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• W2773700017 abstract "•Piezo2 undergoes alternative splicing•Sensory neurons express multiple Piezo2 variants; non-neuronal tissues express one•Different classes of touch neurons express different Piezo2 splice forms•Alternative splicing confers functional differences on Piezo2 Piezo2 is a mechanically activated ion channel required for touch discrimination, vibration detection, and proprioception. Here, we discovered that Piezo2 is extensively spliced, producing different Piezo2 isoforms with distinct properties. Sensory neurons from both mice and humans express a large repertoire of Piezo2 variants, whereas non-neuronal tissues express predominantly a single isoform. Notably, even within sensory ganglia, we demonstrate the splicing of Piezo2 to be cell type specific. Biophysical characterization revealed substantial differences in ion permeability, sensitivity to calcium modulation, and inactivation kinetics among Piezo2 splice variants. Together, our results describe, at the molecular level, a potential mechanism by which transduction is tuned, permitting the detection of a variety of mechanosensory stimuli. Piezo2 is a mechanically activated ion channel required for touch discrimination, vibration detection, and proprioception. Here, we discovered that Piezo2 is extensively spliced, producing different Piezo2 isoforms with distinct properties. Sensory neurons from both mice and humans express a large repertoire of Piezo2 variants, whereas non-neuronal tissues express predominantly a single isoform. Notably, even within sensory ganglia, we demonstrate the splicing of Piezo2 to be cell type specific. Biophysical characterization revealed substantial differences in ion permeability, sensitivity to calcium modulation, and inactivation kinetics among Piezo2 splice variants. Together, our results describe, at the molecular level, a potential mechanism by which transduction is tuned, permitting the detection of a variety of mechanosensory stimuli. Living organisms detect and respond to many types of mechanical force. For instance, our sensory systems allow us to identify objects by their tactile features (Gibson, 1962Gibson J.J. Observations on active touch.Psychol. Rev. 1962; 69: 477-491Crossref PubMed Scopus (874) Google Scholar), coordinate controlled movement (Chesler et al., 2016Chesler A.T. Szczot M. Bharucha-Goebel D. Čeko M. Donkervoort S. Laubacher C. Hayes L.H. Alter K. Zampieri C. Stanley C. et al.The Role of PIEZO2 in human mechanosensation.N. Engl. J. Med. 2016; 375: 1355-1364Crossref PubMed Scopus (185) Google Scholar), and enjoy the pleasure associated with interactive social touch (McGlone et al., 2014McGlone F. Wessberg J. Olausson H. Discriminative and affective touch: sensing and feeling.Neuron. 2014; 82: 737-755Abstract Full Text Full Text PDF PubMed Scopus (515) Google Scholar). Several gene families have been identified as sensors of mechanical stimuli; the transient receptor potential (TRP), mechanosensitive ion channels (MSCs), and degenerin (DEG) epithelial sodium channels (ENaCs) in flies, bacteria, and worms (Goodman et al., 2002Goodman M.B. Ernstrom G.G. Chelur D.S. O’Hagan R. Yao C.A. Chalfie M. MEC-2 regulates C. elegans DEG/ENaC channels needed for mechanosensation.Nature. 2002; 415: 1039-1042Crossref PubMed Scopus (270) Google Scholar, Sukharev et al., 1994Sukharev S.I. Blount P. Martinac B. Blattner F.R. Kung C. A large-conductance mechanosensitive channel in E. coli encoded by mscL alone.Nature. 1994; 368: 265-268Crossref PubMed Scopus (585) Google Scholar, Walker et al., 2000Walker R.G. Willingham A.T. Zuker C.S. A Drosophila mechanosensory transduction channel.Science. 2000; 287: 2229-2234Crossref PubMed Scopus (526) Google Scholar) and the Piezo receptors in multiple phyla (Coste et al., 2010Coste B. Mathur J. Schmidt M. Earley T.J. Ranade S. Petrus M.J. Dubin A.E. Patapoutian A. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1435) Google Scholar). The Piezo genes, Piezo1 and Piezo2, encode exceptionally large mechanosensitive ion channels predicted to contain >14 transmembrane domains per monomer (Coste et al., 2015Coste B. Murthy S.E. Mathur J. Schmidt M. Mechioukhi Y. Delmas P. Patapoutian A. Piezo1 ion channel pore properties are dictated by C-terminal region.Nat. Commun. 