Channel-like slippage modes in the human anion/proton exchanger ClC-4

The ClC family encompasses two classes of proteins with distinct transport functions: anion channels and transporters. ClC-type transporters usually mediate secondary active anion–proton exchange. However, under certain conditions they assume slippage mode behavior in which proton and anion transport are uncoupled, resulting in passive anion fluxes without associated proton movements. Here, we use patch clamp and intracellular pH recordings on transfected mammalian cells to characterize exchanger and slippage modes of human ClC-4, a member of the ClC transporter branch. We found that the two transport modes differ in transport mechanisms and transport rates. Nonstationary noise analysis revealed a unitary transport rate of 5 × 105 s−1 at +150 mV for the slippage mode, indicating that ClC-4 functions as channel in this mode. In the exchanger mode, unitary transport rates were 10-fold lower. Both ClC-4 transport modes exhibit voltage-dependent gating, indicating that there are active and non-active states for the exchanger as well as for the slippage mode. ClC-4 can assume both transport modes under all tested conditions, with exchanger/channel ratios determined by the external anion. We propose that binding of transported anions to non-active states causes transition from slippage into exchanger mode. Binding and unbinding of anions is very rapid, and slower transitions of liganded and non-liganded states into active conformations result in a stable distribution between the two transport modes. The proposed mechanism results in anion-dependent conversion of ClC-type exchanger into an anion channel with typical attributes of ClC anion channels

On the problem of supersonic gas flow in two-dimensional channel with the oscillating upper wall

In the present paper we solve the problem of supersonic gas flow in two-dimensional channel with the moving upper wall making oscillations according to the harmonic law. In order to get a numerical solution for gas dynamics equations we have implemented a difference scheme with space and time approximation of the first order and one with space approximation of the second order. Depending on a type of harmonic law and initial gas inflow conditions, the peculiarities of angle-shock wave propagation in moving curvilinear domains have been investigated. It has been determined that the increase of oscillation amplitude causes the increase of shock wave intensity. It has been shown that under particular oscillation amplitude the moving wall has practically no effect on the flow within the domain

Mutations associated with Dent's disease affect gating and voltage dependence of the human anion/proton exchanger ClC-5

Dent’s disease is associated with impaired renal endocytosis and endosomal acidification. It is linked to mutations in the membrane chloride/proton exchanger ClC-5, however, a direct link between localization in the protein and functional phenotype of the mutants has not been established until now. Here, two Dent’s disease mutations, G212A and E267A, were investigated using heterologous expression in HEK293T cells, patch-clamp measurements and confocal imaging. WT and, mutant ClC-5 exhibited mixed cell membrane and vesicular distribution. Reduced ion currents were measured for both mutants and both exhibited reduced capability to support endosomal acidification. Functionally, mutation G212A was capable of mediating anion/proton antiport but dramatically shifted the activation of ClC-5 towards more depolarized potentials. The shift can be explained by impeded movements of the neighboring gating glutamate Gluext, a residue that confers major part of the voltage dependence of ClC-5 and serves as a gate at the extracellular entrance of the anion transport pathway. Cell surface abundance of E267A was reduced by ~50% but also dramatically increased gating currents were detected for this mutant and accordingly reduced probability to undergoing cycles associated with electrogenic ion transport. Structurally, the gating alternations correlate to the proximity of E267A to the proton glutamate Gluin that serves as intracellular gate in the proton transport pathway and regulates the open probability of ClC-5. Remarkably, two other mammalian isoforms, ClC-3 and ClC-4, also differ from ClC-5 in gating characteristics affected by the here investigated disease-causing mutations. This evolutionary specialization, together with the functional defects arising from mutations G212A and E267A, demonstrate that the complex gating behavior exhibited by most of the mammalian CLC transporters is an important determinant of their cellular function

On the functional consequences of epilepsy-causing mutations located in ion channels and the role of cytoplasmic protein regions in fast and slow inactivation of voltage-gated sodium channels

