Altering intracellular pH reveals the kinetic basis of intraburst gating in the CFTR Cl− channel

KEY POINTS: The cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in the genetic disease cystic fibrosis (CF), forms a gated pathway for chloride movement regulated by intracellular ATP. To understand better CFTR function, we investigated the regulation of channel openings by intracellular pH. We found that short‐lived channel closures during channel openings represent subtle changes in the structure of CFTR that are regulated by intracellular pH, in part, at ATP‐binding site 1 formed by the nucleotide‐binding domains. Our results provide a framework for future studies to understand better the regulation of channel openings, the dysfunction of CFTR in CF and the action of drugs that repair CFTR gating defects. ABSTRACT: Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP‐gated Cl(−) channel defective in the genetic disease cystic fibrosis (CF). The gating behaviour of CFTR is characterized by bursts of channel openings interrupted by brief, flickery closures, separated by long closures between bursts. Entry to and exit from an open burst is controlled by the interaction of ATP with two ATP‐binding sites, sites 1 and 2, in CFTR. To understand better the kinetic basis of CFTR intraburst gating, we investigated the single‐channel activity of human CFTR at different intracellular pH (pH(i)) values. When compared with the control (pH(i) 7.3), acidifying pH(i) to 6.3 or alkalinizing pH(i) to 8.3 and 8.8 caused small reductions in the open‐time constant (τ(o)) of wild‐type CFTR. By contrast, the fast closed‐time constant (τ(cf)), which describes the short‐lived closures that interrupt open bursts, was greatly increased at pH(i) 5.8 and 6.3. To analyse intraburst kinetics, we used linear three‐state gating schemes. All data were satisfactorily modelled by the C(1) ↔ O ↔ C(2) kinetic scheme. Changing the intracellular ATP concentration was without effect on τ(o), τ(cf) and their responses to pH(i) changes. However, mutations that disrupt the interaction of ATP with ATP‐binding site 1, including K464A, D572N and the CF‐associated mutation G1349D all abolished the prolongation of τ(cf) at pH(i) 6.3. Taken together, our data suggest that the regulation of CFTR intraburst gating is distinct from the ATP‐dependent mechanism that controls channel opening and closing. However, our data also suggest that ATP‐binding site 1 modulates intraburst gating

Direct Sensing of Intracellular pH by the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Cl− Channel*♦

In cystic fibrosis (CF), dysfunction of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl− channel disrupts epithelial ion transport and perturbs the regulation of intracellular pH (pHi). CFTR modulates pHi through its role as an ion channel and by regulating transport proteins. However, it is unknown how CFTR senses pHi. Here, we investigate the direct effects of pHi on recombinant CFTR using excised membrane patches. By altering channel gating, acidic pHi increased the open probability (Po) of wild-type CFTR, whereas alkaline pHi decreased Po and inhibited Cl− flow through the channel. Acidic pHi potentiated the MgATP dependence of wild-type CFTR by increasing MgATP affinity and enhancing channel activity, whereas alkaline pHi inhibited the MgATP dependence of wild-type CFTR by decreasing channel activity. Because these data suggest that pHi modulates the interaction of MgATP with the nucleotide-binding domains (NBDs) of CFTR, we examined the pHi dependence of site-directed mutations in the two ATP-binding sites of CFTR that are located at the NBD1:NBD2 dimer interface (site 1: K464A-, D572N-, and G1349D-CFTR; site 2: G551D-, K1250M-, and D1370N-CFTR). Site 2 mutants, but not site 1 mutants, perturbed both potentiation by acidic pHi and inhibition by alkaline pHi, suggesting that site 2 is a critical determinant of the pHi sensitivity of CFTR. The effects of pHi also suggest that site 2 might employ substrate-assisted catalysis to ensure that ATP hydrolysis follows NBD dimerization. We conclude that the CFTR Cl− channel senses directly pHi. The direct regulation of CFTR by pHi has important implications for the regulation of epithelial ion transport

Effect of Third-Particle Material and Contact Mode on Tribology Contact Characteristics at Interface

