Fig. 3.

Identification and characterization of PKD2 hydrophobic gate. a Each single underlined aa of human PKD2 was mutated to N. Representative I–V curves were obtained in oocytes expressing WT or a mutant PKD2, as indicated, in the presence of the divalent free Na-containing solution (in mM): 100 NaCl, 2 KCl, 10 HEPES at pH 7.5. The gain-of-function mutant F604P (in the S5 helix) serves as a positive control and water-injected oocytes as a negative control (Ctrl). b Averaged currents at +80 mV obtained under the same experimental conditions as in a in expressing or control oocytes, as indicated. Currents were averaged from 10–13 oocytes from at least three batches. Data are presented as mean ± SEM. c Upper panel, averaged currents obtained as in panel a for PKD2 WT, A678 mutants, L677N or F604P (n = 8–13). Data are presented as mean ± SEM. Lower panel, western blot of surface biotinylated and total protein of PKD2 WT or indicated mutants. d Upper panel, the PKD2 L677 was replaced with different aa as indicated. Shown are averaged currents obtained as in a, with F604P as a positive control (n = 10–14). Data are presented as mean ± SEM. Lower panel, western blot showing total protein of PKD2 WT or indicated mutants present in the injected oocytes. e Representative I-V curves for F604P/L677 double mutants, as indicated, under the same condition as in a. f Averaged currents at +80 mV recorded from oocytes injected with cRNA of the indicated PKD2 WT or F604P/L677 double mutants (n = 8–12). Data are presented as mean ± SEM. g Averaged currents at +80 mV obtained from oocytes expressing PKD2 WT, or indicated single or double mutants (n = 9–12). Data are presented as mean ± SEM. h Averaged currents at +80 mV obtained from oocytes expressing PKD2 WT, L677N or L677N/N681L mutant. Data are presented as mean ± SEM