Nonselective cation channel of hST. (a) Single channel currents in asymmetrical KCl (150/15 mM). Tracings were filtered at 50 Hz to show the unitary conductance in the absence of subconductance states. Holding potentials are indicated on the right. (b) Current-to-voltage (I/V) relationship in asymmetrical KCl (n = 10) was fitted with the Goldman-Hodgkin-Katz (GHK) equation (solid line). (c) Multiple conductance substates (Top) were identified as mean vs. variance plots (Middle), and low variance histograms (Bottom). (d) Single channel currents in symmetrical KCl (150 mM). (e) I/V relationships in KCl (filled circles, n = 6), Na⁺/K⁺ (open triangles, n = 6) and K⁺/Ca²⁺ (open circles, n = 3). (f) Single channel currents in K⁺/Na⁺ (n = 6), and K⁺/Ca²⁺ (g, n = 3). (h) Inhibition of (cis) K⁺ currents by addition of Ca²⁺ in trans (n = 3).

Inhibition of cation channels, and expression of polycystin-2 in hST. Single channel K⁺ currents were inhibited by La³⁺ (a, n = 3), by amiloride (b, n = 5), and anti-polycystin-2 antibody (1:10 dilution, n = 3) added to the cis side of the chamber (c). (d) RT-PCR with PKD2 (≈570 bp, lane B) but not PKDL primers (lane C). Positive control was mouse kidney (lane A). (e) Western blot analysis of ≈110-kDa protein corresponding to polycystin-2 (lane B, arrow). Lane A indicates fetal rat kidney as positive control. (f) Hematoxylin & eosin staining of hST, indicating apical syncytial epithelial cells (arrows), and vessels (arrow heads) (×180). (g) Polycystin-2 immunolocalized to the apical membrane (arrows).

Expression of polycystin-2 in Sf9 insect cells. (a) Western blots of Sf9 cell extracts infected either with baculovirus lacking the polycystin-2 cDNA insert (lanes 1 and 2) or containing (lanes 3 and 4) polycystin-2 cDNA viral inserts from two independent clones. The position of recombinant polycystin-2 (≈110 kDa) is indicated by the arrow. Arrowheads indicate the position of GIBCO/BRL molecular weight markers (from top to bottom 97.4 kDa, 68 kDa, and 43 kDa, respectively). Extracts from ≈10⁶ cells are shown in each lane. (b) Single channel currents of cell-attached (c/a) patches of infected Sf9 cells. Channel activity was observed in the presence of CsCl (150 mM) in the pipette. Data are representative of nine experiments. (c) Single channel currents of excised inside-out patches. Data in Cs⁺/Na⁺ (150 mM) are representative of 35 experiments. (d Left) Single channel currents of infected Sf9 cells showed subconductance states (expanded tracing at the bottom), and further indicated by all-point histogram (Right). Subconductance states are indicated by asterisks, and main conductance by the arrow. (e) Polycystin-2-mediated single K⁺ currents were blocked by anti-flag antibody (n = 3). (f) Current-to-voltage relationships of most frequent single channel conductances, with average conductances of 16 pS (open circles, n = 8), 32 pS (open triangles, n = 10), 78 pS (open squares, n = 7), and 264 pS (open diamonds, n = 3).

Recombinant polycystin-2 is a nonselective cation channel. (a) Single K⁺ currents of flag-tagged purified polycystin-2 obtained in symmetrical K⁺. (b) Current-to-voltage relationships in asymmetrical K⁺ (Upper, n = 5), and symmetrical K⁺ (Lower, n = 5). (c) Polycystin-2 K⁺ channel activity was inhibited by amiloride (n = 18). (d) Polycystin-2-mediated single K⁺ currents were inhibited by anti-polycystin-2 antibody (1:10 dilution, n = 3). (e) Comparison of most frequent single channel conductance substates in hST and flag-tagged polycystin-2. (f) Dose-response of amiloride. The effect of increasing concentrations of amiloride on the known single-current for hST (filled circles, n = 10), and Sf9 purified material, n = 5, as fitted to the equation 1 − ([Amil]/[Amil] + K_i), where [Amil] = molar concentration of amiloride, obtaining a K_i of 27.0 and 79.4 μM, respectively.

Cation selective channel activity of in vitro translated polycystin-2. (a) In vitro translated S³⁵-labeled polycystin-2 identified by autoradiography after SDS/PAGE. Lane 1, control in the absence of PKD2-containing plasmid. Lane 2, polycystin-2 (110 kDa). Luciferase, with an expected 61 kDa was used as positive control (data not shown). (b) Single channel currents of in vitro translated polycystin-2 in a chemical K⁺ gradient (n = 24). Different conductance states are shown (Top and Middle). (c) Conductance substates are also observed in single channel tracings (n = 3). (d) Current-to-voltage relationships in asymmetrical K⁺. Single channel conductance substates are shown. (e) Small subconductance states can be observed in large conductance openings by mean vs. variance analysis as in Fig. 1c.