Cl? Channels in Basolateral TAL Membranes. XIX. Cytosolic Cl? Regulates mmClC-Ka and mcClC-Ka Channels

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 mmClC-Ka channels: increased open-time probability (P o) 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 mcClC-Ka channels, namely: no augmentation of P o 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 mmClC-Ka but not mRNA levels encoding mcClC-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 mmClC-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 mmClC-Ka synthesis.