2015; 6: 7223Crossref PubMed Scopus (134) Google Scholar, Ge et al., 2015Ge J. Li W. Zhao Q. Li N. Chen M. Zhi P. Li R. Gao N. Xiao B. Yang M. Architecture of the mammalian mechanosensitive Piezo1 channel.Nature. 2015; 527: 64-69Crossref PubMed Scopus (262) Google Scholar). Expression of Piezo proteins is sufficient to confer mechanically evoked ionic currents to cells (Coste et al., 2010Coste B. Mathur J. Schmidt M. Earley T.J. Ranade S. Petrus M.J. Dubin A.E. Patapoutian A. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1435) Google Scholar), and these molecules are believed to be intrinsically gated by force (Syeda et al., 2016Syeda R. Florendo M.N. Cox C.D. Kefauver J.M. Santos J.S. Martinac B. Patapoutian A. Piezo1 channels are inherently mechanosensitive.Cell Rep. 2016; 17: 1739-1746Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar). Piezo2 is most highly expressed in sensory ganglia, although it has also been reported to be found in lung, bladder, skin, and bone (Coste et al., 2010Coste B. Mathur J. Schmidt M. Earley T.J. Ranade S. Petrus M.J. Dubin A.E. Patapoutian A. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1435) Google Scholar, Ikeda et al., 2014Ikeda R. Cha M. Ling J. Jia Z. Coyle D. Gu J.G. Merkel cells transduce and encode tactile stimuli to drive Aβ-afferent impulses.Cell. 2014; 157: 664-675Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, Woo et al., 2014Woo S.H. Ranade S. Weyer A.D. Dubin A.E. Baba Y. Qiu Z. Petrus M. Miyamoto T. Reddy K. Lumpkin E.A. et al.Piezo2 is required for Merkel-cell mechanotransduction.Nature. 2014; 509: 622-626Crossref PubMed Scopus (436) Google Scholar). Mice and humans lacking functional Piezo2 exhibit severe deficits in detection of vibration, fine touch, hair movement, proprioception, and breathing regulation (Chesler et al., 2016Chesler A.T. Szczot M. Bharucha-Goebel D. Čeko M. Donkervoort S. Laubacher C. Hayes L.H. Alter K. Zampieri C. Stanley C. et al.The Role of PIEZO2 in human mechanosensation.N. Engl. J. Med. 2016; 375: 1355-1364Crossref PubMed Scopus (185) Google Scholar, Nonomura et al., 2017Nonomura K. Woo S.H. Chang R.B. Gillich A. Qiu Z. Francisco A.G. Ranade S.S. Liberles S.D. Patapoutian A. Piezo2 senses airway stretch and mediates lung inflation-induced apnoea.Nature. 2017; 541: 176-181Crossref PubMed Scopus (210) Google Scholar, Ranade et al., 2014Ranade S.S. Woo S.H. Dubin A.E. Moshourab R.A. Wetzel C. Petrus M. Mathur J. Bégay V. Coste B. Mainquist J. et al.Piezo2 is the major transducer of mechanical forces for touch sensation in mice.Nature. 2014; 516: 121-125Crossref PubMed Scopus (464) Google Scholar). These results point to Piezo2 as being crucial in the detection of a variety of mechanical stimuli known to be encoded by several classes of mechanosensory neurons. For example, gentle touch is mediated by low threshold mechanoreceptors (LTMRs) with nerve endings in the skin whereas proprioceptors target muscles and tendons. LTMRs themselves are quite diverse, differing from one another based on the type of skin they innervate (e.g., glabrous and hairy skin), the size of receptive fields (wide and narrow field), their adaption properties (slow and rapid), and the morphology of their afferent end organs (Pacinian and Meissner’s corpuscles, Ruffini endings, and Merkel cell complexes; Abraira and Ginty, 2013Abraira V.E. Ginty D.D. The sensory neurons of touch.Neuron. 2013; 79: 618-639Abstract Full Text Full Text PDF PubMed Scopus (779) Google Scholar, Usoskin et al., 2015Usoskin D. Furlan A. Islam S. Abdo H. Lönnerberg P. Lou D. Hjerling-Leffler J. Haeggström J. Kharchenko O. Kharchenko P.V. et al.Unbiased classification of sensory neuron types by large-scale single-cell RNA sequencing.Nat. Neurosci. 2015; 18: 145-153Crossref PubMed Scopus (1124) Google Scholar). We were curious how a single molecule might function in such morphologically diverse settings to permit the detection of a wide range of mechanical stimuli. In particular, we focused on whether alternative splicing, a commonly used mechanism for genes to produce molecular and functional diversity (Lipscombe and Andrade, 2015Lipscombe D. Andrade A. Calcium channel CaVα1 splice isoforms - tissue specificity and drug action.Curr. Mol. Pharmacol. 