Characterization of the chloride conductance in porcine renal brush-border membrane vesicles (original) (raw)
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The Journal of General Physiology, 2003
The distal-convoluted tubule (DCT) of the kidney absorbs NaCl mainly via an Na ϩ -Cl Ϫ cotransporter located at the apical membrane, and Na ϩ , K ϩ ATPase at the basolateral side. Cl Ϫ transport across the basolateral membrane is thought to be conductive, but the corresponding channels have not yet been characterized. In the present study, we investigated Cl Ϫ channels on microdissected mouse DCTs using the patch-clamp technique. A channel of ف 9 pS was found in 50% of cell-attached patches showing anionic selectivity. The NP o in cell-attached patches was not modified when tubules were preincubated in the presence of 10 Ϫ 5 M forskolin, but the channel was inhibited by phorbol ester (10 Ϫ 6 M). In addition, NP o was significantly elevated when the calcium in the pipette was increased from 0 to 5 mM ( NP o increased threefold), or pH increased from 6.4 to 8.0 ( NP o increased 15fold). Selectivity experiments conducted on inside-out patches showed that the Na ϩ to Cl Ϫ relative permeability was 0.09, and the anion selectivity sequence Cl Ϫ ف I Ϫ Ͼ Br Ϫ ف NO 3
Brush-border membrane cation conducting channels from rat kidney proximal tubules
American Journal of Physiology-Renal Physiology, 1989
This is a description and kinetic characterization of cation channels from rat kidney brush-border membrane vesicles and from apical membranes of proximal tubule cells in culture. Channel activity was demonstrated and characterized in both artificial phospholipid bilayers and in tissue culture. Intermediate conductance, approximately 50 pS, cation-selective channels were observed by both methods. Channels were characterized by a Na permeability (PNa)/K permeability (PK) of 1-5:1. Open-channel current-voltage curves were linear in symmetric 300 mM NaCl. In tissue culture the gating kinetics are described by two open-time constants and two closed-time constants. Channel activity was neither voltage nor Ca2+ dependent and the probability of being in the open state ranged from 0.6 to 0.95. In tissue culture experiments the channel demonstrated nonstationary gating activity. A second, 15-pS cation channel, seen in planar bilayers, demonstrated a higher selectivity for Na+ with a (PNa/PK ...
Similar chloride channels in the connecting tubule and cortical collecting duct of the mouse kidney
AJP: Renal Physiology, 2006
Using the patch-clamp technique, we investigated chloride channels on the basolateral membrane of the connecting tubule (CNT) and cortical collecting duct (CCD). We found a ~10-pS channel in CNT cell-attached patches. Replacing NaCl by Na-gluconate in the pipette shifted the reversal potential by +25 mV, whereas NMDG-chloride had no effect, indicating anion selectivity. On inside-out patches, we determined a selectivity sequence of Cl -> Br -~ NO 3 -> F, which is compatible with that of ClC -K2, a ClC chloride channel present in the distal nephron. In addition, the NPo measured in cell-attached patches was significantly increased when the calcium concentration or the pH in the pipette was increased, which is another characteristic of the ClC -K. These findings suggest that this channel is underlain by ClC -K2. A similar chloride channel was found in CCD patches. Since CNT and CCD are heterogeneous tissues, we studied the cellular distribution of the chloride channel using recording conditions ( KCl-rich solution in the pipette) that allowed us to detect simultaneously chloride channels and inwardly-rectifying potassium channels. We detected chloride channels alone in 45 and 42%, and potassium channels alone in 51 and 58% of CNT and CCD patches respectively. Chloride and potassium channels were recorded simultaneously from two patches (4% of patches) in the CNT and from no patch in the CCD.
Journal of Membrane Biology, 2001
We have used perforated patch clamp and Fura-2 microfluorescence measurements to study Ca 2+activated Cl − currents in cultured mouse renal inner medullary collecting duct cells (mIMCD-3). The conductance was spontaneously active under resting conditions and whole cell currents were time and voltageindependent with an outwardly rectifying current-voltage relationship. The channel blockers DIDS, niflumic acid, glybenclamide and NPPB reversibly decreased the basal currents, whereas the sulfhydryl agent, DTT produced an irreversible inhibition. Increasing or decreasing extracellular calcium produced parallel changes in the size of the basal currents. Variations in external Ca 2+ were associated with corresponding changes in free cytosolic Ca 2+ concentration. Increasing cytosolic Ca 2+ by extracellular ATP or ionomycin, further enhanced Cl − conductance, with whole cell currents displaying identical biophysical properties to the basal currents. However, the agonist-stimulated currents were now increased by DTT exposure, but still inhibited by the other channel blockers. Using RT-PCR, three distinct mRNA transcripts belonging to the CLCA family of Ca 2+ -activated Cl − channel proteins were identified, two of which represent novel sequences. Whether different channels underlie the basal and agonist-stimulated currents in mIMCD-3 cells is unclear. Our findings establish a novel link between alterations in external and internal Ca 2+ and the activity of Ca 2+ -activated Cl − transport in these cells.
Epithelial chloride channel. Development of inhibitory ligands
The Journal of General Physiology, 1987
A B S T R A C T Chloride channels are present in the majority of epithelial cells, where they mediate absorption or secretion of NaCl. Although the absorptive and secretory channels are well characterized in terms of their electrophysiological behavior, there is a lack of pharmacological ligands that can aid us in further functional and eventually molecular characterization. To obtain such ligands, we prepared membrane vesicles from bovine kidney cortex and apical membrane vesicles from trachea and found that they contain a chloride transport process that is electrically conductive. This conductance was reduced by preincubating the vesicles in media containing ATP or ATP-~/-S, but not/3methylene ATP, which suggests that the membranes contain a kinase that can close the channels. We then screened compounds derived from three classes: indanyloxyacetic acid (IAA), anthranilic acid (AA), and ethacrynic acid. We identified potent inhibitors from the IAA and the AA series. We tritiated IAA-94 and measured binding of this ligand to the kidney cortex membrane vesicles and found a high-affinity binding site whose dissociation constant (0.6 vM) was similar to the inhibition constant (1 ~tM). There was a good correlation between the inhibitory potency of several IAA derivatives and their efficacy in displacing [SH]IAA-94 from its binding site. Further, other chloride channel inhibitors, including AA derivatives, ethacrynic acid, bumetanide, and DIDS, also displaced the ligand from its binding site. A similar conductance was found in apical membrane vesicles from bovine trachea that was also inhibited by IAA-94 and AA-130B, but the inhibitory effects of these compounds were weaker than their effects on the renal cortex channel. The two drugs were also less potent in displacing [SH]IAA-94 from the tracheal binding site.