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TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells.

Quick K, Zhao J, Eijkelkamp N, Linley JE, Rugiero F, Cox JJ, Raouf R, Gringhuis M, Sexton JE, Abramowitz J, Taylor R, Forge A, Ashmore J, Kirkwood N, Kros CJ, Richardson GP, Freichel M, Flockerzi V, Birnbaumer L, Wood JN - Open Biol (2012)

Bottom Line: Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents.Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation.FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines.

View Article: PubMed Central - PubMed

Affiliation: Molecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK.

ABSTRACT
Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.

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Heterologous expression of TRPC3 with or without TRPC6 confers mechanosensitivity on some cell lines. Cells were transfected with constructs encoding TRPC3-IRES-GFP + TRPC6-IRES-DsRed2, TRPC3-IRES-GFP alone, TRPC6-IRES-DsRed2 alone or IRES-GFP and/or IRES-DsRed2. Cells were selected based on their fluorescence and voltage-clamped at −70 mV in whole-cell configuration. The magnitude of mechanically evoked currents in (a) HEK293a cells, (b) CHO K1 ((a,b): black dots, empty vector (n = 9); red circles, TRPC3/TRPC6 (n = 9)) or (c) ND-C cells, a sensory neuron cell line is presented (black dots, empty vector (n = 33); red circles, TRPC3/TRPC6 (n = 11); green triangles, TRPC3 (n = 12); blue diamonds, TRPC6 (n = 13)). (d) Exemplar traces from (c) induced by a 9.9 μm membrane displacement (top plot: movement of the probe). ND-C cell were transfected with TRPC3 and TRPC6 and voltage-clamped at −70 mV in whole-cell configuration. Mechanically activated currents were elicited by an approximately 14 µm displacement of a glass probe at 10 μm distance from the cell (black lines, empty vector; red lines, TRPC3/TRPC6; green lines, TRPC3; blue lines, TRPC6). (e) Normalized current amplitudes from three cells before, during and 30 s after application of SKF96365. (f) Normalized current amplitudes (n = 7) before and after application of FM1-43. (g) Representative current traces before (black), during (red) and 30 s after (blue) application of 100 μM SKF96365 or 15 μM FM1-43. Top plot shows movement of displacement of glass probe. Data represent mean ± s.e.m. **p < 0.01; ***p < 0.001 (see also electronic supplementary material, figure S4).
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RSOB120068F8: Heterologous expression of TRPC3 with or without TRPC6 confers mechanosensitivity on some cell lines. Cells were transfected with constructs encoding TRPC3-IRES-GFP + TRPC6-IRES-DsRed2, TRPC3-IRES-GFP alone, TRPC6-IRES-DsRed2 alone or IRES-GFP and/or IRES-DsRed2. Cells were selected based on their fluorescence and voltage-clamped at −70 mV in whole-cell configuration. The magnitude of mechanically evoked currents in (a) HEK293a cells, (b) CHO K1 ((a,b): black dots, empty vector (n = 9); red circles, TRPC3/TRPC6 (n = 9)) or (c) ND-C cells, a sensory neuron cell line is presented (black dots, empty vector (n = 33); red circles, TRPC3/TRPC6 (n = 11); green triangles, TRPC3 (n = 12); blue diamonds, TRPC6 (n = 13)). (d) Exemplar traces from (c) induced by a 9.9 μm membrane displacement (top plot: movement of the probe). ND-C cell were transfected with TRPC3 and TRPC6 and voltage-clamped at −70 mV in whole-cell configuration. Mechanically activated currents were elicited by an approximately 14 µm displacement of a glass probe at 10 μm distance from the cell (black lines, empty vector; red lines, TRPC3/TRPC6; green lines, TRPC3; blue lines, TRPC6). (e) Normalized current amplitudes from three cells before, during and 30 s after application of SKF96365. (f) Normalized current amplitudes (n = 7) before and after application of FM1-43. (g) Representative current traces before (black), during (red) and 30 s after (blue) application of 100 μM SKF96365 or 15 μM FM1-43. Top plot shows movement of displacement of glass probe. Data represent mean ± s.e.m. **p < 0.01; ***p < 0.001 (see also electronic supplementary material, figure S4).

