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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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Electrophysiological characterization of sensory neurons of single TRPC3 and TRPC6 knock-out mice. Mechanically evoked currents recorded from small-diameter DRG neurons with broad action potentials using the whole-cell patch clamp technique and classified based on their adaptation kinetic to a static mechanical stimulus applied to the soma. Currents are defined as rapidly adapting (RA), intermediately adapting (IA) and slowly adapting (SA). (a) The proportion of neurons expressing each current type from WT mice, TRPC3 knock-out mice and TRPC6 knock-out mice is shown. TRPC6 knock-out mice were statistically indistinguishable from WT (red, no response; black, RA; dark grey, IA; light grey, SA). (b) The magnitude of mechanically evoked peak currents in small-diameter DRG neurons voltage clamped at −70 mV. No significant difference between genotypes was observed (ANOVA, p > 0.05). Data are expressed as mean ± s.e.m.; **p < 0.01 (black bars, WT; green bars, TRPC3 KO; blue bars, TRPC6 KO).
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RSOB120068F3: Electrophysiological characterization of sensory neurons of single TRPC3 and TRPC6 knock-out mice. Mechanically evoked currents recorded from small-diameter DRG neurons with broad action potentials using the whole-cell patch clamp technique and classified based on their adaptation kinetic to a static mechanical stimulus applied to the soma. Currents are defined as rapidly adapting (RA), intermediately adapting (IA) and slowly adapting (SA). (a) The proportion of neurons expressing each current type from WT mice, TRPC3 knock-out mice and TRPC6 knock-out mice is shown. TRPC6 knock-out mice were statistically indistinguishable from WT (red, no response; black, RA; dark grey, IA; light grey, SA). (b) The magnitude of mechanically evoked peak currents in small-diameter DRG neurons voltage clamped at −70 mV. No significant difference between genotypes was observed (ANOVA, p > 0.05). Data are expressed as mean ± s.e.m.; **p < 0.01 (black bars, WT; green bars, TRPC3 KO; blue bars, TRPC6 KO).

Mentions: DRG neurons in culture express three types of non-selective cationic currents on mechanical stimulation [26,27]. Large-diameter neurons with narrow action potentials all express rapidly adapting (RA) mechanosensitive currents [26], while small-diameter neurons with broad action potentials may be mechano-insensitive or express SA, intermediately adapting (IA) or RA currents [26]. Using the perforated patch configuration of the whole-cell recording technique, neurons were mechanically distended with a fire-polished pipette [26]. Single TRPC3 or TRPC6 knock-out mice had equivalent numbers of mechanically non-responsive small-diameter DRG neurons as control mice (figure 3a). Interestingly, small-diameter neurons from single TRPC3 knock-out mice had 61 per cent fewer RA mechanically activated currents than WT controls (p < 0.01, figure 3a), and this loss of RA currents correlated with a doubling (97%) of the number of neurons that express IA currents (p < 0.01), suggesting that a population of RA current-expressing neurons had switched to an IA phenotype on deletion of TRPC3. The RA neurons had decay kinetics that were best described by a double exponential (τ-fast = 3.3 ms; τ-slow = 45 ms), whereas IA currents had decay kinetics that were best described by a mono-exponential fit. The phenotypic shift from RA to IA is therefore very clear and marked. Decay kinetics of IA neurons from WT or TRPC3 knock-out mice was not significantly different (τ WT = 118 ± 30 ms, τ TRPC3 knock-out = 107 ± 20 ms). Overall, there was no difference in the decay kinetics of RA, IA or SA currents in DRG neurons from any of the genotypes studied. When we used a defined mechanical stimulus to evoke inward currents in TRPC3 and TRPC6 knock-out mice, we found that the average peak currents were statistically indistinguishable from WT mice (figure 3b).Figure 3.


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)

Electrophysiological characterization of sensory neurons of single TRPC3 and TRPC6 knock-out mice. Mechanically evoked currents recorded from small-diameter DRG neurons with broad action potentials using the whole-cell patch clamp technique and classified based on their adaptation kinetic to a static mechanical stimulus applied to the soma. Currents are defined as rapidly adapting (RA), intermediately adapting (IA) and slowly adapting (SA). (a) The proportion of neurons expressing each current type from WT mice, TRPC3 knock-out mice and TRPC6 knock-out mice is shown. TRPC6 knock-out mice were statistically indistinguishable from WT (red, no response; black, RA; dark grey, IA; light grey, SA). (b) The magnitude of mechanically evoked peak currents in small-diameter DRG neurons voltage clamped at −70 mV. No significant difference between genotypes was observed (ANOVA, p > 0.05). Data are expressed as mean ± s.e.m.; **p < 0.01 (black bars, WT; green bars, TRPC3 KO; blue bars, TRPC6 KO).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

RSOB120068F3: Electrophysiological characterization of sensory neurons of single TRPC3 and TRPC6 knock-out mice. Mechanically evoked currents recorded from small-diameter DRG neurons with broad action potentials using the whole-cell patch clamp technique and classified based on their adaptation kinetic to a static mechanical stimulus applied to the soma. Currents are defined as rapidly adapting (RA), intermediately adapting (IA) and slowly adapting (SA). (a) The proportion of neurons expressing each current type from WT mice, TRPC3 knock-out mice and TRPC6 knock-out mice is shown. TRPC6 knock-out mice were statistically indistinguishable from WT (red, no response; black, RA; dark grey, IA; light grey, SA). (b) The magnitude of mechanically evoked peak currents in small-diameter DRG neurons voltage clamped at −70 mV. No significant difference between genotypes was observed (ANOVA, p > 0.05). Data are expressed as mean ± s.e.m.; **p < 0.01 (black bars, WT; green bars, TRPC3 KO; blue bars, TRPC6 KO).
Mentions: DRG neurons in culture express three types of non-selective cationic currents on mechanical stimulation [26,27]. Large-diameter neurons with narrow action potentials all express rapidly adapting (RA) mechanosensitive currents [26], while small-diameter neurons with broad action potentials may be mechano-insensitive or express SA, intermediately adapting (IA) or RA currents [26]. Using the perforated patch configuration of the whole-cell recording technique, neurons were mechanically distended with a fire-polished pipette [26]. Single TRPC3 or TRPC6 knock-out mice had equivalent numbers of mechanically non-responsive small-diameter DRG neurons as control mice (figure 3a). Interestingly, small-diameter neurons from single TRPC3 knock-out mice had 61 per cent fewer RA mechanically activated currents than WT controls (p < 0.01, figure 3a), and this loss of RA currents correlated with a doubling (97%) of the number of neurons that express IA currents (p < 0.01), suggesting that a population of RA current-expressing neurons had switched to an IA phenotype on deletion of TRPC3. The RA neurons had decay kinetics that were best described by a double exponential (τ-fast = 3.3 ms; τ-slow = 45 ms), whereas IA currents had decay kinetics that were best described by a mono-exponential fit. The phenotypic shift from RA to IA is therefore very clear and marked. Decay kinetics of IA neurons from WT or TRPC3 knock-out mice was not significantly different (τ WT = 118 ± 30 ms, τ TRPC3 knock-out = 107 ± 20 ms). Overall, there was no difference in the decay kinetics of RA, IA or SA currents in DRG neurons from any of the genotypes studied. When we used a defined mechanical stimulus to evoke inward currents in TRPC3 and TRPC6 knock-out mice, we found that the average peak currents were statistically indistinguishable from WT mice (figure 3b).Figure 3.

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