Kinetics and regulation of a Ca2+-activated Cl- conductance in mouse renal inner medullary collecting duct cells.

Using the whole cell patch-clamp technique, a Ca2+-activated Cl- conductance (CaCC) was transiently activated by extracellular ATP (100 microM) in primary cultures of mouse inner medullary collecting duct (IMCD) cells and in the mouse IMCD-K2 cell line. ATP also transiently increased intracellular Ca2+ concentration ([Ca2+]i) from 100 nM to peak values of approximately 750 nM in mIMCD-K2 cells, with a time course similar to the ATP-induced activation and decay of the CaCC. Removal of extracellular Ca2+ had no major effect on the peak Cl- conductance or the increase in [Ca2+]i induced by ATP, suggesting that Ca2+ released from intracellular stores directly activates the CaCC. In mIMCD-K2 cells, a rectifying time- and voltage-dependent current was observed when [Ca2+]i was fixed via the patch pipette to between 100 and 500 nM. Maximal activation occurred at approximately 1 microM [Ca2+]i, with currents losing any kinetics and displaying a linear current-voltage relationship. From Ca2+-dose-response curves, an EC50 value of approximately 650 nM at -80 mV was obtained, suggesting that under physiological conditions the CaCC would be near fully activated by mucosal nucleotides. Noise analysis of whole cell currents in mIMCD-K2 cells suggests a single-channel conductance of 6-8 pS and a density of approximately 5,000 channels/cell. In conclusion, the CaCC in mouse IMCD cells is a low-conductance, nucleotide-sensitive Cl- channel, whose activity is tightly coupled to changes in [Ca2+]i over the normal physiological range.

The voltage-dependent Cl(-) channel ClC-5 and plasma membrane Cl(-) conductances of mouse renal collecting duct cells (mIMCD-3).

1. We have tested the hypothesis that the voltage-dependent Cl(-) channel, ClC-5 functions as a plasma membrane Cl(-) conductance in renal inner medullary collecting duct cells. 2. Full-length mouse kidney ClC-5 (mClC-5) was cloned and transiently expressed in CHO-K1 cells. Fast whole-cell patch-clamp recordings confirmed that mClC-5 expression produces a voltage-dependent, strongly outwardly rectifying Cl(-) conductance that was unaffected by external DIDS. 3. Slow whole-cell recordings, using nystatin-perforated patches from transfected CHO-K1 cells, also produced voltage-dependent Cl(-) currents consistent with ClC-5 expression. However, under this recording configuration an endogenous DIDS-sensitive Ca(2+)-activated Cl(-) conductance was also evident, which appeared to be activated by green fluorescent protein (GFP) transfection. 4. A mClC-5-GFP fusion protein was transiently expressed in CHO-K1 cells; confocal laser scanning microscopy (CLSM) showed localization at the plasma membrane, consistent with patch-clamp experiments. 5. Endogenous expression of mClC-5 was demonstrated in mouse renal collecting duct cells (mIMCD-3) by RT-PCR and by immunocytochemistry. 6. Using slow whole-cell current recordings, mIMCD-3 cells displayed three biophysically distinct Cl(-)-selective currents, which were all inhibited by DIDS. However, no cells exhibited whole-cell currents that had mClC-5 characteristics. 7. Transient transfection of mIMCD-3 cells with antisense mClC-5 had no effect on the endogenous Cl(-) conductances. Transient transfection with sense mClC-5 failed to induce the Cl(-) conductance seen in CHO-K1 cells but stimulated levels of the endogenous Ca(2+)-activated Cl(-) conductance 24 h post-transfection. 8. Confocal laser scanning microscopy of mIMCD-3 cells transfected with mClC-5-GFP showed that the protein was absent from the plasma membrane and was instead localized to acidic endosomal compartments. 9. These data discount a major role for ClC-5 as a plasma membrane Cl(-) conductance in mIMCD-3 cells but suggest a role in endosomal function.

The swelling-activated anion conductance in the mouse renal inner medullary collecting duct cell line mIMCD-K2.

