Mechanosensitive PIEZO ion channels are evolutionarily conserved proteins whose presence is critical for normal physiology in multicellular organisms. Here we show that, in addition to mechanical stimuli, PIEZO channels are also powerfully modulated by voltage and can even switch to a purely voltage-gated mode. Mutations that cause human diseases, such as xerocytosis, profoundly shift voltage sensitivity of PIEZO1 channels toward the resting membrane potential and strongly promote voltage gating. Voltage modulation may be explained by the presence of an inactivation gate in the pore, the opening of which is promoted by outward permeation. Older invertebrate (fly) and vertebrate (fish) PIEZO proteins are also voltage sensitive, but voltage gating is a much more prominent feature of these older channels. We propose that the voltage sensitivity of PIEZO channels is a deep property co-opted to add a regulatory mechanism for PIEZO activation in widely different cellular contexts.

The skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. Indeed, some mechanoreceptors can detect even nanometer-scale movements. Movement is transformed into electrical signals via the gating of mechanically activated ion channels at sensory endings in the skin. The sensitivity of Piezo mechanically gated ion channels is controlled by stomatin-like protein-3 (STOML3), which is required for normal mechanoreceptor function. Here we identify small-molecule inhibitors of STOML3 oligomerization that reversibly reduce the sensitivity of mechanically gated currents in sensory neurons and silence mechanoreceptors in vivo. STOML3 inhibitors in the skin also reversibly attenuate fine touch perception in normal mice. Under pathophysiological conditions following nerve injury or diabetic neuropathy, the slightest touch can produce pain, and here STOML3 inhibitors can reverse mechanical hypersensitivity. Thus, small molecules applied locally to the skin can be used to modulate touch and may represent peripherally available drugs to treat tactile-driven pain following neuropathy.

37Mechanoelectrical transduction is a cellular signalling pathway where physical stimuli are 38 converted into electro-chemical signals by mechanically activated ion channels. We describe 39 here the presence of mechanically activated currents in melanoma cells that are dependent on 40 TMEM87a, which we have renamed Elkin1. Heterologous expression of this protein in 41 PIEZO1-deficient cells, that exhibit no baseline mechanosensitivity, is sufficient to 42 reconstitute mechanically activated currents. Melanoma cells lacking functional Elkin1 43 exhibit defective mechanoelectrical transduction, decreased motility and increased 44 dissociation from organotypic spheroids. By analysing cell adhesion properties, we 45 demonstrate that Elkin1 deletion is associated with increased cell-substrate adhesion and 46 decreased homotypic cell-cell adhesion strength. We therefore conclude that Elkin1 supports 47 a PIEZO1-independent mechanoelectrical transduction pathway and modulates cellular 48 adhesions and regulates melanoma cell migration and cell-cell interactions. 49 50 130 occurred within the stimulus range, allowing us to use a Boltzmann sigmoidal fit to determine 131 the MA current sensitivity. Half-maximal activation of MA currents was seen with 132 approximately 18 nm of substrate deflection (Effective deflection ED50; standard error = 133 20.5 nm). These data indicate a correlation between migratory properties and the MA current 134 sensitivity to deflections applied at cell-substrate contact points. The robust MA current 135 activation observed in cells cultured on LM511 also provided an excellent system to 136 investigate the molecules required for this mechanoelectrical transduction. 137 5 138 157 1999), indicating that neither likely mediates the deflection-evoked currents in WM266-4 158 cells (Figure 1-figure supplement 2). We then examined the proteomics data for proteins of 159 unknown function with 4 or more predicted transmembrane (TM) domains. We prioritised 160 the investigation of Elkin1 due to its expression in melanoma cells but not healthy 161 melanocytes, its expression in additional mechanosensitive cells (Alveolar Type II cells) and 162 its upregulation in additional human cancers (Human Protein Atlas (Uhlén et al., 2005) 163 available from www.proteinatlas.org). We generated miRNA constructs targeting Elkin1 and 164 found that knockdown of Elkin1 transcript resulted in a dramatic reduction in MA currents to 165 deflections up to 1000 nm (Figure 2A,B). These data suggested that Elkin1 contributes to 166 MA currents in melanoma cells. 167 168 Three human isoforms (representing splice variants) of Elkin1 have been identified: isoforms 169 1 and 3 (555 and 494 aa respectively), contain 6 predicted TM domains (Figure 2C). Isoform 170 2 (181 aa) does not contain any predicted TM domains and was not examined in this study.171 6We cloned hsElkin1-iso1 and hsElkin1-iso3 from WM266-4 cDNA and generated C-terminal 172 GFP fusion constructs. We confirmed the plasma membrane localisation of these two 17...

