Anion Permeation in Human ClC-4 Channels

Abstract

ClC-4 and ClC-5 are mammalian ClC isoforms with unique ion conduction and gating properties. Macroscopic current recordings in heterologous expression systems revealed very small currents at negative potentials, whereas a substantially larger instantaneous current amplitude and a subsequent activation were observed upon depolarization. Neither the functional basis nor the physiological impact of these channel features are currently understood. Here, we used whole-cell recordings to study pore properties of human ClC-4 channels heterologously expressed in tsA201 or HEK293 cells. Variance analysis demonstrated that the prominent rectification of the instantaneous macroscopic current amplitude is due to a voltage-dependent unitary current conductance. The single channel amplitudes are very small, i.e., 0.10 +/- 0.02 pA at +140 mV for external Cl(-) and internal I(-). Conductivity and permeability sequences were determined for various external and internal anions, and both values increase for anions with lower dehydration energies. ClC-4 exhibits pore properties that are distinct from other ClC isoforms. These differences can be explained by assuming differences in the size of the pore narrowing and the electrostatic potentials within the ion conduction pathways.

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

“…Similar values were reported previously for the two other isoforms ClC-4 and ClC-5. 28,38,39,44 Nonstationary noise analysis can also be used to determine the number of proteins in the membrane and the apparent open probability of the investigated channels. 45 Unfortunately, in the case of ClC-3, the current variances depended linearly on the current amplitudes ( Figure 3c).…”

Glutamate Modifies Ion Conduction and Voltage-dependent Gating of Excitatory Amino Acid Transporter-associated Anion Channels

“…The pcDNA3.1-rEAAT4 construct was kindly provided by Dr. J. Rothstein, The Johns Hopkins University, Baltimore (17). Transient transfection of tsA201 cells using the Ca 3 (PO 4 ) 2 technique was performed as described previously (18). To identify cells with a high probability of expressing recombinant transporters, cells were cotransfected with a plasmid encoding the CD8 antigen and incubated 5 min before use with polystyrene microbeads precoated with anti-CD8 antibodies (Dynabeads M-450 CD8, Dynal, Great Neck, NY) (19).…”

“…For each construct, two independent recombinants were examined and exhibited indistinguishable functional properties. Oligoclonal cell lines stably expressing hEAAT2 were obtained by selection for resistance to the aminoglycoside antibiotic geneticin (G418, Roche Applied Science) as described previously (18). The stable cell lines so obtained exhibited electrical properties indistinguishable from transiently transfected tsA201 cells.…”

“…Reversal potentials were only used for further analysis when the cell exhibited a whole-cell conductance that was at least three times larger than the mean value for untransfected cells under identical conditions. Permeability ratios were calculated from reversal potential measurements under biionic conditions using the Goldman-Hodgkin-Katz equation as described (18) ( Tables I and II (Fig. 7A) was determined by plotting the normalized instantaneous current amplitude at ϩ 90 mV after 0.2-s prepulses to different voltages versus the preceding potential.…”

“…Noise Analysis-Non-stationary noise analysis (23, 24) was performed using an EPC10 amplifier (HEKA Electronics, Lambrecht, Germany) equipped with a 16-bit AD/DA converter as described (18). Currents were filtered at 10 kHz and digitized with a sampling rate of 50 kHz.…”