Piezo1-dependent stretch-activated channels are inhibited by Polycystin-2 in renal tubular epithelial cells

Abstract

Mechanical forces associated with fluid flow and/or circumferential stretch are sensed by renal epithelial cells and contribute to both adaptive or disease states. Non-selective stretch-activated ion channels (SACs), characterized by a lack of inactivation and a remarkably slow deactivation, are active at the basolateral side of renal proximal convoluted tubules. Knockdown of Piezo1 strongly reduces SAC activity in proximal convoluted tubule epithelial cells. Similarly, overexpression of Polycystin-2 (PC2) or, to a greater extent its pathogenic mutant PC2-740X, impairs native SACs. Moreover, PC2 inhibits exogenous Piezo1 SAC activity. PC2 coimmunoprecipitates with Piezo1 and deletion of its N-terminal domain prevents both this interaction and inhibition of SAC activity. These findings indicate that renal SACs depend on Piezo1, but are critically conditioned by PC2.

Figure 1

Native SACs in PCT epithelial cells. (A) Top trace: Cell-attached patch-clamp recording of SACs at a holding potential of −80 mV on the basolateral side of a freshly isolated proximal convoluted tubule. Middle trace: same patch at a holding potential of 0 mV. Bottom trace illustrates the pressure pulses. (B) Top trace: SAC activity recorded in a cultured isolated wild-type PCT cell at a holding potential of −80 mV. Middle trace: same patch at a holding potential of 0 mV. Bottom trace illustrates the pressure pulses. (C) Single channel currents recorded at −80 mV and elicited by a continuous −20 mm Hg pressure stimulation. I–V curve of SACs in PCT cells recorded at −20 mm Hg (linear regression). The single channel conductance of SACs in PCT is 31.8±0.3 pS (n=5) and the extrapolated reversal potential around 0 mV. (D) Pressure-effect curves in control conditions (black traces), in the presence of 50 μm ruthenium red (red traces) or in the presence of 5 μM GsMTx-4 L optical isomer (magenta traces). GsMTx-4 might act as a gating modifier with partition in the lipid bilayer and shift of the pressure-effect curve to stronger stimuli [40]. PCT, proximal convoluted tubule; SAC, stretch-activated ion channel.

Figure 2

Endogenous SAC activity in PCT epithelial cells critically depends on Piezo1. (A) Quantitative PCR expression of Piezo1 and Piezo2 normalized to Topoisomerase 1 (TOP1) expression in whole kidney, isolated proximal tubules and immortalized PCT cells. (B) Pressure-effect curves for mean SAC activity recorded in cultured PCT cells either transfected with a non-targeting siRNA (NT; P_0.5=−21.8±2.8 mm Hg, k=9.7±3.4, n=68) or with two different siRNAs directed against Piezo1 (si1Piezo1; n=41 and si2Piezo1; n=28). PCT, proximal convoluted tubule; SAC, stretch-activated ion channel; siRNA, short interfering RNA.

Figure 3

Endogenous SAC activity in PCT epithelial cells is conditioned by PC2 and PC2-740x. Mean (n=44) cell-attached SAC current (in black) at a holding potential of −80 mV in cultured PCT cells transfected with a mock expression EGFP vector. (B) Mean (n=28) cell-attached SAC current (in red) at a holding potential of −80 mV in cultured PCT cells transfected with PC2-740X ires2-EGFP. In A and B, bottom traces illustrate the pressure pulses. Same scales for A and B. (C) Pressure-effect curve for SAC activity in wild-type PCT cells transfected with either the mock EGFP vector (in black: P_0.5=−16.0±1.7 mm Hg, k=10.4±2.4 or with PC2 ires2-EGFP in magenta: P_0.5=−32.8±12.8 mm Hg, k=15.8±10.6 or with PC2-740X ires2-EGFP in red: P_0.5=−9.6±1.9 mm Hg, k=7.7±1.3). EGFP, enhanced green fluorescent protein; PC2, Polycystin-2; PCT, proximal convoluted tubule; SAC, stretch-activated ion channel.