Swelling-activated Cl(-) currents (I(Cl,swell)) have been characterized in a mouse renal inner medullary collecting duct cell line (mIMCD-K2). Currents activated by exposing the cells to hypotonicity exhibited characteristic outward rectification and time- and voltage-dependent inactivation at positive potentials and showed an anion selectivity of I(-) > Br(-) > Cl(-) > Asp(-). NPPB (100 microm) inhibited the current in a voltage independent manner, as did exposure to 10 microm tamoxifen and 500 microm niflumic acid (NFA). In contrast, DIDS (100 microm) blocked the current with a characteristic voltage dependency. These characteristics of I(Cl, swell) in mIMCD-K2 cells are essentially identical to those of heterologously expressed cardiac CLC-3. A defining feature of CLC-3 is that activation of PKC by PDBu inhibits the conductance. In mIMCD-K2 cells preincubation with PDBu (100 nm) prevented the activation of I(Cl,swell) by hypotonicity. However, PDBu inhibition of I(Cl,swell) was reversed after PDBu withdrawal, but this was refractory to subsequent PDBu inhibition. Activation of either the cystic fibrosis transmembrane conductance regulator (CFTR) or Ca(2+) activated Cl(-) conductance (CaCC), which are coexpressed in mIMCD-K2 cells prior to PDBu treatment, abolished the PDBu inhibition of I(Cl,swell). Control of I(Cl,swell) by PKC therefore depends on the physiological status of the cell. In intact mIMCD-K2 layers in Ussing chambers, forskolin stimulation of an inward short-circuit current (due to transepithelial Cl(-) secretion via apical CFTR) was inhibited by cell swelling upon hypotonic exposure at the basolateral surface. Activation of I(Cl,swell) is therefore capable of regulating transepithelial Cl(-) secretion and suggests that I(Cl,swell) is located at the basolateral membrane. PDBu exposure prior to or during hypotonic challenge was ineffective in reversing the swelling-activated inhibition of Cl(-) secretion, but tamoxifen (100 microm) abolished the hypotonic inhibition of forskolin-stimulated short-circuit current (I(sc)). RT-PCR analysis confirmed expression of mRNA for members of the CLC family, including both CLC-2 and 3, in the mIMCD-K2 cell line.

Ca2+ and cAMP-activated Cl- conductances mediate Cl- secretion in a mouse renal inner medullary collecting duct cell line.

1. The nature of Cl- conductance(s) participating in transepithelial anion secretion by renal inner medullary collecting duct (IMCD, mIMCD-K2 cell line) was investigated. 2. Extracellular ATP (100 microM) stimulated a transient increase in both whole-cell Cl- conductance and intracellular free Ca2+. In contrast, ionomycin (10-100 nM) caused a sustained increase in whole-cell Cl- conductance. Pre-loading cells with the Ca2+ buffer BAPTA abolished the ATP-dependent responses and delayed the onset of the increase observed with ionomycin. 3. The Ca2+-activated whole-cell Cl- current stimulated by ATP (peak) and ionomycin (maximal) displayed (i) a linear steady-state current-voltage relationship and (ii) time and voltage dependence with slow activation at +80 mV and slow inactivation at -80 mV. In BAPTA-loaded cells, ionomycin-elicited whole-cell currents exhibited pronounced outward rectification with time-dependent activation/inactivation. 4. Ca2+-activated and forskolin-activated Cl- conductances co-exist since ATP activation of whole-cell current occurred during a maximal stimulation by forskolin in single cell recordings. 5. In IMCD epithelial layers, ATP and ionomycin stimulated an inward short circuit current (Isc) dependent upon basal medium Na+ and Cl-/HCO3- but independent of the presence of apical bathing medium Na+ and Cl-/HCO3-. This was identical to forskolin stimulation and consistent with transepithelial anion secretion. 6. PCR amplification of reverse-transcribed mRNA using gene-specific primers demonstrated expression of both cystic fibrosis transmembrane conductance regulator (CFTR) mRNA and Ca2+-activated Cl- channel (mCLCA1) mRNA in mIMCD-K2 cells. 7. Ca2+ and forskolin-activated Cl- conductances participate in anion secretion by IMCD.

Bradykinin regulation of salt transport across mouse inner medullary collecting duct epithelium involves activation of a Ca(2+)-dependent Cl(-) conductance.

