Functional activity and expression of calcium-activated chloride channel TMEM16A in epididymal principal cells. (Aa) Whole-cell current of a principal cell showing sensitivity toward the putative CaCC blocker niflumic acid (NFA; 10–100 µM). Currents were in response to the repeatedly applied depolarizing episodes from a holding potential of −60 to 60 mV. Bath solution was normal PSS, and pipette solution was K⁺ based with 0.1 mM EGTA. (Ab and Ac) Typical current tracing from a principal cell in response to a series of 500-ms, 20-mV increment voltages steps applied between −120 and 60 mV evoked from the holding potential −60 mV before (Ab) and after (Ac) the addition of 100 µM niflumic acid. (B) Current responses of a principal cell obtained in the control condition with 2.5 mM Ca²⁺ in the bathing solution (Ba) and in the nominally extracellular Ca²⁺-free condition (0 mM [Ca²⁺]_o; Bb). The dialysed pipette solution was K⁺ based with 0.1 mM EGTA. (Bc) The subtraction revealed that the extracellular Ca²⁺-sensitive currents were mainly the outwardly rectifying current with time-dependent activating kinetics at positive potentials as well as a small transient decaying current at negative potentials. (Bd) Current tracings recorded from a principal cell in response to a hyperpolarizing step from −60 to −120 mV before and after removal of extracellular Ca²⁺. Dotted lines indicate the zero current levels. (Be) I-V plots for the conditions as described in Ba–Bc. Data were measured at the end of testing pulses. Error bars represent SEM. (C) Conventional RT-PCR detection of the TMEM16A mRNA in the rat epididymis and efferent duct. CD, cauda epididymides; CPS, corpus; CPT, caput; ED, efferent duct; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IS, initial segment; M, marker; NTC, no template control. (D–G) Confocal immunofluorescent images showing the localization of TMEM16A protein (red) in the apical pole of principal cells of the rat interzone and cauda epididymides and in the ciliated cells of the efferent duct. Weak immunoreactivity was also detected in smooth muscle cells and in some interstitial cells, but negligible immunoreactivity was detected in principal cells of the caput and corpus epididymis. (H) Western blot detection of TMEM16A protein using the same antibody for immunofluorescent labeling in the crude protein extracts from rat trachea (Trach.) and epididymis (Epid.). The DNA of epithelial cells and sperm is labeled with DAPI in blue. L, lumen. Bar, 50 µm.