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

Lookup NU author(s): Dr Stefan Boese, Dr Michael Glanville, Dr Michael Gray, Professor Nicholas Simmons


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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- > B1- > Asp-. NPPB (100 μM) inhibited the current in a voltage independent manner, as did exposure to 10 μM tamoxifen and 500 μM niflumic acid (NFA). In contrast, DIDS (100 μM) 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 Ca2+ 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 suggest 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 μM) 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.

Publication metadata

Author(s): Glanville M; Gray MA; Boese SH; Simmons NL

Publication type: Article

Publication status: Published

Journal: Journal of Membrane Biology

Year: 2000

Volume: 177

Issue: 1

Pages: 51-64

ISSN (print): 0022-2631

ISSN (electronic): 1432-1424

Publisher: Springer New York LLC


DOI: 10.1007/s002320001099

PubMed id: 10960153


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