The role of Drosophila Piezo in mechanical nociception

Abstract

Transduction of mechanical stimuli by receptor cells is essential for senses such as hearing, touch and pain^1,2,3,4. Ion channels have a role in neuronal mechanotransduction in invertebrates¹; however, functional conservation of these ion channels in mammalian mechanotransduction is not observed. For example, no mechanoreceptor potential C (NOMPC), a member of transient receptor potential (TRP) ion channel family, acts as a mechanotransducer in Drosophila melanogaster⁵ and Caenorhabditis elegans^6,7; however, it has no orthologues in mammals. Degenerin/epithelial sodium channel (DEG/ENaC) family members are mechanotransducers in C. elegans⁸ and potentially in D. melanogaster⁹; however, a direct role of its mammalian homologues in sensing mechanical force has not been shown. Recently, Piezo1 (also known as Fam38a) and Piezo2 (also known as Fam38b) were identified as components of mechanically activated channels in mammals¹⁰. The Piezo family are evolutionarily conserved transmembrane proteins. It is unknown whether they function in mechanical sensing in vivo and, if they do, which mechanosensory modalities they mediate. Here we study the physiological role of the single Piezo member in D. melanogaster (Dmpiezo; also known as CG8486). Dmpiezo expression in human cells induces mechanically activated currents, similar to its mammalian counterparts¹¹. Behavioural responses to noxious mechanical stimuli were severely reduced in Dmpiezo knockout larvae, whereas responses to another noxious stimulus or touch were not affected. Knocking down Dmpiezo in sensory neurons that mediate nociception and express the DEG/ENaC ion channel pickpocket (ppk) was sufficient to impair responses to noxious mechanical stimuli. Furthermore, expression of Dmpiezo in these same neurons rescued the phenotype of the constitutive Dmpiezo knockout larvae. Accordingly, electrophysiological recordings from ppk-positive neurons revealed a Dmpiezo-dependent, mechanically activated current. Finally, we found that Dmpiezo and ppk function in parallel pathways in ppk-positive cells, and that mechanical nociception is abolished in the absence of both channels. These data demonstrate the physiological relevance of the Piezo family in mechanotransduction in vivo, supporting a role of Piezo proteins in mechanosensory nociception.

Main

D. melanogaster is widely used to study mechanotransduction, and genetic studies have identified several ion channels that are essential for mechanosensation^5,9,12,13,14. However, none of the identified proteins have been shown to be activated by mechanical force when expressed in heterologous systems. Because expression of mouse Piezo proteins in a variety of mammalian cells induces mechanically activated currents¹⁰, we investigated whether the Drosophila counterpart is also sufficient to induce mechanosensitivity. Similar to its mammalian counterparts, the Dmpiezo gene is predicted to consist of a large number of transmembrane domains (39; Supplementary Fig. 1). Although fly and mammalian piezo genes do not exhibit extensive sequence conservation (24% identity), expression of Dmpiezo in cultured human cells induced mechanically activated cationic currents, suggesting a role of Dmpiezo in mechanotransduction¹¹.

To characterize Dmpiezo expression in flies we used a fusion between the Dmpiezo enhancer/promoter region and GAL4 (DmpiezoP–GAL4). Four independent DmpiezoP–GAL4 transgenic insertions were examined to avoid insertional effects on GAL4 expression. We used green fluorescent protein (GFP) regulated by upstream activating sequence elements (UAS) (UAS-GFP) for labelling cells, except for arborized neurons that were optimally visualized using the membrane-targeted UAS-CD8::GFP. We found fluorescent labelling induced by Dmpiezo enhancer/promoter region in all types of sensory neurons and several non-neuronal tissues in both adults and larvae (Supplementary Fig. 2). This diverse pattern of Dmpiezo expression observed in Drosophila is in accord with the expression of Piezo1 and Piezo2 in mice¹⁰.

