Plasma membrane mechanical stress activates TRPC5 channels

Abstract

Mechanical forces exerted on cells impose stress on the plasma membrane. Cells sense this stress and elicit a mechanoelectric transduction cascade that initiates compensatory mechanisms. Mechanosensitive ion channels in the plasma membrane are responsible for transducing the mechanical signals to electrical signals. However, the mechanisms underlying channel activation in response to mechanical stress remain incompletely understood. Transient Receptor Potential (TRP) channels serve essential functions in several sensory modalities. These channels can also participate in mechanotransduction by either being autonomously sensitive to mechanical perturbation or by coupling to other mechanosensory components of the cell. Here, we investigated the response of a TRP family member, TRPC5, to mechanical stress. Hypoosmolarity triggers Ca2+ influx and cationic conductance through TRPC5. Importantly, for the first time we were able to record the stretch-activated TRPC5 current at single-channel level. The activation threshold for TRPC5 was found to be 240 mOsm for hypoosmotic stress and between -20 and -40 mmHg for pressure applied to membrane patch. In addition, we found that disruption of actin filaments suppresses TRPC5 response to hypoosmotic stress and patch pipette pressure, but does not prevent the activation of TRPC5 by stretch-independent mechanisms, indicating that actin cytoskeleton is an essential transduction component that confers mechanosensitivity to TRPC5. In summary, our findings establish that TRPC5 can be activated at the single-channel level when mechanical stress on the cell reaches a certain threshold.

Fig 1. Hypoosmolarity induces Ca²⁺ influx and whole-cell currents through TRPC5.

(A-B) representative time-series traces showing [Ca²⁺]_i responses to hypotonicity (210 mOsm) in TRPC5-HEK cells. Blue bars on top indicate duration of hypoosmolarity. Lanthanum (La³⁺, 100 μM) directly potentiates TRPC5 activity and was used to show functionality of the express TRPC5. T5E3 (4 μg/ml) is TRPC5-specific blocking antibody and was applied extracellularly to inhibit channel activity (B). (C) quantification of [Ca²⁺]_i response to 210 mOsm at different conditions. (D-E), representative time-series traces showing [Ca²⁺]_i responses to carbachol (Cch, 100 μM) and 210 mOsm when TRPC5-HEK cells were treated by 10 μM of U73122 (D) or U73343 (E) for 30 minutes prior to the recordings. (F) quantification of [Ca²⁺]_i response to 210 mOsm as in (D and E). (G) whole-cell I-V relationships of a representative TRPC5-HEK cell under isotonic (300 mOsm) and hypotonic (240 mOsm) conditions. (H) I-V relationships of hypoosmolarity-activated currents in cells pretreated with preimmune IgG (control IgG, 15 μg/ml) or T5E3 (15 μg/ml). The curves were obtained by subtracting the basal currents at isotonic condition from that at hypotonic condition. (I) summary of data showing hypoosmolarity-activated whole-cell current density at ±80 mV in vector stably-transfected HEK293 cells, TRPC5-HEK cells and TRPC5-HEK cells treated with 2APB (75 μM), preimmune IgG (+control IgG) and T5E3. Values represent mean ±SEM.

Fig 2. Pipette pressure activates TRPC5 on single-channel membrane patch.

(A) schematic diagram depicting single-channel current measurement in TRPC5-expressing CHO-K1 cells. Membrane stretch was elicited by applying suction through the patch pipette, as indicated by red arrow. (B) a representative cell-attached recording (n = 11) of TRPC5-expressing CHO-K1 cell showing changes in channel activity when -40 mmHg pipette pressure was applied (suction) and subsequently released (release). The pipette holding potential was -60 mV. (C) representative traces showing single-channel activities in vector transfected (Vector) and TRPC5-expressing (TRPC5) CHO-K1 cells at 0 mmHg and -40 mmHg pipette pressure with cell-attached configuration. Channel activities were also recorded in TRPC5-expressing cells pretreated with 10 μM BAPTA-AM to buffer cytosolic Ca²⁺ fluctuation (TRPC5+BAPTA-AM). The pipette holding potentials were -60 mV. (D) channel open probability (NPo) values over time for the stretch-activated channel under 0 mmHg and -40 mmHg. Shown are analyses from the same cell-attached patch applied with the indicated pipette pressures for 90 seconds. (E) quantification of the single-channel open probabilities (NPo) of the channel activities recorded on TRPC5-expressing CHO-K1 cells as in (C). (F) single-channel I-V relationships of the stretch-activated channels in cell-attached configuration. The bath solution was 130 mM K⁺ solution (High K ⁺, red circle) for dissipating the membrane potential. The slope conductance is 39 ± 2 pS. (G) schematic diagram showing the two-step backfilling protocol. Patch pipettes were backfilled with T5E3 (15 μg/ml) using a two-step protocol. T5E3 eventually diffused to pipette tip to inhibit TRPC5. Only the patched membrane is depicted for simplicity. Recordings were performed with cell-attached configuration. (H) representative traces showing the stretch (-40 mmHg)-activated channel in the presence of preimmune IgG (15 μg/ml) or T5E3 (15 μg/ml) immediately (0 min) and 10 minutes (10 min) after gigaseal formation. The pipette holding potentials were -60 mV. (I) summary of single-channel open probabilities (NPo) as in (H).

Fig 3. Threshold of TRPC5 mechanosensitivity.

(A) representative time-series traces showing [Ca²⁺]_i in TRPC5-expressing HEK293 cells in response to different osmolarities. Blue bar on top indicated duration of hypoosmolarity. (B) quantification of the [Ca²⁺]_i response at different osmolarities. *, p<0.05 compared to 270 mOsm. (C) representative traces showing the stretch-activated channel under different pipette pressures from a single cell-attached patch of TRPC5-expressing CHO-K1 cell. (D) quantification of single-channel open probabilities (NPo) of under different pipette pressure as in (C). *, p<0.05 compared to 0 mmHg.

Fig 4. Actin filament is essential to TRPC5 mechanosensitivity to hypoosmotic stress.

(A) a representative time-series trace showing [Ca²⁺]_i responses to different osmolarities in TRPC5-expressing HEK293 cells that were pretreated with 25 μM cytochalasin D for 45 min. (B) summary showing effect of 25 μM cytochalasin D treatment on the [Ca²⁺]_i responses to hypoosmolarity. (C) representative time-series traces showing [Ca²⁺]_i responses to 100 μM LaCl₃ (La³⁺, arrow indicates time of addition) in cells that were treated with or without 25 μM cytochalasin D. (D) summary of the [Ca²⁺]_i responses to 100 μM carbachol (Cch) or 100 μM LaCl₃ (La ³⁺). n.s. denotes "not significant". E, a representative cell-attached patch of TRPC5-expressing CHO-K1 cells pretreated with 5 μM cytochalasin D at 0 mmHg and -40 mmHg pipette pressure. F, quantifications of channel open probability (NPo) from patches with or without cytochalasin D treatment. NPo of cytochalasin D treated patches is significantly lower compared to the untreated at -40 mmHg (*, p<0.05), and is significantly higher compared to that at 0 mmHg (#, p<0.05). (G) representative time-series trace showing [Ca²⁺]_i response to different osmolarities in HEK293 cells expressing ΔC-TRPC5, a truncated form lacking the C-terminal PDZ-binding motif. (H) summary of [Ca²⁺]_i responses to different osmolarities in ΔC-TRPC5-expressing HEK293 cells. *, p<0.05 compared to 270 mOsm.