The mechanism by which bradykinin regulates renal epithelial salt transport has been investigated using a mouse inner medullary renal collecting duct cell-line mIMCD-K2. Using fura-2 loaded mIMCD-K2 cells bradykinin (100 nM) has been shown to induce a transient increase in intracellular Ca(2+) via activation of bradykinin B2 receptors localized to both the apical and basolateral epithelial cell surfaces. In mIMCD-K2 epithelial cell-layers clamped in Ussing chambers, 100 nM bradykinin via apical and basolateral bradykinin B2 receptors stimulated a transient increase in inward short-circuit current (I:(sc)) of similar duration to the increase in intracellular Ca(2+). Replacements of the bathing solution Na(+) by the impermeant cation, N-methyl-D-glucamine and of Cl(-) and HCO(3)(-) by the impermeant anion gluconate at either the apical (no reduction) or basal bathing solutions (abolition of the response) are consistent with the bradykinin-stimulated increase in inward I:(sc) resulting from basal to apical Cl(-) (anion) secretion. Using the slow whole cell configuration of the patch-clamp technique, bradykinin was shown to activate a transient Cl(-) selective whole cell current which showed time-dependent activation at positive membrane potentials and time-dependent inactivation at negative membrane potentials. These currents were distinct from those activated by forskolin (CFTR), but identical to those activated by exogenous ATP and are therefore consistent with bradykinin activation of a Ca(2+)-dependent Cl(-) conductance. The molecular identity of the Ca(2+)-dependent Cl(-) conductance has been investigated by an RT - PCR approach. Expression of an mRNA transcript with 96% identity to mCLCA1/2 was confirmed, however an additional but distinct mRNA transcript with only 81% of the identity to mCLCA1/2 was identified.

Single-channel properties of swelling-activated anion conductance in rat inner medullary collecting duct cells.

Single-channel properties of the volume-activated outwardly rectifying Cl- conductance of rat IMCD cells were studied in primary cultures by means of the patch-clamp technique in the whole cell and in the outside-out configuration. Measurements were performed by noise analysis and in single-channel recordings during voltage-induced current inactivation and reactivation and in long-lasting experiments at constant membrane voltages. Unitary conductances could be defined for the voltage range of -100 to -50 mV and between +50 and +120 mV and chord conductances of 34.1 and 76.6 pS, respectively, can be calculated. The overall current-to-voltage relationship very much resembles that of the macroscopic Cl- conductance and the open probability of the activated channel is close to unity (Po = 0.98-0.99). The channel exhibits many similarities to volume-activated outwardly rectifying Cl- channels found in other systems although certain species differences do exist.

Taurine permeation through swelling-activated anion conductance in rat IMCD cells in primary culture.

Whole cell recordings were performed on rat inner medullary collecting duct (IMCD) cells in primary culture. With 140 mmol/l CsCl in bath and pipette we find within 10 min a 60-fold increase in membrane conductance from 0.02 +/- 0.003 to 1.2 +/- 0.1 nS/pF when bath osmolarity is decreased from 600 to 500 mosmol/l. The effect is due to the activation of an outwardly rectifying anion conductance with the anion selectivity SCN- > I- > NO-3 > Br- > Cl- > F- > isethionate > gluconate > or = aspartate > or = glutamate. A relative permeability of the organic osmolyte taurine to Cl- (Ptaurine: PCl-) of 0.15 was detected. With taurine in pipette and bath, the channel exhibits a nearly identical activation and sensitivity profile to a variety of anion channel blockers as under symmetrical Cl- conditions. Furthermore, the 50% inhibitory concentration value for the effect of 5-nitro-2-(3-phenylpropylamino)benzoate on both currents is virtually identical. We conclude that hypotonic stress increases the anion conductance of rat IMCD cells and that this anion conductance mediates taurine efflux.

Osmoregulation in the renal papilla: membranes, messengers and molecules.

This contribution summarizes recent progress in the understanding of the molecular basis of the release of organic osmolytes that occurs when inner medullary cells are confronted with a drop in osmolarity in their environment. For sorbitol release across the basolateral membrane an increase in intracellular calcium seems to be the prominent signal, initiated by G-protein activation, followed by phosphatidylcholine phospholipase activation and generation of arachidonic acid. The increase in betaine permeability is also G-protein dependent but calcium independent, and is restricted to the basal-lateral cell face. Myo-inositol and glycerophosphorylcholine efflux are calcium and G-protein independent and occur both across the apical and basolateral membrane, although to a different extent. Taurine release is also calcium and G-protein independent; a swelling-activated anion channel at the basolateral membrane represents the major efflux pathway.