Presence of subconductance states in the PC2 channel. (A) Single-channel currents of endogenous hST PC2. Holding potential is 60 mV in asymmetrical K⁺. (B) Three intermediate subconductance levels are identifiable in the all-point histogram. C, closing state; O₁–O₄, four sub-conductance states. (C) Expanded single-channel current subconductance states of PC2 from region indicated with horizontal line in (A). (D) Examples of subconductance states in the purified PC2 reconstituted protein as in (26). Spontaneous, full channel closures (arrow) are followed by ‘staircase’ activation of the four subconductance states, revealing four identical functional subunits (dashed lines). This is further evidenced by the mean versus variance analysis of the tracing (Right). (E) The mean activation was calculated by averaging kinetics after full closures. Voltage-dependence of subconductance states (open circles, 40 mV, compared with filled symbols, 0 mV, P < 0.02). Data are the average of nine and seven sojourns, from five experiments, respectively. Averages do not display discrete steps, indicating the variable time spent by the channel at each subconductance level. There is a kinetic delay only observed at 0 mV. (F) Spontaneous subconductance states in PC2 (Top, dashed lines) can be further modified by reduction in cytoplasmic pH (cis chamber), which decreases PC2 channel activity as previously reported (26). Solid lines indicate the respective averages at normal pH (7.25, also see E). (G) The quaternary structure of PC2 was confirmed by gel electrophoresis of affinity purified PC2 under denaturing (Left) and non-denaturing (Right) conditions. The presence of 440 kDa protein complexes (tetramers) were observed under non-denaturing conditions, in contrast to denaturing conditions, which only showed monomers (∼110 kDa), dimers (∼210 kDa) and a cleaved form of PC2 (∼70 kDa). Lane 1 indicates purified protein and lane 2 the whole cell lysate in the native gel (Right).

Electrophysiology of the TRPC1 channel. (A) Single-channel currents of affinity-purified TRPC1 at different holding potentials in asymmetrical K⁺. (B) Current-to-voltage relationship for the TRPC1, indicating a single-channel conductance of 5.0 ± 0.7 pS (n = 5), and a bigger conductance of 22.8 ± 2.0 pS (n = 5). (C) Representative tracing showing a TRPC1 channel complex displays four subconductance states at +100 mV. (D) All-point histogram of (C) shows the presence of four subconductance states in TRPC1. (E) Effect of amiloride (200 µM, trans side) on TRPC1 single-channel currents. Addition of amiloride to the trans chamber was without the effect on the purified channel. Representative tracings from n = 10. (F) Lowering the cytoplasmic pH had no effect on the TRPC1 single-channel currents. The cytoplasmic pH was reduced by addition of 1.0 N HCl to the cis chamber. Data representative of n = 7 experiments.

Electrophysiological features of the PC2/TRPC1 hetero-complex. PC2/TRPC1 hetero-complex displayed two conductances (A and B), which correspond to the smaller TRPC1 and bigger PC2 channels. (C) Shows the all point histogram of the extended tracing (B). (D) The I–V relationship of PC2/TRPC1 hetero-complex shows a conductance (red lines and circle) between those of homomeric PC2 (157 pS, higher grey dashed line) (26) and homomeric TRPC1 (∼23 pS, lower green line indicates the fitted I–V relationship for TRPC1 homomers as shown in (Fig. 2B). There is also a lower conductance present in the PC2/TRPC1 hetero-complexes (the blue triangles and line), which is close to the conductance of homomeric TRPC1. The black square represents PC2/TRPC1 complexes showing a higher conductance, closer to PC2 contribution alone. (E) The bar graph for the conductance of PC2, TRPC1 and PC2/TRPC1 complexes indicates that the single-channel conductance of the heterocomplex is statistically different from either homomer alone. * indicates P < 0.001.

Electrophysiological features of the PC2/TRPC1 hetero-complex. Effect of amiloride. (A) Single-channel currents of reconstituted PC2/TRPC1 channel complexes in asymmetrical K⁺. Note the flickering in the large conductance deflections, consistent with PC2 contribution, contrasting the much longer open probability of the small conductance states observed in the TRPC1 channel contribution to the complex. (B) Expanded single-channel currents unmask the large conductance PC2, and smaller conductance TRPC1 under control conditions. (C) PC2 conductance was blocked by amiloride but TRPC1 conductance persisted. All-point histograms to the right side of the panels indicate the channel conductances under each condition. Data are representative of n = 7 independent experiments.