Figure 4

Exogenous Piezo1 activity is conditioned by PC2-740X in PCT or COS-7-transfected cells. (A) The top trace in black shows the mean (n=22) cell-attached SAC current at a holding potential of −80 mV in Piezo1 ires EGFP-transiently transfected PCT cells together with a DsRed mock vector. The middle trace in red shows the mean (n=12) cell-attached SAC current at a holding potential of −80 mV in Piezo1 ires EGFP-transfected PCT cells together with PC2-740X ires2-DsRed. Bottom traces illustrate the pressure pulses. (B) Pressure-effect curves for SACs/Piezo1 activity (as indicated) in transfected PCT cells (control PCT cells transfected with a mixture of mock vectors EGFP and DsRed: P_0.5=−25.3±2.0 mm Hg, k=6.3±1.1; Piezo1 ires EGFP+mock vector DsRed: P_0.5=−32.7±4.2 mm Hg, k=12.9±3.6; Piezo1 ires EGFP+PC2-740X ires2-DsRed: P_0.5=−41.1±1.6 mm Hg, k=9.0±0.8). n-values are indicated on the right side of the graph. (C) The top trace in black shows the mean (n=32) cell-attached SAC current at a holding potential of −80 mV in Piezo1 ires EGFP-transfected COS-7 cells together with a mock vector DsRed. The middle trace in red shows the mean (n=49) cell-attached SAC current at a holding potential of −80 mV in Piezo1 ires EGFP-transfected COS-7 cells together with PC2-740X ires2-DsRed. Bottom traces illustrate the pressure pulses. Same scales for A and C. (D) Pressure-effect curves for SACs/Piezo1 activity (as indicated) in transfected COS-7 cells (control COS cells transfected with a mixture of mock vectors EGFP and DsRed: P_0.5 =−10.9±1.5 mm Hg, k=3.0±4.0; Piezo1 ires EGFP+mock vector DsRed: P_0.5=−14.9±1.5 mm Hg, k=8.5±1.9; Piezo1 ires EGFP+PC2-740X ires2-DsRed: P_0.5=−16.9±1.2 mm Hg, k=10.2±1.6; Piezo1 ires EGFP+TRPC1 ires2-DsRed: P_0.5=−14.6±2.1 mm Hg, k=9.3±3.0). n-values are indicated on the right side of the graph. In A and C, only patches in which the whole pressure range could be applied without rupture were included. EGFP, enhanced green fluorescent protein; PC2, Polycystin-2; PCT, proximal convoluted tubule; SAC, stretch-activated ion channel.

Figure 5

PC2 and PC2 mutants coimmunoprecipitation with Piezo1 in transiently transfected COS cells. (A) Western blot analysis of lysates and immunoprecipitates from COS cells cotransfected with Piezo1-HA and either NH2 (ΔN203) or COOH terminal deletions (from −740X to −690X) in MYC-PC2 (as indicated) show that the NH2 terminus of PC2 is necessary for immunoprecipitation with Piezo1-HA, while the COOH terminus is not required (bottom most panel). Neither MYC-Kv2.1 nor MYC-Kv9.3 (used as negative controls) immunoprecipitate with Piezo-HA. Immunoprecipitation is absent without an HA tag on Piezo1. Blots were probed with 3F10 (Probe HA) or with 9E10 (Probe MYC). Inputs and IPs are as indicated. (B) Inhibition of Piezo1 by PC2 in cotransfected COS cells. Deletion of the N-terminal domain of PC2 (ΔN203) prevents Piezo1 inhibition (Piezo1-EGFP+mock vector DsRed: P_0.5=−21.6±1.4 mm Hg, k=6.8±1.2; Piezo1-EGFP+MYC-PC2 ires2-DsRed: P_0.5=−39.9±2.8 mm Hg, k=12.2±2.2; Piezo1-EGFP+MYC-PC2ΔN203 ires2-DsRed: P_0.5=−18.9±1.5 mm Hg, k=8.2±1.8). All currents were normalized to the fitted value for Piezo1-EGFP+mock vector DsRed at −80 mm Hg. n-values are indicated on the right side of the graph. EGFP, enhanced green fluorescent protein; HA, haemagglutinin; SAC, stretch-activated ion channel; PC2, Polycystin-2; .