2015; 8: 22-31Crossref PubMed Scopus (24) Google Scholar, Pan et al., 2008Pan Q. Shai O. Lee L.J. Frey B.J. Blencowe B.J. Deep surveying of alternative splicing complexity in the human transcriptome by high-throughput sequencing.Nat. Genet. 2008; 40: 1413-1415Crossref PubMed Scopus (2543) Google Scholar, Wang et al., 2008Wang E.T. Sandberg R. Luo S. Khrebtukova I. Zhang L. Mayr C. Kingsmore S.F. Schroth G.P. Burge C.B. Alternative isoform regulation in human tissue transcriptomes.Nature. 2008; 456: 470-476Crossref PubMed Scopus (3567) Google Scholar), is used to regulate Piezo2 function. We find that Piezo2 undergoes a surprisingly extensive alternative splicing, which is used to generate unique isoforms that are found in specific tissues and cell types. We identify two previously unannotated exons and 16 isoforms of Piezo2 that are specifically enriched in mouse sensory neurons. Importantly, we demonstrate that Piezo2 variants exhibit major differences in three key biophysical properties: their rates of inactivation; ion permeability; and modulation by intracellular calcium. Given that distinct classes of sensory neurons express select classes of Piezo2, we identify alternative splicing as an important determinant in mechanosensory specialization. Piezo2 has been reported to be expressed in sensory ganglia and in non-neuronal tissues, including lung and bladder (Coste et al., 2010Coste B. Mathur J. Schmidt M. Earley T.J. Ranade S. Petrus M.J. Dubin A.E. Patapoutian A. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1435) Google Scholar). We therefore used in situ hybridization (ISH) to fully characterize the expression of Piezo2 in these tissues (Figure 1). As expected, Piezo2 was detectable in discrete cells in bladder and lung, whereas it is expressed at very high levels in a large proportion of neurons in the trigeminal ganglion (TG) (Figure 1A). Double-label ISH experiments showed that Piezo2 is co-expressed with genes found in LTMRs (Figures 1B and 1C). By contrast, Piezo2 is virtually absent from TRPM8-expressing neurons (2% TRPM8-neurons; Figures 1B and 1C), consistent with the proposed role of these neurons as dedicated cold sensors (Knowlton et al., 2013Knowlton W.M. Palkar R. Lippoldt E.K. McCoy D.D. Baluch F. Chen J. McKemy D.D. A sensory-labeled line for cold: TRPM8-expressing sensory neurons define the cellular basis for cold, cold pain, and cooling-mediated analgesia.J. Neurosci. 2013; 33: 2837-2848Crossref PubMed Scopus (183) Google Scholar, Pogorzala et al., 2013Pogorzala L.A. Mishra S.K. Hoon M.A. The cellular code for mammalian thermosensation.J. Neurosci. 2013; 33: 5533-5541Crossref PubMed Scopus (138) Google Scholar). Interestingly, despite the apparent lack of mechanical pain abnormalities in individuals with non-functional Piezo2 (Chesler et al., 2016Chesler A.T. Szczot M. Bharucha-Goebel D. Čeko M. Donkervoort S. Laubacher C. Hayes L.H. Alter K. Zampieri C. Stanley C. et al.The Role of PIEZO2 in human mechanosensation.N. Engl. J. Med. 2016; 375: 1355-1364Crossref PubMed Scopus (185) Google Scholar) and similar absence of pain phenotypes in Piezo2-null mice (Ranade et al., 2014Ranade S.S. Woo S.H. Dubin A.E. Moshourab R.A. Wetzel C. Petrus M. Mathur J. Bégay V. Coste B. Mainquist J. et al.Piezo2 is the major transducer of mechanical forces for touch sensation in mice.Nature. 2014; 516: 121-125Crossref PubMed Scopus (464) Google Scholar), the majority of non-peptidergic nociceptors marked by the expression of the Mas-related G-protein receptor D (Mrgprd) also express Piezo2 (76% Mrgprd neurons; Figures 1B and 1C). Piezo2 partially overlapped with TH, a proposed marker for C-fiber low threshold mechano receptors (C-LTMRs), supporting the idea that neurons expressing this gene are more heterogeneous than previously assumed (Nguyen et al., 2017Nguyen M.Q. Wu Y. Bonilla L.S. von Buchholtz L.J. Ryba N.J.P. Diversity amongst trigeminal neurons revealed by high throughput single cell sequencing.PLoS ONE. 2017; 12: e0185543Crossref PubMed Scopus (30) Google Scholar). Furthermore, Piezo2 was also found in subsets of TRPV1- and TRPA1-positive