Mentions: We examined the mechanosensitivity of cell lines over-expressing TRPC3, TRPC6 or both channels using the same methods that demonstrated a deficit in TRPC3/TRPC6 DKO sensory neurons. When the channels were over-expressed either singly or in combination in HEK293a cells or chinese hamster ovary (CHO) cells, no mechanically evoked inward currents could be detected (figure 8a,b). However, when the two channels were expressed in ND-C cells [40], a sensory neuron-derived cell line, RA currents could be detected even after small mechanical displacements (approx. 1 µm), while no currents were evoked in control transfected ND-C cells at membrane displacements less than 8 µm (figure 8c,d). Mechanical stimulation of TRPC3-expressing cells evoked an inward current that developed within milliseconds and increased in size with the strength of the mechanical distension (figure 8c,d). The decay kinetics of the mechanically activated currents in TRPC3-expressing cells was best described by a mono-exponential fit (τ = 37.2 ± 6.7 ms, n = 12). Co-expression of TRPC6 with TRPC3 shifted the stimulus response curve to the left, resulting in a lower threshold of activation and increased currents in response to mechanical stimuli. Additionally, the decay kinetics of mechanically activated currents in TRPC3 and TRPC6-expressing cells were significantly faster compared with cells expressing TRPC3 alone (τ = 20.1 ± 1.2 ms, n = 10, p < 0.05). TRPC6 expression alone did not confer increased mechanosensitivity on ND-C cells (figure 8c,d). We confirmed that the expressed currents were potentially due to TRPC3 and TRPC6 expression, using SKF96365, a blocker with some selectivity for TRPC channels, that reversibly blocked mechanically gated currents in ND-C cells transfected with TRPC3 and TRPC6 (figure 8f,g). The fluorescent styryl dye FM1-43 is a permeant blocker of mechanosensitive channels in both the cochlea and sensory neurons. We found that TRPC3/TRPC6-dependent mechanosensitive inward currents were also blocked by micromolar FM1-43 concentrations (figure 8e,g).Figure 8.


TRPC3 and TRPC6 are essential for normal mechanotransduction in subsets of sensory neurons and cochlear hair cells.

Quick K, Zhao J, Eijkelkamp N, Linley JE, Rugiero F, Cox JJ, Raouf R, Gringhuis M, Sexton JE, Abramowitz J, Taylor R, Forge A, Ashmore J, Kirkwood N, Kros CJ, Richardson GP, Freichel M, Flockerzi V, Birnbaumer L, Wood JN - Open Biol (2012)

Heterologous expression of TRPC3 with or without TRPC6 confers mechanosensitivity on some cell lines. Cells were transfected with constructs encoding TRPC3-IRES-GFP + TRPC6-IRES-DsRed2, TRPC3-IRES-GFP alone, TRPC6-IRES-DsRed2 alone or IRES-GFP and/or IRES-DsRed2. Cells were selected based on their fluorescence and voltage-clamped at −70 mV in whole-cell configuration. The magnitude of mechanically evoked currents in (a) HEK293a cells, (b) CHO K1 ((a,b): black dots, empty vector (n = 9); red circles, TRPC3/TRPC6 (n = 9)) or (c) ND-C cells, a sensory neuron cell line is presented (black dots, empty vector (n = 33); red circles, TRPC3/TRPC6 (n = 11); green triangles, TRPC3 (n = 12); blue diamonds, TRPC6 (n = 13)). (d) Exemplar traces from (c) induced by a 9.9 μm membrane displacement (top plot: movement of the probe). ND-C cell were transfected with TRPC3 and TRPC6 and voltage-clamped at −70 mV in whole-cell configuration. Mechanically activated currents were elicited by an approximately 14 µm displacement of a glass probe at 10 μm distance from the cell (black lines, empty vector; red lines, TRPC3/TRPC6; green lines, TRPC3; blue lines, TRPC6). (e) Normalized current amplitudes from three cells before, during and 30 s after application of SKF96365. (f) Normalized current amplitudes (n = 7) before and after application of FM1-43. (g) Representative current traces before (black), during (red) and 30 s after (blue) application of 100 μM SKF96365 or 15 μM FM1-43. Top plot shows movement of displacement of glass probe. Data represent mean ± s.e.m. **p < 0.01; ***p < 0.001 (see also electronic supplementary material, figure S4).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3376737&req=5

