Formation of a new receptor-operated channel by heteromeric assembly of TRPP2 and TRPC1 subunits

Abstract

Although several protein-protein interactions have been reported between transient receptor potential (TRP) channels, they are all known to occur exclusively between members of the same group. The only intergroup interaction described so far is that of TRPP2 and TRPC1; however, the significance of this interaction is unknown. Here, we show that TRPP2 and TRPC1 assemble to form a channel with a unique constellation of new and TRPP2/TRPC1-specific properties. TRPP2/TRPC1 is activated in response to G-protein-coupled receptor activation and shows a pattern of single-channel conductance, amiloride sensitivity and ion permeability distinct from that of TRPP2 or TRPC1 alone. Native TRPP2/TRPC1 activity is shown in kidney cells by complementary gain-of-function and loss-of-function experiments, and its existence under physiological conditions is supported by colocalization at the primary cilium and by co-immunoprecipitation from kidney membranes. Identification of the heteromultimeric TRPP2/TRPC1 channel has implications in mechanosensation and cilium-based Ca(2+) signalling.

Figure 1

Differential activation mechanisms of transient receptor potential ion channel complexes. (A) Schematic diagram of an outside-out perforated microvesicle obtained with amphotericin-B (Ampho-B) in the patch pipette solution. (B–E) Single-channel activities from cells expressing (B) TRPP2, (C) PKD1/TRPP2, (D) TRPC1 and (E) TRPP2/TRPC1. Each microvesicle was exposed to the MR3 antibody (1/100 for 2 min) or BK (500 nM for 30 s). Traces are shown at different timescales. V_h=−40 mV. The cell in (E) was microinjected with 300 ng/μl and 100 ng/μl of TRPP2 and TRPC1 complementary DNAs, respectively. (F) Amplitude histograms of cation channel currents for the cells shown in (C–E). Bottom-right panel: superimposed Gaussian curves for PKD1/TRPP2, TRPC1 and TRPP2/TRPC1 channels showing open-state amplitude levels of −5.55, −2.01 and −0.79 pA, respectively. Vertical bars indicate the number of events. BK, bradykinin; PKD, polycystin; TRP, transient receptor potential.

Figure 2

Single-channel conductance of TRPP2 and TRPC1 channel complexes. (A) Scatter graph showing single-channel amplitude in microvesicles from cells expressing TRPP2, PKD1/TRPP2, TRPC1, TRPC1/TRPP2, TRPC1/TRPP2-D511V and PKD1/TRPP2-D511V. Cells were intranuclearly microinjected with 100 ng/μl (1) or 300 ng/μl (3) complementary DNA. The mean±SEM is shown. V_h=−40 mV. The grey bar indicates the limit of resolution at the filter frequencies used. (B,C) Mean pooled I–V relationships of (B) PKD1/TRPP2 and PKD1/TRPP2-D511V channels and (C) TRPC1 and TRPC1/TRPP2 channels. Each data point represents the mean±SEM of 10–17 microvesicles. Mean unitary conductances of PKD1/TRPP2, TRPC1 and TRPC1/TRPP2 channels were 142±5, 16±1 and 40±5 pS, respectively. Dashed lines represent 95% confidence limits. BK, bradykinin; MR3, PKD1 specific antibody; PKD, polycystin; TRP, transient receptor potential; TRPP2-D511V, channel-dead mutant.

Figure 3

Differential effects of amiloride on TRPP and TRPC1 channels. Inhibition of (A) PKD1/TRPP2, (B) TRPC1 and (C) TRPP2/TRPC1 cation channels by 100 μM amiloride (Amil.). Sample traces obtained from the same recordings are given at two different time resolutions. Amplitude histograms in the absence and presence of amiloride are also given. V_h=−40 mV. (D) Conductances of PKD1/TRPP2, TRPC1 and TRPP2/TRPC1 channels were plotted against the decrease in single-channel amplitude induced by amiloride. Each data point is the mean±SEM of 4–6 microvesicles. BK, bradykinin; Ctr, control; PKD, polycystin; TRP, transient receptor potential.

Figure 4

Functional expression of transfected TRPP2/TRPC1 in mIMCD3 kidney cells. (A–D) Oxo-M-induced normalized currents in (A) untransfected cells and in cells expressing (B) m1AChR/TRPP2, (C) m1AChR/TRPC1 and (D) m1AChR/TRPP2/TRPC1. Currents were recorded using a voltage clamp ramp protocol. Oxo-M, 10 μM; amiloride, 500 μM. Each panel represents the mean±SEM of 5–7 cells. (E–H) Mean I–V relationships for basal currents (black), Oxo-M-induced current (green) and post-amiloride current (red) in (E) untransfected cells and in cells expressing (F) m1AChR/TRPP2, (G) TRPC1 and (H) TRPP2/TRPC1. Black, green and red arrows in (A–D) indicate the time points at which I–V curves were taken. (I–K) Summary data of normalized currents before the application of (I) 10 μM Oxo-M (basal), (J) net Oxo-M-induced inward currents and (K) percentage block of maximum current (after application of Oxo-M) by amiloride (500 μM) obtained from untransfected cells (1), TRPP2 (2), TRPC1 (3) or TRPP2/TRPC1 (4) expressing mIMCD3 cells (n=5–7). The red lines indicate current densities of untransfected cells. ^*P<0.05. (L,M) I–V curves derived from cells co-transfected with TRPP2 (n=7), TRPC1 (n=7) or TRPP2/TRPC1 (n=7) in the presence of (L) 135 mM extracellular Na⁺ or (M) 10 mM CaCl₂. (N) Na⁺ and Ca²⁺ permeability profiles of TRPP2, TRPC1 and TRPP2/TRPC1 in mIMCD3 cells. mIMCD, murine inner medullary collecting duct; Oxo-M, oxotremorine-M; TRP, transient receptor potential.

Figure 5

Functional expression of endogenous TRPP2/TRPC1 in mIMCD3 kidney cells. (A–H) Representative voltage step currents (A–D) and averaged I–V curves (E–H) of (A,E) untransfected and cells transfected with (B,F) TRPP2-D511V, (C,G) shRNAi^TRPC1 and (D,H) I-mfa. I–V curves were constructed from 7 to 8 cells. (I) Effect of amiloride (500 μM) on basal currents (at −100 mV) of mIMCD3 cells transfected with CD8 (mock, n=5), TRPP2-D511V (n=7), shRNAi^TRPC1 (n=7) or TRPP2-D511V and shRNAi^TRPC1 (n=7). (J) Summary data of amiloride-sensitive current in cell groups shown in (I). ^*P<0.05. (K) Time course of 10 μM Oxo-M-induced current (at −100 mV) in mIMCD3 cells transfected with CD8 (mock, n=6), TRPP2-D511V (n=7), shRNAi^TRPC1 (n=7) or TRPP2-D511V and shRNAi^TRPC1 (n=6). (L) Summary data of Oxo-M-induced current in cell groups shown in (K). ^*P<0.05. mIMCD, murine inner medullary collecting duct; Oxo-M, oxotremorine-M; shRNAi; TRPC1-specific short hairpin RNA interference construct; TRP, transient receptor potential; D511V, construct-dead mutant.