We created Dmpiezo knockout flies in which all 31 coding exons were deleted using genomic recombination¹⁵ (Fig. 1a, see Supplementary Fig. 3 for details). The knockout flies were viable, fertile and did not show a lack of coordination or a defect in bristle mechanoreceptor potential (Supplementary Fig. 4). We studied whether Dmpiezo knockout larvae have mechanical nociception deficits by using a mechanically induced escape behaviour assay^9,14,16. Stimulation with von Frey filaments that ranged from 2–60 milliNewton (mN) demonstrated that Dmpiezo knockout larvae have a severe response deficit over a wide range (Fig. 1b). Repeated stimulations of the same larvae resulted in comparable responsiveness in both wild-type and Dmpiezo knockout, indicating that the stimuli did not induce considerable damage to the sensory system (Fig. 1c, d). A 153 ± 11.0 mN filament elicited responses only to the first of three stimulations in wild-type larvae, arguing that this amount of force is damaging (data not shown). For further experiments, we chose to stimulate the larvae using a 45 mN filament, which has been used in a previous study¹⁴, and elicits a substantial response in both wild-type and Dmpiezo mutant larvae. Thirty four ± 4.4% of Dmpiezo knockout larvae showed a response to 45 mN filament stimulation, compared to over 80% of wild-type or heterozygote larvae (Fig. 1e). As a control for the genetic background, we used larvae that carry the Dmpiezo knockout allele on one chromosome and a deficiency in which the entire Dmpiezo genomic region is deleted on the homologous chromosome. The defect in the trans-heterozygous larvae was similar to the knockout homozygote phenotype (51 ± 3.9%, P = 0.091). In contrast, Dmpiezo knockout larvae were indistinguishable from wild type in an assay for responses to high temperature, a different noxious stimulus that elicits the same escape response¹⁴ (Fig. 1f). Therefore, Dmpiezo knockout larvae retain a normal ability to elicit the escape behaviour in response to noxious stimuli, whereas Dmpiezo is specifically required for the mechanical modality of nociception. To evaluate the possible role of Dmpiezo in other modes of larval mechanical sensing, we tested the sensitivity of Dmpiezo knockout to gentle touch, which is mediated through ciliated neurons^17,18. We observed no defect in the sensitivity of Dmpiezo knockout larvae to innocuous gentle touch (Fig. 1g).

Figure 1: Mechanical nociception defect in Dm piezo knockout larvae.

a, Genomic map showing wild-type Dmpiezo gene (top) and engineered Dmpiezo knockout (bottom). Yellow and black boxes represent coding and non-coding exons, respectively. The segment deleted from the left arm of chromosome 2 (2L) in Dmpiezo knockout is marked with a grey box. b, Mechanical nociception assay using a range of stimulus forces in wild-type (WT) and Dmpiezo knockout larvae. n = 40 from four independent experiments. *P < 0.05, **P < 0.01 from two-tailed paired Student t-test. c, d, Mechanical nociception assay using repeated stimuli of the same larvae. n = 40. KO, knockout. e, Mechanical nociception assay using a 45 mN von Frey filament with wild type (+/+), heterozygous knockout (+/−), heterozygous deficiency (Def/+), homozygous KO (−/−) and trans-heterozygous KO (Def/−). n > 85. ***P < 0.001. f, Thermal nociception assay using heated probe (45 °C). n = 60. g, Gentle touch assay¹⁷. For details about the Kernan score, see Methods. n > 150. Error bars indicate mean ± s.e.m. NS, not significant.

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A mechanical nociception phenotype was previously observed in mutants of ppk, a DEG/ENaC channel⁹ and painless (pain), a TRPA ion channel¹⁴. The specificity of Dmpiezo knockout to mechanical nociception resembles the phenotype of ppk, as pain is also essential for sensing thermal nociception¹⁴. We therefore tested the role of Dmpiezo in ppk-positive cells using ppk-GAL4, which labels subclasses of multidendritic neurons^19,20. The multidendritic neurons are non-ciliated receptor cells that tile the body wall of the larvae and respond to a variety of external stimuli such as mechanical forces, temperature and light^9,14,16,21. We used enhanced (E)GFP driven directly by the regulatory regions of the ppk gene (ppk-EGFP)²² together with a red fluorescent protein expression in Dmpiezo-positive cells to probe Dmpiezo and ppk co-expression. Indeed, we did observe that all ppk-positive cells also expressed Dmpiezo (Fig. 2a). Next we used ppk-GAL4 to drive the expression of Dmpiezo RNA interference (RNAi) to test whether Dmpiezo function is specifically required in ppk-expressing cells. The restricted knockdown of Dmpiezo resulted in a mechanical nociceptive phenotype (Fig. 2b) similar to the phenotype observed in Dmpiezo knockout larvae (Fig. 1e). In a complementary approach, we used ppk-GAL4-driven expression of Dmpiezo complementary DNA in an attempt to rescue the mechanical nociception phenotype of Dmpiezo knockout larvae. We used a fusion between DmPiezo and GFP to monitor expression levels in ppk cells and DmPiezo localization within the neurons. GFP–DmPiezo fusion protein induces mechanically activated currents in human cell lines, similar to untagged DmPiezo, confirming functionality (Supplementary Fig. 5a–c). When expressed in Drosophila, GFP–DmPiezo fluorescence was present throughout cell bodies, axons and dendritic arborizations of ppk-positive neurons (Supplementary Fig. 5d). Importantly, expression of GFP–DmPiezo in ppk-positive neurons alone was sufficient to rescue the mechanical nociception defect of Dmpiezo knockout larvae (Fig. 2c). These data suggest that Dmpiezo functions in ppk-positive neurons to mediate mechanical nociception.