neurons (32% and 60%, respectively; data not shown). Piezo2 is found in LTMRs that rapidly signal hair movement, proprioceptors that detect muscle contraction, and cells in lung and bladder that respond relatively slowly to radial stretch. Given the differences in these mechanosensory tasks, we hypothesized that Piezo2 might, at the molecular level, be differentially regulated by sensory and non-sensory tissues. To investigate the possibility of alternative splicing underlying this process, we sequenced Piezo2 mRNA from the lung and bladder and compared it with Piezo2 from the TG (Figure 2). The sequences of Piezo2 amplified from lung and bladder were the same as those reported previously (Coste et al., 2010Coste B. Mathur J. Schmidt M. Earley T.J. Ranade S. Petrus M.J. Dubin A.E. Patapoutian A. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1435) Google Scholar). Remarkably, sequencing Piezo2 mRNA from TG revealed the existence of multiple novel variants that included two previously unannotated exons (exons 18 and 35). In total, we found that five exons (that we named E18, E19, E33, E35, and E40) are alternatively spliced in mouse sensory ganglion neurons (Figure 3A). Importantly, usage of these exons does not generate translational frameshifts in the coding sequence of Piezo2 transcripts and, as expected for bone fide exons, the genomic sequences surrounding these exons contain consensus splice acceptor and donor sequences (Trapnell et al., 2009Trapnell C. Pachter L. Salzberg S.L. TopHat: discovering splice junctions with RNA-seq.Bioinformatics. 2009; 25: 1105-1111Crossref PubMed Scopus (8995) Google Scholar). In addition, ISH with probes designed to specifically detect these exons corroborated their expression in sensory neurons (Figure S1A).Figure 3Sequences Encoded by Alternately Spliced Exons Have Intracellular LocationsShow full caption(A) Alignment of the coding sequence of alternatively spliced exons from multiple species shows their high level of sequence similarity; amino acids identical to those of human Piezo2 are shaded gray. Numbering below sequence refers to the position in V16 mouse Piezo2 sequence.(B) Schematic representation of the proposed structure of Piezo2, based on sequence alignment of Piezo2 and Piezo1 and the predicted membrane topology of Piezo1 (Coste et al., 2015Coste B. Murthy S.E. Mathur J. Schmidt M. Mechioukhi Y. Delmas P. Patapoutian A. Piezo1 ion channel pore properties are dictated by C-terminal region.Nat. Commun. 2015; 6: 7223Crossref PubMed Scopus (134) Google Scholar). The proposed positions of sequences encoded by alternatively spliced exons are indicated in blue as well as the approximate positions of amino acid for V16 mouse Piezo2.(B–E) The predicted intracellular location of alternatively spliced exons was confirmed by HA-epitope-tagging experiments.(C–E) Fields of HEK293 cells transfected with HA-epitope-tagged Piezo2 constructs were immune stained, live (upper panels) and following permeabilization (lower panels). HA epitope was engineered into E10 (C), E18 and E19 (D), and E40 (E). HA epitope was detected (red) only after membrane disruption in cells expressing E10 (HA), E18 (HA), and E40 (HA), confirming the sequences they encode have an intracellular location.(F) As expected, cells expressing extracellular N-terminally tagged TacR1 produced detectable epitope staining in both permeabilized and non-permeabilized cells. Note, all epitope-tagged Piezo2 constructs retained normal mechanically activated ion channel activity.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Alignment of the coding sequence of alternatively spliced exons from multiple species shows their high level of sequence similarity; amino acids identical to those of human Piezo2 are shaded gray. Numbering below sequence refers to the position in V16 mouse Piezo2 sequence. (B) Schematic representation of the proposed structure of Piezo2, based on sequence alignment of Piezo2 and Piezo1 and the predicted membrane topology of Piezo1 (Coste et al., 2015Coste B. Murthy S.E. Mathur J. Schmidt M. Mechioukhi Y. Delmas P. Patapoutian A. Piezo1 ion channel pore properties are dictated by C-terminal region.Nat. Commun. 