Fig. 1: 7β,27-DHC activates the polycystin complex on the plasma membrane.
From: Cilia-enriched oxysterol 7β,27-DHC is required for polycystin ion channel activation
A Strategy to express the polycystin complex in the plasma membrane. The black dotted arrow indicates ciliary trafficking of PC-1 with endogenous signal peptide and PC-2. Red arrow indicates redirected plasma membrane trafficking using a IgG kappa-derived secretion peptide on PC-128. B Immunofluorescent staining of HEK293 and IMCD-3 cells overexpressing the polycystin complex. Surface HA staining indicates that polycystin complex accumulates in plasma membrane of HEK cells (Top) or cilia (IMCD-3 cells, bottom). Green, surface HA; Red, total HA or cilia marker Arl13b; Magenta, PC-2; Images are representative for at least three independent experiments. scale bar = 10 µM. C Structure of 7β,27-dihydrocholesterol (DHC). D Whole-cell patch-clamp traces recorded from parental HEK293 cells or overexpressing sPC-1/2, PC-1 or PC-2. Voltage step pulse from −100 mV to +180 mV in +20 mV increments with 0 mV holding potential and 5 μM 7β,27-DHC in the pipette solution. E, F Current (I)–voltage (V) relationship and current density for HEK293 cells overexpressing sPC-1/2, PC-1 or PC-2. Each value was obtained at the maximum current (pA) from −100 mV to +180 mV and divided by capacitance (pF); sPC-1/2 (red, n = 16), PC-1 (orange, n = 12), PC-2 (green, n = 10), and HEK293 (n = 15). F Quantification of current density I (pA/pF) at +180 mV. Two-tailed unpaired Student’s t-test *P < 0.05. G Effect of extracellular application of 7β,27-DHC on polycystin channel activation. 5 μM 7β,27-DHC was applied in bath solution. Ramp pulse applied from −100 mV to +100 mV with 500 ms duration. Each value (pA) at −100 mV (gray square) and +100 mV (gray blank square) is divided by capacitance (pF) and plotted over time. Orange line indicates holding potential (0 mV). H, I Extracellular NMDG inhibits 7β,27-DHC evoked PC−1/2 currents. Blank and black squares indicate the current density (pA/pF) obtained at +100 mV and −100 mV, respectively. i Left. I–V relationship of polycystin currents in presence of Na+ (red) and NMDG (gray). Blue insert shows reversal potential shift as the extracellular solution changes from Na+ to NMDG+. Right. (E) I (pA/pF) at +100 mV and −100 mV before (red) and after (gray) NMDG perfusion. n = 5. Two-tailed paired Student’s t-test, *P < 0.05. Error bars: mean ± SEM.
