(A) Illustration of PC-1 and PC-2F604P topology highlighting extracellular HA and secretion sequence (indicated with an s prefix) in PC-1. (B) Expression and immunostaining of HEK293 cells transfected with sPC-1/PC-2, HAPC-1/PC-2 with endogenous leader sequence, sPC-1/PC-2F604P, and sPC-1 alone. Left column, representative images of HA surface staining (see methods). Middle column, total staining for PC-2 or PC-1. Right column, merged images. Scale bar: 10 μm. (C) Quantification of relative anti-HA surface staining shown in B. ***: p<0.0001. (D) Representative currents from each construct recorded in the whole cell patch clamp configuration in response to voltage step pulses (left). (E) Whole-cell I-V relationship of sPC-1/PC-2F604P (n = 6, red) and PC-1/PC-2F604P (n = 10, white). Arrow indicates the expanded view of I-V relationship between membrane potentials of −50 mV and +50 mV. (F) Current densities for sPC-1/PC-2F604P, HAPC-1/PC-2F604P, sPC-1/PC-2, and PC-2F604P alone at +100 mV. (G) Relative open probability of sPC-1/PC-2F604P at −80 mV calculated using a Boltzmann distribution fitted to I/Imin of tail current amplitudes, black line (n = 6, red). (H) Top view of the human PC-1/PC-2 polycystin complex using pymol (Su et al., 2018). Positively charged R4100, R4107, and H4111 are highlighted in red and displayed in stick shape (top). Side view of selectivity filter showing the location of three positively charged amino acids on PC-1 TM-11 (bottom). (I) Representative whole cell currents of sPC-1pore/PC-2F604P. (J) I-V relationship of sPC-1/PC-2F604P (n = 6, red) and sPC-1pore/PC-2F604P (n = 6, blue). Arrow indicates the expanded view of I-V relationship between −50 mV and +50 mV. Current densities of sPC-1/PC-2F604P and sPC-1pore/PC-2F604P were compared at +100 mV holding potential. (K) Relative open probability analysis of sPC-1/PC-2F604P (n = 6, red) and sPC-1pore/PC-2F604P (n = 6, blue) during a −80 mV tail pulse. Statistical significance is indicated by the background. ***p<0.001 (dark gray), **p<0.01 (gray), *p<0.05 (light gray). Statistical analysis was computed using student t-test unpaired one-pair.