2015; 6: 7223Crossref PubMed Scopus (134) Google Scholar). The proposed positions of sequences encoded by alternatively spliced exons are indicated in blue as well as the approximate positions of amino acid for V16 mouse Piezo2. (B–E) The predicted intracellular location of alternatively spliced exons was confirmed by HA-epitope-tagging experiments. (C–E) Fields of HEK293 cells transfected with HA-epitope-tagged Piezo2 constructs were immune stained, live (upper panels) and following permeabilization (lower panels). HA epitope was engineered into E10 (C), E18 and E19 (D), and E40 (E). HA epitope was detected (red) only after membrane disruption in cells expressing E10 (HA), E18 (HA), and E40 (HA), confirming the sequences they encode have an intracellular location. (F) As expected, cells expressing extracellular N-terminally tagged TacR1 produced detectable epitope staining in both permeabilized and non-permeabilized cells. Note, all epitope-tagged Piezo2 constructs retained normal mechanically activated ion channel activity. To precisely quantify the extent of alternative splicing, we turned to a recently developed NextGen sequencing methodology (single molecule real-time sequencing [SMRT] sequencing; Eid et al., 2009Eid J. Fehr A. Gray J. Luong K. Lyle J. Otto G. Peluso P. Rank D. Baybayan P. Bettman B. et al.Real-time DNA sequencing from single polymerase molecules.Science. 2009; 323: 133-138Crossref PubMed Scopus (2434) Google Scholar), which enabled long sequencing reads (3–3.5 kbp) of thousands of Piezo2 transcripts from multiple mouse tissues; TG (n = 1,721 full-length reads of middle region); lung (n = 4,800); and bladder (n = 2,373). Figure 2 shows the sequence reads of Piezo2 from TG, lung, and bladder aligned against the coding sequence of Piezo2 (also see Figure S1B and Table S1). Remarkably, this type of comparison revealed that lung and bladder principally express a single form of Piezo2; we name this V2 (approximately 80% of reads), which contains E33 but lacks the other 4 alternatively spliced exons (Figures 2C–2E). By contrast, the same comparison showed that TG neurons express at least 17 different splice variants of Piezo2 (Figures 2B and 2E). Certain splice forms were absent from the TG, lung, and bladder, demonstrating that splicing is selective (Figure 2E). Unbiased analysis of sequencing results for other regions of Piezo2 failed to reveal additional sites of alternative splicing except for a sixth alternatively spliced exon, E10, which is alternatively spliced in the lung and bladder (Figure S1B). E10 is expressed in all TG neurons and is apparently not alternatively spliced in this tissue. We next sought to determine the positions of alternately spliced exons within the Piezo2 protein. Little is known about the structure of Piezo2, so we took advantage of the fact that its sequence can be broadly aligned to Piezo1. Based on known and predicted membrane topologies of Piezo1 (Coste et al., 2015Coste B. Murthy S.E. Mathur J. Schmidt M. Mechioukhi Y. Delmas P. Patapoutian A. Piezo1 ion channel pore properties are dictated by C-terminal region.Nat. Commun. 2015; 6: 7223Crossref PubMed Scopus (134) Google Scholar), we expected exons E10, E18, E19, E33, E35, and E40 to be found within three intracellular loops (Figure 3B). To confirm our hypothesis, we performed live cell staining experiments using constructs where epitopes were inserted at exons E10, E18, or E40, locations found not to interfere with functional responses. Our results show that all the alternatively spliced exons are located in intracellular domains (Figures 3C–3E). Intriguingly, exons 33, 35, and 40 are found in an exceptionally large loop toward the C terminus of the receptor close to where the pore domain is located (Coste et al., 2015Coste B. Murthy S.E. Mathur J. Schmidt M. Mechioukhi Y. Delmas P. Patapoutian A. Piezo1 ion channel pore properties are dictated by C-terminal region.Nat. Commun. 2015; 6: 7223Crossref PubMed Scopus (134) Google Scholar, Ge et al., 2015Ge J. Li W. Zhao Q. Li N. Chen M. Zhi P. Li R. Gao N. Xiao B. Yang M. Architecture of the mammalian mechanosensitive Piezo1 channel.Nature. 