Ion channels provide the basis for excitability in nerve and muscle cells. This thesis presents the functional characterization of K+ and Na+ channel mutations causing inherited epilepsies and a structure-function study about the role of two cytoplasmic protein regions of the voltage-gated Na+ channel. Three epilepsy causing mutations 2513delG in the KCNQ2 channel (causing benign familial neonatal convulsions) and T685M and R1460H in the voltage-gated Na+ channel (associated with generalized epilepsy with febrile seizures plus) were expressed and functionally characterized. For all three mutations changes were found explaining the occurrence of epileptic seizures. Fast inactivation in voltage-gated Na+ channels is believed to function in the so-called "hinged-lid" fashion - a hydrophobic particle of three amino acids (IFM) occludes the pore from the intracellular site of the membrane. Possible binding sites for the inactivation particle are the D4/S6 segment and the D4/S4-S5 interloop. Two mutations in the intracellular loop D4/S4-S5 (L1482C/A) were investigated. Both mutations introduced prominent effects on fast and slow inactivation, demonstrating that D4/S4-S5 loop is involved in the regulation of the before mentioned processes. The applied thermodynamic analysis showed no functional cooperativity of D4/S4-S5 region and the inactivation particle in fast inactivation. To investigate in detail the role of segment D4/S6 in Na+ channel gating, the amino acids at positions F1586, V1589, M1592 and I1596 were substituted by cysteines and the effects of the mutations and application of MTS reagents, covalently binding to cysteines, were studied. All gating transitions, following activation were strongly affected, demonstrating a central functional role of segment D4/S6 in the gating of voltage-dependent Na+ channels. Additionally, the reported effects propose that the slow inactivation gate in Na+ channels contains the cytoplasmic part of segment D4/S6

ClC-3 Is an Intracellular Chloride/Proton Exchanger with Large Voltage-Dependent Nonlinear Capacitance

The chloride/proton exchangers ClC-3, ClC-4 and ClC-5 are localized in distinct intracellular compartments and regulate their luminal acidity. We used electrophysiology combined with fluorescence pH measurements to compare the functions of these three transporters. Since the expression of WT ClC-3 in the surface membrane was negligible, we removed an N-terminal retention signal for standard electrophysiological characterization of this isoform. This construct (ClC-313–19A) mediated outwardly rectifying coupled Cl–/H+ antiport resembling the properties of ClC-4 and ClC-5. In addition, ClC-3 exhibited large electric capacitance, exceeding the nonlinear capacitances of ClC-4 and ClC-5. Mutations of the proton glutamate, a conserved residue at the internal side of the protein, decreased ion transport but increased nonlinear capacitances in all three isoforms. This suggests that nonlinear capacitances in mammalian ClC transporters are regulated in a similar manner. However, the voltage dependence and the amplitudes of these capacitances differed strongly between the investigated isoforms. Our results indicate that ClC-3 is specialized in mainly performing incomplete capacitive nontransporting cycles, that ClC-4 is an effective coupled transporter, and that ClC-5 displays an intermediate phenotype. Mathematical modeling showed that such functional differences would allow differential regulation of luminal acidification and chloride concentration in intracellular compartments

Metabolic energy sensing by mammalian CLC anion/proton exchangers

CLC anion/proton exchangers control the pH and [Cl−] of the endolysosomal system that is essential for cellular nutrient uptake. Here, we use heterologous expression and whole‐cell electrophysiology to investigate the regulation of the CLC isoforms ClC‐3, ClC‐4, and ClC‐5 by the adenylic system components ATP , ADP , and AMP . Our results show that cytosolic ATP and ADP but not AMP and Mg2+‐free ADP enhance CLC ion transport. Biophysical analysis reveals that adenine nucleotides alter the ratio between CLC ion transport and CLC gating charge and shift the CLC voltage‐dependent activation. The latter effect is suppressed by blocking the intracellular entrance of the proton transport pathway. We suggest, therefore, that adenine nucleotides regulate the internal proton delivery into the CLC transporter machinery and alter the probability of CLC transporters to undergo silent non‐transporting cycles. Our findings suggest that the CBS domains in mammalian CLC transporters serve as energy sensors that regulate vesicular Cl−/H+ exchange by detecting changes in the cytosolic ATP /ADP /AMP equilibrium. Such sensing mechanism links the endolysosomal activity to the cellular metabolic state