Figure 2: Dm piezo functions in ppk -positive type II sensory neurons.

a, Double fluorescence labelling using ppk-EGFP (green) and DmpiezoP-GAL4 that drives the expression of the nucleus targeted UAS-DsRed-NLS (red). A representative high-magnification image shows one ppk-positive neuron (arrow). All three ppk-positive cells in each hemisegment expressed Dmpiezo in all segments. b, Mechanical nociception assay with Dmpiezo knockdown larvae in ppk-expressing cells by ppk-GAL4 and UAS-Dmpiezo-RNAi. n > 85, ***P < 0.001. c, Mechanical nociception assay in rescued Dmpiezo knockout. GFP–DmPiezo was expressed in ppk-cells using ppk-GAL4 and UAS-GFP–DmPiezo. n > 60. **P < 0.01. Error bars indicate mean ± s.e.m.

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To test if the ppk-positive neurons respond to mechanical stimuli and if Dmpiezo mediates such responses, we performed electrophysiological recordings from isolated cells. Larvae that had GFP labelling in ppk-positive neurons were dissociated using enzymatic digestion and mechanical trituration. Plated fluorescent neurons were then tested using patch-clamp recordings in the cell-attached configuration, and they were stimulated using negative pressure through the recording pipette¹⁰. Stimulating wild-type neurons resulted in a current that was rapidly activated and had a half-maximal activation (P₅₀) of 27.6 ± 7.6 mm Hg (Fig. 3). These currents were not observed in the Dmpiezo knockout mutant neurons (Fig. 3). Therefore, ppk-positive neurons, which mediate the avoidance response to noxious stimuli, display Dmpiezo-dependent, mechanically activated currents.

Figure 3: Dm piezo mediates mechanically activated currents in ppk -positive neurons.

a, Representative currents elicited by negative pipette pressure (0 to −60 mm Hg, Δ10 mm Hg) in cell-attached configuration at −80 mV in wild type (left) and Dmpiezo^−/− (right). b, Average peak current–pressure relationship of stretch-activated currents in wild type (n = 12 cells) and Dmpiezo^−/− (n = 13 cells). Data points are mean ± s.e.m. fitted with a Boltzmann equation. **P < 0.01, ***P < 0.001, Mann–Whitney test.

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Silencing of ppk cells resulted in complete abolition of noxious mechanosensation (Supplementary Fig. 6), in accord with the severe defect previously observed¹⁶. In contrast, only a moderate deficit is observed upon eliminating or knocking down ppk in the same cells⁹, suggesting that there are multiple pathways for mechanical sensing. We tested mechanical nociception in larvae that are deficient in Dmpiezo and either pain or ppk to gain insight into cellular pathways that involve mechanotransduction in these cells. Once again, we used a 45 mN filament, enabling us to monitor both Dmpiezo-dependent and independent mechanisms (Fig. 1b). The Dmpiezo::pain double mutant had a defect that was comparable to each one of the mutants separately, suggesting that Dmpiezo and pain might function in the same pathway (Fig. 4a). Larvae that are heterozygous for both Dmpiezo and pain showed a response deficit whereas each one of them separately was normal (Fig. 4b), further demonstrating their role in a common signalling mechanism. Remarkably, combining both Dmpiezo and ppk knockdowns resulted in a nearly complete abolishment of responses to noxious mechanical stimuli (Fig. 4c). Importantly, responses to noxious temperatures and touch were normal in larvae with both Dmpiezo and ppk knocked down (Fig. 4d, e). These data indicate that Dmpiezo and ppk function in two parallel pathways in ppk-positive sensory neurons, and that together they constitute the response to noxious mechanical stimuli. There could be many reasons why the mechanically activated currents we observe are entirely dependent on DmPiezo (Fig. 3). This could either be because PPK responds to a different modality of mechanical stimulus or due to the specific experimental settings (for example, level of applied forces, solutions, applied voltage). Future experiments should resolve this issue.

