Fig. 4: E208 and R581 within PC-2 are key amino acids required for 7β,27-DHC-dependent activation.
From: Cilia-enriched oxysterol 7β,27-DHC is required for polycystin ion channel activation
A, B Mutation of E208A or R581A does not affect plasma membrane or ciliary trafficking of the polycystin complex. Live cell immunostaining of HEK cells (top: DOX-induced, bottom: DOX-uninduced) stably expressing sPC-1/PC-2, sPC-1/PC-2E208A or sPC-1/PC-2R581A using an anti-HA antibody (green), indicative of sPC-1 trafficking. Data represent one intensity value per image. sPC-1/PC-2 (+Dox: n = 3, −Dox: n = 1), sPC-1/PC-2E208A (+Dox: n = 5, −Dox: n = 1), sPC-1/PC-2R581A (+Dox: n = 5, −Dox: n = 1). Two-tailed unpaired student’s T-test. C sPC-1/PC-2 E208A or R581A are insensitive to 7β,27-DHC-dependent activation. Whole-cell patch-clamp recordings of HEK cells stably expressing sPC-1/PC-2, sPC-1/PC-2E208A or sPC-1/PC-2R581A and respective double mutant with additional GOF F604P. The same voltage step pulse protocol from Fig. 1D was applied to test binding mutants. sPC-1/2E208A and sPC-1/2R581A were tested with 5 μM 7β,27-DHC added to the intracellular solution, while the binding mutants with F604P (sPC-1/2E208A-F604P and sPC-1/2R581A-F604P) were tested without 5 μM 7β,27-DHC. D Comparison of current density at + 180 mV from whole-cell patch-clamp recordings of the two oxysterol-binding mutants. The current amplitudes at +180 mV with E208A (green, n = 10) and R581A (yellow, n = 11) were compared to those of the double mutants, E208A-F604P (green, n = 6) and R581A-F604P (yellow, n = 5). Two-tailed unpaired Student’s t-test *P < 0.05, ***P < 0.0001. E, F Immunofluorescent imaging analysis of ciliary expression of sPC-1 with PC-2E208A or PC-2R581A. Ciliary localization of sPC-1 co-expressed with PC-2, PC-2E208A and PC-2R581A was confirmed using anti-PC-2 (red) and anti-acetylated tubulin antibodies (cilia marker, green). Each n indicates the number of cilia for each group. PC-2 (n = 328), PC-2E208A (n = 289) and PC-2R581A (n = 374). **P < 0.005 between the indicated groups tested by One-way ANOVA. G Representative excised ciliary single channel recordings. Single channel recordings of parent PC-2 knockout IMCD-3 cells or overexpressing sPC-1 with PC-2, PC-2E208A or PC-2R581A without intracellular 7β,27-DHC application are shown at +100 mV and −100 mV. Error bar, mean ± SEM.