Electrophysiological features of the PC2/TRPC1 hetero-complex. Effect of lowering pH. The PC2/TRPC1 hetero-complexes were insensitive to changes in pH (cis chamber). Data were obtained in the presence of KCl chemical gradient. Lowering the pH to either 6.4 (A) or 5.9 (B) had no effect on the channel activity of PC2/TRPC1 hetero-complexes. Data are representative of n = 7.

AFM imaging of PC2. (A) PC2 was imaged in flattened, PC2-containing proteoliposomes, onto freshly cleaved mica in saline solution. (B) Upper panel, PC2 channels were identified in AFM scans as round structures, often displaying a central indentation, and a tetra-lobular structure. Lower panel (1–3) showed three expanded PC2 structures highlighted in the circles on the upper panel. (C) Cross-section histograms from B1 – B3 indicate the height and diameter of the contributing monomers, and the central indentation. Three such measurements are superimposed in the graph. (D) Population analysis of channel units indicates that the monomers segregated in a scattered population (n = 107). (E) Analysis of the distribution of diameters indicates two peaks, suggesting asymmetry in the shape of the channels. (F) Further analysis of the distribution of heights indicates three peaks.

Effect of anti-PC2 antibody on AFM imaging of PC2. (A) PC2 monomers were identified in AFM scans (Scale bar, 500 nm) as round structures and the exposure to the anti-PC2 antibody (Zymed Catalog No. 52-5217) increased both the height and the diameter of PC2. (B) The cross sections at the lines indicated in (A) show the binding of PC2 antibody increased the apparent diameter and the height of PC2. Data are representative of two-paired experiments.

Changes in AFM imaging of PC2 at low pH. (A) PC2 was imaged in flattened, PC2-containing proteoliposomes, onto freshly cleaved mica in saline solution (pH 7.4). (B) Lowering the sample's pH (∼6.1) decreased PC2's extra-membrane size. Data are representative of three-paired experiments.

AFM imaging of TRPC1. (A) TRPC1 channels were identified in AFM scans (Scale bar, 500 nm) from flattened, TRPC1-containing proteoliposomes, placed onto freshly cleaved mica in saline solution as tetra-lobular structures as shown in the panel at the bottom. (B) The tetrameric structure can be easily distinguished in higher magnification scans (B panels 1,2). (C) Cross-section histograms from dashed lines in (B) indicate the height and diameter of the contributing monomers, and the central indentation. Two such measurements are superimposed (B panels 1,2) in the graph. (D) Population analysis of channel units indicates the average diameter and height, respectively (n = 105). (E) Analysis of the distribution of diameters indicates two peaks, suggesting asymmetry in the shape of the channels. (F) Further analysis of the distribution of heights indicates three peaks.

Distribution of the molecular volumes of PC2 and TRPC1 homomeric complexes. (A) The height–diameter relationship of PC2 (n = 97). Complexes with height higher than 2 nm were excluded from the analysis. A linear fitting showed no/weak linear correlation between height and diameter (R² = 0.0934). (B) Histogram of calculated molecular volume of PC2 molecular complexes. (C) Height–diameter relationship for TRPC1 (n = 94). Note the complexes with height higher than 5 nm were excluded from the analysis. A linear fitting also revealed no/weak correlation between height and diameter (R² = 0.161). (D) Histogram of calculated molecular volumes of TRPC1 complexes.

AFM imaging of PC2/TRPC1 hetero-complexes. (A) PC2/TRPC1 hetero-multimers were prepared by mixing (1:1) affinity purified PC2 and TRPC1 proteins, which were incorporated into liposomes. PC2/TRPC1-containing proteoliposomes were placed and flattened onto freshly cleaved mica in saline solution. The amplitude (Left), height (Middle) and the perspective (Right) images show the structure of both lipid and channel complexes at low magnification. (B) The height (Left) and perspective (Right) images at high magnification show that the hetero-multimeric channel complexes displayed lax, tetra-lobular shapes. These PC2/TRPC1 channel complexes had four contributing lobules of different height and a central indentation. The circles are examples of PC2/TRPC1 hetero-tetramers; the squares and hexagons highlight the TRPC1 homo-tetramer and PC2 homo-tetramer, respectively. (C) The height and diameter of the monomeric units in the PC2/TRPC1 channel complex could be easily identified in the cross-section histograms (insert, dashed lines). The higher peak correlates with TRPC1 monomers, whereas the lower peak with PC2 monomers.