Primary cultured type II alveolar epithelial cells grown to confluence on nonporous surfaces form many small fluid-filled hemicysts or domes. These domes are generally thought to result from active transport of solutes from the medium above the cell monolayer to the substratum, with water following passively. We have investigated the characteristics of active transport by primary cultured monolayers of type II alveolar epithelial cells from rat lungs. Changes in dome density were measured after exposure to metabolic inhibitors, Na+ or Cl- transport inhibitors, and low-Na+ or low-Cl- culture media. Metabolic and Na+ transport inhibitors, and low-Na+ medium, lead to disappearance of domes, whereas Cl- transport inhibitors and low-Cl- medium seem to have no effect on dome density. These results suggest the presence of a Na+-dependent active transepithelial transport process across the monolayer, which is responsible for the formation of domes. This finding implies that absorption of fluid by mammalian alveolar epithelium in vivo may be important in the maintenance of normal lung fluid balance.

Mechanosensitive PIEZO ion channels are evolutionarily conserved proteins whose presence is critical for normal physiology in multicellular organisms. Here we show that, in addition to mechanical stimuli, PIEZO channels are also powerfully modulated by voltage and can even switch to a purely voltage gated mode. Mutations that cause human diseases such as Xerocytosis profoundly shift voltage sensitivity of PIEZO1 channels towards the resting membrane potential and strongly promote pure voltage gating. Our data may be explained by the presence of an inactivation gate in the pore, the opening of which is promoted by outward permeation.Invertebrate (fly) and vertebrate (fish) PIEZO proteins are also voltage sensitive but voltage gating is a much more prominent feature of these older channels. We propose that the voltage sensitivity of PIEZO channels is a deep property co-opted to add a regulatory mechanism for PIEZO activation in widely different cellular contexts.(OH), C-terminal extracellular domain (CED), inner helix (IH) and intracellular C-terminal domain (CTD) (Ge et al., 2015). The peripheral regions of the protein are composed of the extracellular "blade" domains, 12 peripheral helices (PHs) in each subunit and intracellular "beam" and "anchor" domains. The PIEZO1 structure has facilitated biophysical exploration of the ion channel pore (Lewis and Grandl, 2015; Wu et al., 2016; Zhao et al., 2016) and experiments with chimeric structures have suggested that it is the N-terminal non-pore containing region that confers mechanosensitivity on the channel (Zhao et al., 2016). However recent experiments suggested that this subject needs further validation (Dubin et al., 2017).Biophysical investigation of PIEZO channels have focused mostly on their activation by mechanical forces. For example, proteins like STOML3 have been identified that can dramatically increase the sensitivity of PIEZO channel to mechanical force (Poole et al., 2014; Wetzel et al., 2016). However, the role of membrane voltage in modulating PIEZO channel activity has barely been addressed despite the fact that both mammalian stretch-activated potassium channels (Brohawn et al., 2014; Dedman et al., 2009; Honore et al., 2002) as well as bacterial stretch-activated ion channels are clearly voltage sensitive (Bass et al., 2002). Here we asked whether voltage can modulate or even gate vertebrate and invertebrate PIEZO channels.We show that both PIEZO1 and PIEZO2 show significant voltage sensitivity that is dependent on critical residues in the pore lining region of the channel. We provide evidence for an inactivation gate that closes following inward permeation and under physiological conditions renders >90% of the channels unavailable for opening by mechanical force. Outward permeation of the channel is sufficient to lead to a slow conformational change that opens the inactivation gate. Pathological human mutations in Piezo1 primarily weaken the inactivation gate and render channels insensitive to voltage modulation. The same mutations also allow...