Tentonin 3/TMEM150c Confers Distinct Mechanosensitive Currents in Dorsal-Root Ganglion Neurons with Proprioceptive Function

Abstract

Touch sensation or proprioception requires the transduction of mechanical stimuli into electrical signals by mechanoreceptors in the periphery. These mechanoreceptors are equipped with various transducer channels. Although Piezo1 and 2 are mechanically activated (MA) channels with rapid inactivation, MA molecules with other inactivation kinetics have not been identified. Here we report that heterologously expressed Tentonin3 (TTN3)/TMEM150C is activated by mechanical stimuli with distinctly slow inactivation kinetics. Genetic ablation of Ttn3/Tmem150c markedly reduced slowly adapting neurons in dorsal-root ganglion neurons. The MA TTN3 currents were inhibited by known blockers of mechanosensitive ion channels. Moreover, TTN3 was localized in muscle spindle afferents. Ttn3-deficient mice exhibited the loss of coordinated movements and abnormal gait. Thus, TTN3 appears to be a component of a mechanosensitive channel with a slow inactivation rate and contributes to motor coordination. Identification of this gene advances our understanding of the various types of mechanosensations, including proprioception.

Endogenous Piezo1 Can Confound Mechanically Activated Channel Identification and Characterization

“…1a, right). These results confirm the findings of the two previous reports (Hong et al, 2016; Zhao et al, 2016) of increased MA channel activity in HEK cells transfected with these putative mechanosensors.…”

“…We are confident that Piezo1-deficient HEK cells are healthy and capable of sustaining MA channel function, as Piezo1 overexpression in naïve HEK and HEK-P1KO cells gave rise to MA currents that were indistinguishable from each other. Furthermore, both cell lines maintained good holding currents at strong stimuli just prior to “losing” the cell, and the strongest displacements achievable in our dataset were similar to, if not higher than, those reported previously (Hong et al, 2016; Zhao et al, 2016). As an alternative hypothesis, one could imagine that endogenous inactivated Piezo1 in HEK-P1KO cells, if present, is acting as a dominant negative to suppress Piezo1-ASIC1 activity.…”

“…Currents inactivated slowly (Fig1.d, e left), similar to those observed in vector control-transfected HEK cells (Fig.1b, left). Among the responding cells, more robust responses to mechanical stimuli were observed in cells expressing TMEM150c (P< 0.02; Student’s t -test) and Piezo1-ASIC1 (P< 0.05) compared to vector-transfected HEK cells as previously reported (Hong et al, 2016; Zhao et al, 2016). It is also interesting that the apparent threshold for responsive vector-transfected HEK cells (12.4 ± 1.1 μm, n=11) was about twice that observed for cells expressing either TMEM150c or Piezo1-ASIC1.…”

“…Recording pipettes contained the CsCl-based intracellular solution described in Coste et al, 2010 with some exceptions. Mg-ATP was 2 or 4 mM and Na 2 -GTP was 200 or 400 μM, to mimic conditions described in (Hong et al, 2016; Zhao et al, 2016). The intracellular solution used at Duke differed in that EGTA was 1 mM and no Ca 2+ was added, MgCl 2 was 2 rather than 1 mM, and Na 2 GTP was 300μM; the extracellular solution contained 5 instead of 10 mM glucose, and 5 mM TEA-Cl was added.…”

“…Two recent reports claim that heterologous expression of particular constructs in HEK cells was sufficient to mediate MA currents dependent on the exogenous protein. In one, Tentonin 3 (TTN3)/TMEM150c was proposed to be a component of a MA channel based on several experiments, including the finding that heterologous expression of TMEM150c in HEK cells gives rise to MA currents (Hong et al, 2016). In the other, a chimera (mPiezo1(1-2190)-mASIC1, “Piezo1-ASIC1”) which combined the large N-terminal “propeller” domain of Piezo1 that lacks the pore (Coste et al, 2015; Ge et al, 2015; Zhao et al, 2016) with the pore-forming domain of the mechanically-insensitive, sodium-selective ASIC1 was reported to give rise to MA currents when transfected in HEK cells (Zhao et al, 2016).…”