A moving pair with two-body contact is the ideal situation assumed in previous analyses. However, all moving pairs are in a three-body contact state at the start of operation or immediately after the start of operation, such as bearings, ball-screws, gears and engines. This work studies the influence of wear particles (SUJ2), environmental particles (SiO2 and Al2O3) and nano-additives (CuO) on the tribological contact characteristics under different particle concentrations, particle sizes, surface roughnesses and contact modes. The three-body microcontact analysis revealed that the differences in the real contact area, particle contact area and separation of the four-particle materials in the three-body s–s and p–s contact modes are rather small. Under the three-body hybrid contact mode, the difference is relatively large and the sequence of the real contact area value obtained due to the elastic modulus for the four-particle material at this interface is Al2O3 > SUJ2 > CuO > SiO2. The order of the other two contact characteristics is reversed. The difference increases as the particle size or particle concentration increases. The order of the critical load required to transform three kinds of contact modes is SiO2 > CuO > SUJ2 > Al2O3. On the nearly initial three-body hybrid contact mode, the plastic contact area ratio at the interface first increases to a critical value and then decreases as the load increases because the original plastic contact spot area and contact spot number increases with the increase in load. At the same time, the elasto-plastic contact area ratio decreases to a low value and then increases. The elastic contact area ratio at the interface decreases as the load increases. Among the four third-particle materials, the experimental results and theoretical predictions show that the environmental particles, Al2O3, cause the maximum friction and wear observed at the interface

The Analysis of Three-Body Contact Temperature under the Different Third Particle Size, Density, and Value of Friction

Recently, many studies have investigated the friction, wear, and temperature characteristics of the interface between two relative movements. Such analyses often set the coefficient of friction as a fixed value and are analyzed in cases of two-body contact; however, the interface is often a three-body contact and the coefficient of friction varies depending on the operating conditions. This is a significant error in the analysis of contact characteristics, therefore, in this study, the actual interface and the change of the coefficient of friction were analyzed based on three-body micro-contact theory where the contact temperature was also analyzed and the difference between the generally assumed values were compared. The results showed that under three-body contact, the coefficient of total friction increased with an increase in particle size; and at a different particle size and area density of particles, the surface contact temperature increased with the plasticity index and load increases, and the particle contact temperature increased with the increasing particle size. The surface temperature rise was mainly affected by the ratio of the average temperature between surface 1 and surface 2 to the multiplication between the 100th root of the area density of particles and the square root of the equivalent surface roughness (Ts1s2_ave*/ηa0.01σ0.5) and the ratio of the 10th root of the mean particle diameter to the 100th root of the equivalent surface roughness (xa0.1/σ0.001). Particle temperature was mainly affected by the ratio of the 10th root of the mean particle diameter to the 100th root of the equivalent surface roughness (xa0.1/σ0.001) and the area density of particles ηa. Our study indicated that when the contact of surface with surface and the contact of the particles with the surface, the resulting heat balance was assigned to the particles and the surface in a three-body contact situation. Under this contact behavior, it could avoid a too high a rise in micro-contact temperature to achieve the material failure temperature

The contact temperature and deformation area of asperities on rough surface for three- body contact situation

In the micro-machine or precision machine, particles are often presented at contact interfaces. And these particles will affect the variation of plastic deformation of asperities and the contact temperature between the contact surfaces. In this paper, we used three-body microcontact model and contact temperature theory to evaluate elastic contact area, plastic contact area, elastic-plastically deformed contact area and contact temperature under the different particle sizes, velocities and applied loads conditions. The friction force is one of the main heat resources of contact temperature. Because friction coefficient is variable parameter in this work, the contact temperature rise between the contact surfaces is larger than that of assuming the constant friction coefficient conditions of CrMo steel for the different loads. The contact temperatures of particles and asperity increase when the velocity and applied load increase. The increases of particle size will give rise to the increase of particle temperature and decrease of asperity temperature on rough surface. The plastic deformed contact area increases when the particle size and particle density increase

The contact temperature and deformation area of asperities on rough surface for three- body contact situation

In the micro-machine or precision machine, particles are often presented at contact interfaces. And these particles will affect the variation of plastic deformation of asperities and the contact temperature between the contact surfaces. In this paper, we used three-body microcontact model and contact temperature theory to evaluate elastic contact area, plastic contact area, elastic-plastically deformed contact area and contact temperature under the different particle sizes, velocities and applied loads conditions. The friction force is one of the main heat resources of contact temperature. Because friction coefficient is variable parameter in this work, the contact temperature rise between the contact surfaces is larger than that of assuming the constant friction coefficient conditions of CrMo steel for the different loads. The contact temperatures of particles and asperity increase when the velocity and applied load increase. The increases of particle size will give rise to the increase of particle temperature and decrease of asperity temperature on rough surface. The plastic deformed contact area increases when the particle size and particle density increase