Figure 4: Dm piezo and ppk function in parallel pathways.

a, Mechanical nociception assay using a 45 mN von Frey filament with double-null mutant of Dmpiezo and painless. Single-knockout strains were used as controls and the wild-type strain is w¹¹¹⁸. n > 60. b, Mechanical nociception assay on heterozygous larvae for Dmpiezo and/or pain. n (heterozygote Dmpiezo knockout) = 74 from three trials, n (heterozygote painless¹) = 169 from five trials, n (trans-heterozygote) = 166 from five trials. c, Mechanical nociception assay with ppk and Dmpiezo knockdown. ppk and/or Dmpiezo RNAi were driven by ppk-GAL4. n > 90. *P < 0.5, ***P < 0.001. d, Gentle touch sensitivity assay with ppk and Dmpiezo knockdown. For details about the Kernan score, see Methods. Wild type is w¹¹¹⁸. n > 90. e, Thermal nociception assay using 45 °C probe with ppk and Dmpiezo knockdown. n > 75. Error bars indicate mean ± s.e.m.

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Using the Drosophila model system we have demonstrated that piezo is essential for sensing noxious mechanical stimulus in vivo. This is the first demonstration that a Piezo family member is essential for mechanotransduction in the whole animal. Indeed, Dmpiezo is, to our knowledge, the first eukaryotic excitatory channel component shown to be activated by mechanical force in a heterologous expression system and required for sensory mechanotransduction in vivo. Piezo2 is expressed in mouse dorsal root ganglion neurons that are involved in sensing nociception, and is required for rapidly adapting mechanically activated currents in such isolated neurons¹⁰. This study raises the possibility that mammalian Piezo2 is also required for mechanical pain transduction in vivo. Furthermore, Drosophila genetics can now be used to map cellular pathways involved in piezo-dependent mechanotransduction in sensory neurons and beyond.

Methods Summary

Fly stocks

PiggyBacs (PBac{WH}CG8486-f02291, PBac{RB}CG8486-e00109; Exelixis Collection at the Harvard Medical School), ppk-GAL4 (Bloomington Drosophila Stock Center (BDSC), 32078, 32079), Deficiency (Df(2L)Exel7034/CyO; BDSC, 7807), UAS-Dmpiezo-RNAi (National Institute of Genetics, Japan, 8486R-3), UAS-ppk-RNAi (Vienna Drosophila RNAi Center, 108683), ppk-EGFP5 (ref. 22; Y. N. Jan), painless¹ (BDSC, 27895).

Generating Dmpiezo knockout flies

The Dmpiezo knockout fly was generated by FLP-FRT recombination with two PiggyBac lines as described previously¹⁵. The recombined knockout fly was confirmed by PCR (Supplementary Fig. 3). The genetic background was cleaned using meiotic recombination with w¹¹¹⁸.

Imaging

Fluorescence in adult fly or larva was detected by Nikon C2 Confocal Laser Point Scanning Microscope, Olympus FluoView500 Confocal Microscope or Olympus AX70 microscope.

Cloning

To clone the enhancer/promoter of the Dmpiezo gene, the genomic region between 1.0 kb upstream of the beginning of transcription and the start codon of Dmpiezo was amplified by PCR and cloned into the pPTGAL vector. The GFP–DmPiezo construct has three alanines as a linker between the carboxy-terminal GFP and amino-terminal DmPiezo. The construct was cloned in modified pUAST vector to generate transgenic flies and in modified pIRES2-EGFP vector for electrophysiology recordings.

Behavioural assays and statistics

The mechanical nociception was tested as described previously^9,14,16 using calibrated von Frey filaments. The thermal nociception was tested as described previously¹⁴ using a 45 °C heated metal probe. All error bars represent mean ± s.e.m.

Isolation of ppk-positive neurons

Third instar larvae that had GFP labelling in ppk-positive neurons were dissected, digested with collagenase and mechanically triturated. The cells were collected by centrifugation and plated on a poly-d-lysine-coated glass coverslip. The fluorescent ppk-positive cells were recorded after incubating for 2 h at room temperature (23–25 °C).