RSOB120068F8: Heterologous expression of TRPC3 with or without TRPC6 confers mechanosensitivity on some cell lines. Cells were transfected with constructs encoding TRPC3-IRES-GFP + TRPC6-IRES-DsRed2, TRPC3-IRES-GFP alone, TRPC6-IRES-DsRed2 alone or IRES-GFP and/or IRES-DsRed2. Cells were selected based on their fluorescence and voltage-clamped at −70 mV in whole-cell configuration. The magnitude of mechanically evoked currents in (a) HEK293a cells, (b) CHO K1 ((a,b): black dots, empty vector (n = 9); red circles, TRPC3/TRPC6 (n = 9)) or (c) ND-C cells, a sensory neuron cell line is presented (black dots, empty vector (n = 33); red circles, TRPC3/TRPC6 (n = 11); green triangles, TRPC3 (n = 12); blue diamonds, TRPC6 (n = 13)). (d) Exemplar traces from (c) induced by a 9.9 μm membrane displacement (top plot: movement of the probe). ND-C cell were transfected with TRPC3 and TRPC6 and voltage-clamped at −70 mV in whole-cell configuration. Mechanically activated currents were elicited by an approximately 14 µm displacement of a glass probe at 10 μm distance from the cell (black lines, empty vector; red lines, TRPC3/TRPC6; green lines, TRPC3; blue lines, TRPC6). (e) Normalized current amplitudes from three cells before, during and 30 s after application of SKF96365. (f) Normalized current amplitudes (n = 7) before and after application of FM1-43. (g) Representative current traces before (black), during (red) and 30 s after (blue) application of 100 μM SKF96365 or 15 μM FM1-43. Top plot shows movement of displacement of glass probe. Data represent mean ± s.e.m. **p < 0.01; ***p < 0.001 (see also electronic supplementary material, figure S4).
Mentions: We examined the mechanosensitivity of cell lines over-expressing TRPC3, TRPC6 or both channels using the same methods that demonstrated a deficit in TRPC3/TRPC6 DKO sensory neurons. When the channels were over-expressed either singly or in combination in HEK293a cells or chinese hamster ovary (CHO) cells, no mechanically evoked inward currents could be detected (figure 8a,b). However, when the two channels were expressed in ND-C cells [40], a sensory neuron-derived cell line, RA currents could be detected even after small mechanical displacements (approx. 1 µm), while no currents were evoked in control transfected ND-C cells at membrane displacements less than 8 µm (figure 8c,d). Mechanical stimulation of TRPC3-expressing cells evoked an inward current that developed within milliseconds and increased in size with the strength of the mechanical distension (figure 8c,d). The decay kinetics of the mechanically activated currents in TRPC3-expressing cells was best described by a mono-exponential fit (τ = 37.2 ± 6.7 ms, n = 12). Co-expression of TRPC6 with TRPC3 shifted the stimulus response curve to the left, resulting in a lower threshold of activation and increased currents in response to mechanical stimuli. Additionally, the decay kinetics of mechanically activated currents in TRPC3 and TRPC6-expressing cells were significantly faster compared with cells expressing TRPC3 alone (τ = 20.1 ± 1.2 ms, n = 10, p < 0.05). TRPC6 expression alone did not confer increased mechanosensitivity on ND-C cells (figure 8c,d). We confirmed that the expressed currents were potentially due to TRPC3 and TRPC6 expression, using SKF96365, a blocker with some selectivity for TRPC channels, that reversibly blocked mechanically gated currents in ND-C cells transfected with TRPC3 and TRPC6 (figure 8f,g). The fluorescent styryl dye FM1-43 is a permeant blocker of mechanosensitive channels in both the cochlea and sensory neurons. We found that TRPC3/TRPC6-dependent mechanosensitive inward currents were also blocked by micromolar FM1-43 concentrations (figure 8e,g).Figure 8.

Bottom Line: Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents.Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation.FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines.

View Article: PubMed Central - PubMed

Affiliation: Molecular Nociception Group, Wolfson Institute for Biomedical Research, University College London, London WC1E 6BT, UK.

ABSTRACT
Transient receptor potential (TRP) channels TRPC3 and TRPC6 are expressed in both sensory neurons and cochlear hair cells. Deletion of TRPC3 or TRPC6 in mice caused no behavioural phenotype, although loss of TRPC3 caused a shift of rapidly adapting (RA) mechanosensitive currents to intermediate-adapting currents in dorsal root ganglion sensory neurons. Deletion of both TRPC3 and TRPC6 caused deficits in light touch and silenced half of small-diameter sensory neurons expressing mechanically activated RA currents. Double TRPC3/TRPC6 knock-out mice also showed hearing impairment, vestibular deficits and defective auditory brain stem responses to high-frequency sounds. Basal, but not apical, cochlear outer hair cells lost more than 75 per cent of their responses to mechanical stimulation. FM1-43-sensitive mechanically gated currents were induced when TRPC3 and TRPC6 were co-expressed in sensory neuron cell lines. TRPC3 and TRPC6 are thus required for the normal function of cells involved in touch and hearing, and are potential components of mechanotransducing complexes.

Show MeSH
Related in: MedlinePlus