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

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 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

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

Multiple Discrete Transitions Underlie Voltage-Dependent Activation in CLC Cl−/H+ Antiporters

AbstractMost mammalian chloride channels and transporters in the CLC family display pronounced voltage-dependent gating. Surprisingly, despite the complex nature of the gating process and the large contribution to it by the transport substrates, experimental investigations of the fast gating process usually produce canonical Boltzmann activation curves that correspond to a simple two-state activation. By using nonlinear capacitance measurements of two mutations in the ClC-5 transporter, here we are able to discriminate and visualize discrete transitions along the voltage-dependent activation pathway. The strong and specific dependence of these transitions on internal and external [Cl−] suggest that CLC gating involves voltage-dependent conformational changes as well as coordinated movement of transported substrates

Anion Channels: Regulation of ClC-3 by an Orphan Second Messenger

SummaryClC-3 is a ubiquitously expressed chloride channel isoform whose biological function has been a matter of debate for many years. A recent study reporting its regulation by Ins(3,4,5,6)P4 assigns novel transport functions and cellular roles to ClC-3 and identifies a regulatory pathway that affects epithelial transport and endosomal pH regulation

Anion- and Proton-Dependent Gating of ClC-4 Anion/Proton Transporter under Uncoupling Conditions

AbstractClC-4 is a secondary active transporter that exchanges Cl− ions and H+ with a 2:1 stoichiometry. In external SCN−, ClC-4 becomes uncoupled and transports anions with high unitary transport rate. Upon voltage steps, the number of active transporters varies in a time-dependent manner, resembling voltage-dependent gating of ion channels. We here investigated modification of the voltage dependence of uncoupled ClC-4 by protons and anions to quantify association of substrates with the transporter. External acidification shifts voltage dependence of ClC-4 transport to more positive potentials and leads to reduced transport currents. Internal pH changes had less pronounced effects. Uncoupled ClC-4 transport is facilitated by elevated external [SCN−] but impaired by internal Cl− and I−. Block by internal anions indicates the existence of an internal anion-binding site with high affinity that is not present in ClC channels. The voltage dependence of ClC-4 coupled transport is modulated by external protons and internal Cl− in a manner similar to what is observed under uncoupling conditions. Our data illustrate functional differences but also similarities between ClC channels and transporters