Cl- channels in basolateral TAL membranes. XIX. Cytosolic Cl- regulates mmCIC-Ka and mcCIC-Ka channels

Abstract

We evaluated the effects of culturing mouse MTAL cells under conditions that suppressed steady-state cytosolic Cl- on chloride channels fused into bilayers from basolateral vesicles of cultured MTAL cells. We used two agents to suppress Cl- entry: 10(-6) M PGE2 and 10(-4) M bumetanide. Basolateral Cl- channels from control cultured MTAL cells exhibited the signature characteristics of mmCIC-Ka channels: increased open-time probability (Po) either by raising cytosolic-face [Cl-] or, at 2 mM cytosolic Cl-, by adding (ATP + PKA), and first-order conductance kinetics. Either 10(-6) M PGE2 or 10(-4) M bumetanide in culture media reduced steady-state MTAL cytosolic Cl-. Chloride channels from these cells exhibited characteristics unique to CTAL mcCIC-Ka channels, namely: no augmentation of Po either by raising cytosolic Cl- or with cytosolic (ATP + PKA), and multi-ion occupancy. Semi-quantitative RT-PCR and real-time quantitative PCR showed that culturing MTAL cells with 10(-6) M PGE2 or 10(-4) M bumetanide reduced mRNA levels encoding mmCIC-Ka but not mRNA levels encoding mcCIC-Ka. However, when MTAL cells were cultured under control conditions, and then pre-incubated for 60 minutes with 10(-4) M bumetanide, cytosolic Cl- fell acutely but Cl- channels exhibited characteristics of mmCIC-Ka channels. Thus PGE2 and bumetanide, both of which lower steady-state MTAL cytosolic Cl- concentrations, inhibit either the transcriptional and/or the translational processes for mmCIC-Ka synthesis.