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 mu M) inhibited the current in a voltage independent manner, as did exposure to 10 mu M tamoxifen and 500 mu M niflumic acid (NFA), In contrast, DIDS (100 mu M) 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-CI.swell. Control of I-CI.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 mu M) 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.