The chloride current in urine responses was dependent of a calcium influx. (A) In a representative VSN recorded using perforated patch clamp technique, the urine-induced inward current (V_hold = −80 mV) in 0 Ca²⁺ Ringer's ([Ca²⁺] << 10 nM) was not influenced by 300 µM DIDS (second vs. third traces) but eliminated by replacing extracellular Na⁺ with choline (0 Ca²⁺-0 Na⁺ Ringer's; bottom trace). The interval between urine applications was 2–3 min. (B) In all the VSNs tested, the amplitude of urine-induced inward current in 0 Ca²⁺ Ringer's was the same as that in 0 Ca²⁺ Ringer's plus 300 µM DIDS. The urine response was abolished in 0 Ca²⁺-0 Na⁺ Ringer's. The open circle is the value of the urine response normalized to the initial urine response in Ringer's (control) for each cell. The filled circles and error bars are the mean and SEM for the cells tested in the same conditions. The number of cells is listed on the top. NS, not significant difference, Wilcoxon signed rank test. DU, 1:500 dilute urine.