹ or 6 mmol·L⁻¹ MTSES present in the intracellular solution. Mean of data from 3–8 patches. Mean slope conductance measured under control conditions was 10.0 ± 0.1 pS (n= 10), somewhat larger than the value we measured for wild-type CFTR under identical conditions (∼8.5 pS; Fatehi et al., 2007; Zhou et al., 2007). A small increase in unitary conductance has previously been reported in cys-less CFTR (Mense et al., 2006).

Figure 1

Block of cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ current by intracellular (2-sulphonatoethyl) methanethiosulphonate (MTSES). (A) An example of the leak-subtracted macroscopic current–voltage relationship for cys-less/V510A-CFTR following maximal current stimulation with protein kinase A catalytic subunit (PKA), adenosine 5′-triphosphate (ATP) and pyrophosphate (PPi). Current from the same patch is shown before (control) and immediately following addition of 2 mmol·L⁻¹ MTSES (+MTSES) to the intracellular solution, and also following washing of MTSES from the bath and re-application of PKA, ATP and PPi (wash). (B) A similar example of the current–voltage relationship recorded before (control) and immediately following addition of 2 mmol·L⁻¹[2-(trimethylammonium)ethyl] methanethiosulphonate (+MTSET) to the intracellular solution. (C) Mean fractional current remaining following the addition of different concentrations of MTSES, measured at membrane potentials of −80 mV, −20 mV and +40 mV. Mean of data from 4–5 patches. Each set of data has been fitted by Equation (1) as described in Methods, with the following parameters: at −80 mV, K_d= 1.97 mmol·L⁻¹, n_H= 1.57; at −20 mV, K_d= 5.47 mmol·L⁻¹, n_H= 1.11; and at +40 mV, K_d= 26.1 mmol·L⁻¹, n_H= 0.88. (D) Relationship between mean K_d[estimated using fits such as those shown in (C)] and membrane potential. The data have been fitted by Equation (2), with K_d(0) = 11.2 mmol·L⁻¹ and zδ=−0.52.

Figure 3

Inhibition of single channel current amplitude by (2-sulphonatoethyl) methanethiosulphonate (MTSES) is dependent on extracellular Cl⁻ concentration. (A) An example of single channel currents recorded at a membrane potential of −30 mV with a low extracellular Cl⁻ concentration (4 mmol·L⁻¹). The closed state of the channel is indicated by the line on the far left. Currents were recorded in the absence of MTSES (control) or with 2 or 6 mmol·L⁻¹ MTSES present in the intracellular solution as indicated. (B) Mean unitary current–voltage relationships recorded under these conditions in the absence of MTSES (control) and with 2 mmol·L⁻¹ or 6 mmol·L⁻¹ MTSES present in the intracellular solution. Mean of data from 3–6 patches. Mean slope conductance measured under control conditions was 6.7 ± 0.5 pS (n= 5), again somewhat larger than the value we measured for wild-type cystic fibrosis transmembrane conductance regulator under identical conditions (5.4–5.7 pS; Ge and Linsdell (2006); St. Aubin and Linsdell 2006). (C,D) Mean reduction in unitary current amplitude in response to addition of 2 mmol·L⁻¹ (C) or 6 mmol·L⁻¹ MTSES (D), under conditions of high external (Cl⁻) (154 mmol·L⁻¹; see Figure 2) or low external (Cl⁻) (4 mmol·L⁻¹). Where the voltage ranges studied under different ionic conditions overlap, the fraction of control current remaining was significantly less with 4 mmol·L⁻¹ extracellular Cl⁻ than with 154 mmol·L⁻¹ Cl⁻ (P < 0.05), both at 2 mmol·L⁻¹ and 6 mmol·L⁻¹ MTSES, as indicated by asterisks. These mean data have been fitted by Equation (3) with the following parameters: (C) for 154 mmol·L⁻¹ Cl⁻: K_d(0) = 9.5 mmol·L⁻¹, zδ=−0.51, for 4 mmol·L⁻¹ Cl⁻: K_d(0) = 3.2 mmol·L⁻¹, zδ=−0.34; (D) for 154 mmol·L⁻¹ Cl⁻: K_d(0) = 8.8 mmol·L⁻¹, zδ=−0.63, for 4 mmol·L⁻¹ Cl⁻: K_d(0) = 2.8 mmol·L⁻¹, zδ=−0.27.