Identification of ClC-2 and ClC-K2 chloride channels in cultured rat type IV spiral ligament fibrocytes

Voltage-gated chloride channels (CICs) are important mediators of cellular ion homeostasis and volume regulation. In an earlier study, we used immunohistochemical, Western blot, and reverse transcriptase PCR (RT-PCR) approaches to identify CIC-K variants in types II, IV, and V fibrocytes of the rodent spiral ligament. We have now confirmed the expression of CIC-K2 in these cells by in situ hybridization. All three of these fibrocyte subtypes are thought to be involved in cochlear K+ recycling; thus, it is important to understand the precise mechanisms regulating their membrane conductance and the role played by ClCs in this process. In this study, we report the characterization of a secondary cell line derived from explants from the region of the rat spiral ligament underlying and inferior to the spiral prominence. The cultured cells were immunopositive for vimentin, Na,K/ATPase, Na,K,CI-cotransporter, carbonic anhydrase isozyme II, and creatine kinase isozyme BB, but not for cytokeratins or Ca/ATPase, an immunostaining profile indicative of the type IV subtype. Evaluation of the cultures by RT-PCR and Western blot analysis confirmed the presence of both CIC-2 and -K2. Whole-cell patch clamp recordings identified two biophysically distinct Cl- currents in the cultured cells. One, an inwardly rectifying Cl- current activated by hyperpolarization or decreasing extracellular pH corresponded with the properties of CIC-2. The other, a weak outwardly rectifying Cl- current regulated by extracellular pH, Cl-, and Ca2+ resembled the channel characteristics of CIC-K2 when expressed in Xenopus oocytes. These findings suggest that at least two functionally different chloride channels are involved in regulating membrane anion conductance in cultured type IV spiral ligament fibrocytes.