Electrophysiology

HEK cells were studied in the whole cell configuration using a polished glass probe for stimulation¹⁰ and ppk-positive neurons were stimulated using negative pressure in the cell attached configuration¹⁰.

Online Methods

Fly stocks

We used the following stocks: PiggyBacs (PBac{WH}CG8486-f02291, PBac{RB}CG8486-e00109, Exelixis Collection at the Harvard Medical School), ppk-GAL4 (Bloomington Drosophila Stock Center (BDSC), 32078, 32079), Deficiency (Df(2L)Exel7034/CyO, BDSC, 7807), UAS-Dmpiezo-RNAi (National Institute of Genetics, Japan, 8486R-3), UAS-ppk-RNAi (Vienna Drosophila RNAi Center, 108683), ppk-EGFP5 (ref. 22; Y. N. Jan), painless¹ (BDSC, 27895) and UAS-DsRed-NLS (J. W. Posakony). The following stocks were from BDSC: UAS-GFP, UAS-CD8::GFP, CyO-GFP, w¹¹¹⁸ and Canton-S.

Engineering Dmpiezo knockout flies

The Dmpiezo knockout fly was generated as described in previously described¹⁵. Two PiggyBac lines that carry the FRT sequence were selected for FLP-FRT recombination. PBac{WH}CG8486-f02291 is inserted in the first intron and PBac{RB}CG8486-e00109 in the 3′ untranslated region (UTR) of the Dmpiezo gene. After FLP-FRT recombination, 20 kb of the Dmpiezo gene, including all 31 coding exons, was removed and replaced with 7 kb of PiggyBac insertion that contained the FRT sequence and white gene. The recombined knockout fly was confirmed by PCR reactions (Supplementary Fig. 2). The genetic background was cleaned using meiotic recombination with w¹¹¹⁸.

Molecular biology

To clone the enhancer/promoter of the Dmpiezo gene, the genomic region between 1.0 kb upstream of the beginning of transcription and the start of the Dmpiezo coding region was amplified by PCR using forward primer, 5′-ATCTGGCGGCCGCTATCTATTTTTTAACTAGTGGAAGTCT-3′ and reverse primer, 5′-TTACTGGTACCATGGATGCCTCCGGCGCCGTTCTCCTCCAG-3′. The amplified sequence was cloned into pPTGAL vector (Drosophila Genomic Resource Center, 1225) using NotI and KpnI sites and the sequence was verified.

For rescue experiments, Dmpiezo cDNA was amplified from the plasmid reported in ref. 11, using forward primer 5′-TATTAGCGGCCGCAGTCTTCAGCTATGCGTGCATGGTG-3′ and reverse primer 5′-TAATTCGGTCCGTTATTGCGGTTGCTGTGGCTGCAGTTGCTCCGG-3′ and cloned into a modified pUAST vector using NotI and RsrII. NotI restriction enzyme site was used as a linker by providing three alanine residues between EGFP and DmPiezo. The order of sequences in the pUAST vector is the following: UAS-kozak-EGFP-3×(Ala)-DmPiezo. To generate transgenic flies, DNA was injected into the isogenized w¹¹¹⁸ embryos along with transposase Δ2–3. For the electrophysiology experiment, EGFP–DmPiezo was cloned into mammalian expression vector with CMV promoter.

Behaviour assays

Mechanical nociception was tested as described previously^9,14,16 using calibrated von Frey filaments. Thermal nociception was tested as described previously¹⁴ using a calibrated heated metal probe. For both nociception assays, the number of larvae that showed at least one 360° rotation was counted for each trial. The gentle touch assay was performed and each stimulated larva was scored as described previously¹⁷. 0 = no response, 1 = hesitates, 2 = turns or withdraws anterior segments, 3 = single reverse contractile wave, and 4 = multiple waves. For all behaviour assays each third instar larva was stimulated only once. All data were generated from at least three trials.

The von Frey filaments for larvae behaviour experiments were modified from Touch-Test sensory Evaluator (North Coast Medical) or from monofilament fishing lines. Each monofilament was cut to a length of 18 mm, glued into a pipette tip so that 9 mm of it protruded and mounted on a hand manipulator with a 90° angle. Each von Frey filament was calibrated as described previously⁹. The force of each von Frey stimulator was determined by measuring the weight upon filament bending and converting the value into the force: force (mN) = mass (g) × gravity acceleration constant (g; 9.8). Each stimulator was calibrated 15 times and its mean value was used in figures. The calibrated forces (mean ± s.e.m.) of each stimulator are as follows (in mN): 1.78 ± 0.15, 2.59 ± 0.15, 5.04 ± 0.19, 11.2 ± 0.66 and 59.9 ± 1.79.

