Fig 2: Role of extracellular Ca2+ ions in the PKD2L1 off-response.(A) Representative current recorded at -50 mV in a PKD2L1-expressing oocyte under various extracellular conditions, as indicated. (B) Based on experiments described in panel A, averaged data to show the effect of extracellular EGTA (1 mM). *p = 0.04, unpaired t-test. (C) Dose-dependence of the relative Ca2+ inhibition of the off-response current. Data were averaged from 11 oocytes and the curve is a fit of the data to Equation 1, with Ki value of 167 ± 33 µM.

Fig 3: Roles of Ca2+ ions in the PKD2L1 off-response.(A) Representative tracing recorded at −50 mV, showing the off-response activation and ensuing inactivation in the presence of extracellular Ca2+ (5 mM). (B) Representative current recorded at −50 mV in a PKD2L1-expressing oocyte, showing the inhibition of the off-response current by extracellular Ca2+ (5 mM). (C) Representative current recorded at −50 mV in a PKD2L1-expressing oocyte 1 hr after injection of 50 nL of 50 mM EGTA, showing the effect of extracellular Ca2+ (5 mM). (D) Averaged and normalized off-response currents with EGTA or water (Ctrl) pre-injection. (E) Averaged percentage of the off-response current inhibition by extracellular Ca2+ with EGTA or water pre-injection. ***p = 0.0007, unpaired t-test.

Fig 4: Effects of mutation D523N on the PKD2L1 on- and off-responses.(A) Averaged and normalized on- and off-response currents elicited by extracellular 5 mM Ca2+ and pH 3.0, respectively, from oocytes expressing WT or mutant PKD2L1 voltage clamped at -50 mV. Shown are on- (***p = 3.0e-6, unpaired t-test) and off-response currents (**p = 0.002, unpaired t-test) averaged from different numbers of oocyte, as indicated. (B) Representative off-response I-V curves obtained by a ramp protocol under the same condition as in panel A. (C) Western blotting data showing the protein expression of WT and mutant PKD2L1 in expressing or water-injected (Ctrl) oocytes. (D) Representative immunofluorescence data showing the PM localization of WT and mutant PKD2L1 in oocytes.

Fig 5: Identification of domain C1 of PKD2L1 critical for its channel function.(A) Representative whole-cell current tracings obtained from Xenopus oocytes expressing PKD2L1 truncation mutant T622X, S581X, or deletion mutant ΔC1 (C1 deletion from PKD2L1 WT) using the two-microelectrode voltage clamp technique under similar experimental conditions as those for Fig. 2. (B) Averaged currents obtained from oocytes expressing PKD2L1 WT or truncation/deletion mutants, as indicated. Currents at −50 mV were averaged from indicated numbers of oocytes and normalized to that of PKD2L1 WT. Water-injected oocytes were used as control (Ctrl). ‘***' indicates P ≤ 0.001 when comparing with “WT”. (C) Representative current–voltage relationship curves obtained using a voltage ramp protocol, as indicated in Fig. 2C, before (7.5) and after (7.5+Ca) addition of 5 mM CaCl2. (D) Representative immunofluorescence data showing the plasma membrane expression of mutants T622X, S581X and ΔC1 expressed in oocytes, or those injected with water (Ctrl). (E) Surface membrane expression of mutants T622X, S581X and ΔC1 were averaged from the indicated numbers of oocytes and normalized to that of PKD2L1 WT.