Fluorescence imaging

For identifying tissues or cells expressing fluorescence by the Dmpiezo promoter, both adult flies and third instar larvae carrying DmpiezoP-GAL4 and UAS-GFP, or UAS-CD8::GFP, were dissected or whole-mounted. For double fluorescent labelling in multidendritic neurons, second instar larvae carrying ppk-EGFP, DmpiezoP-GAL4 and UAS-DsRed were whole-mounted. For imaging ppk-cells expressing GFP–DmPiezo, third instar larvae carrying ppk-GAL4 and UAS-GFP–DmPiezo were whole-mounted. Fluorescence images were obtained either by Nikon C2 Confocal Laser Point Scanning Microscope, Olympus FluoView500 Confocal Microscope or Olympus AX70 microscope.

Isolation of larvae ppk-positive neurons

In both wild-type and Dmpiezo knockout larvae, ppk-positive neurons were fluorescently labelled by ppk-EGFP, which is a direct fusion of ppk genomic regulatory regions with EGFP. Third instar larvae were dissected in M3 media containing 10% heat inactivated FBS. Each larva was cut twice and its internal organs were removed. The cleaned body wall was treated with 5 mg ml⁻¹ collagenase type IV at 25 °C for 1 h in serum-free M3 media and washed with serum containing M3 media. The enzyme-treated body wall was triturated with fire-polished Pasteur pipettes in M3 media with 2 mM EGTA and 10% FBS. The cuticle and debris were removed by centrifugation at 40g and the small size cells including neurons were collected by centrifugation at 360g for 10 min. The cell pellet was resuspended with serum containing M3 media and plated into a poly-d-lysine-coated coverslip in a small droplet. After 2 h of incubation at room temperature (23–25 °C), the coverslips were transferred to the electrophysiology rig for recording.

Electrophysiology

For whole-cell recordings in HEK293T cells, patch pipettes had resistances of 2–3 MΩ when filled with an internal solution consisting of (in mM) 133 CsCl, 10 HEPES, 5 EGTA, 1 CaCl₂, 1 MgCl₂, 4 MgATP and 0.4 Na2GTP (pH adjusted to 7.3 with CsOH). The extracellular solution consisted of (in mM) 130 NaCl, 3 KCl, 1 MgCl₂, 10 HEPES, 2.5 CaCl₂, 10 glucose (pH adjusted to 7.3 with NaOH). Mechanical stimulation was achieved using a fire-polished glass pipette (tip diameter 3–4 μm). The probe had a velocity of 1 μm ms⁻¹ during the ramp segment of the command for forward motion and the stimulus was applied for 150 ms.

For cell-attached recordings in ppk-positive dissociated neurons, patch pipettes had resistances of 3–3.5 MΩ when filled with a solution consisting of (in mM) 130 NaCl, 5 KCl, 10 HEPES, 1 CaCl₂, 1 MgCl₂, 10 TEA-Cl (pH 7.3 with NaOH). External solution used to zero the membrane potential consisted of (in mM) 140 KCl, 10 HEPES, 1 MgCl₂, 10 glucose (pH 7.3 with KOH). Membrane patches were stimulated with brief negative pressure pulses through the recording electrode using a Clampex controlled pressure clamp HSPC-1 device (ALA-scientific). Stretch-activated channels were recorded at a holding potential of −80 mV with pressure steps from 0 to −60 mm Hg (−10 mm Hg increments). Current–pressure relationships were fitted with a Boltzmann equation of the form: I(P) = (1 + exp (−(P – P₅₀)/s))−1, where I is the peak of stretch-activated current at a given pressure, P is the applied patch pressure (in mm Hg), P₅₀ is the pressure value that evoked a current value which is 50% of I_max, and s reflects the current sensitivity to pressure.

All experiments were performed at room temperature. Currents were sampled at 50 or 20 kHz and filtered at 5 or 2 kHz. Voltages were not corrected for a liquid junction potential. Leak currents before mechanical stimulations were subtracted off-line from the current traces.

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