2015; 527: 64-69Crossref PubMed Scopus (262) Google Scholar). The preferential expression of multiple splice variants of Piezo2 in sensory neurons versus the highly restricted splicing found in non-neuronal tissue suggested that there might be intrinsic functional differences between isoforms. To test this hypothesis, we compared the properties of the major Piezo2 isoform expressed in non-neuronal cells, V2 (which contains E33 but lacks E18, E19, E35, and E40; see Figures 2E and 4A ), with the properties of the neuronal isoform that is most different from it, V14 (which contains E18, E19, E35, and E40 but lacks E33). We examined the electrophysiological properties of V2 and V14 by heterologously expressing them in HEK293 cells, assessing mechanically activated inward currents generated by membrane indentation. Expression of both V2 and V14 was sufficient to generate large responses, which increased proportionally to stimulus strength and which were not seen in control GFP-transfected cells (Chesler et al., 2016Chesler A.T. Szczot M. Bharucha-Goebel D. Čeko M. Donkervoort S. Laubacher C. Hayes L.H. Alter K. Zampieri C. Stanley C. et al.The Role of PIEZO2 in human mechanosensation.N. Engl. J. Med. 2016; 375: 1355-1364Crossref PubMed Scopus (185) Google Scholar). This control, an exclusion cutoff of <50 pA (unless stated otherwise), and other controls (Experimental Procedures and Figures S2A–S2C) were employed to ensure HEK293 endogenous Piezo1 currents did not contaminate recordings (Dubin et al., 2017aDubin A.E. Murthy S. Lewis A.H. Brosse L. Cahalan S.M. Grandl J. Coste B. Patapoutian A. Editorial note to: endogenous Piezo1 can confound mechanically activated channel identification and characterization.Neuron. 2017; 94: 265Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar, Dubin et al., 2017bDubin A.E. Murthy S. Lewis A.H. Brosse L. Cahalan S.M. Grandl J. Coste B. Patapoutian A. Endogenous Piezo1 can confound mechanically activated channel identification and characterization.Neuron. 2017; 94: 266-270.e3Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Having confirmed that the splice variants were functional, we focused our experiments on three important channel properties that would be predicted to strongly influence neuronal excitability: ion permeability, modulation by Ca2+, and inactivation kinetics. First, we examined ion permeability, looking for differences between V2 and V14. We measured permeability of Ca2+ by these Piezo2 isoforms relative to Cs+ (Coste et al., 2010Coste B. Mathur J. Schmidt M. Earley T.J. Ranade S. Petrus M.J. Dubin A.E. Patapoutian A. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1435) Google Scholar). The relative permeability for Ca2+ of V2 was significantly higher than that of V14 (Figures 4A and 4B; Table S2). This difference in permeability was selective for Ca2+ because we did not find differences in Na+ or Mg2+ permeability (Figures S2D and S2E; Table S2). Splice V16 (Figures S2F and S2G) showed a calcium permeability similar to that of V2, consistent with the presence of exon 33 positively affecting calcium permeability despite being located in an intracellular loop. More importantly, these results provide evidence that alternate splicing of Piezo2 indeed generates receptors expressed by neurons and non-neuronal tissues with distinct pore properties. A recent study reported that Piezo2 function is positively regulated by intracellular Ca2+ and that this may be important for alterations in touch sensitivity under pathological conditions (Eijkelkamp et al., 2013Eijkelkamp N. Linley J.E. Torres J.M. Bee L. Dickenson A.H. Gringhuis M. Minett M.S. Hong G.S. Lee E. Oh U. et al.A role for Piezo2 in EPAC1-dependent mechanical allodynia.Nat. Commun. 2013; 4: 1682Crossref PubMed Scopus (143) Google Scholar). Given the intracellular location of the alternatively spliced-exons-encoded parts of the loops (Figure 3), we hypothesized that, in addition to differences in ion permeability, sensory-neuron-specific variants might be more robustly modulated by intracellular calcium. Therefore, we compared the mechanically evoked currents from V2 and V14 either in the presence or in the absence of high intracellular Ca2+. When nominally divalent free intracellular solutions were used, V2 and V14 displayed similar responses to mechanical force. Strikingly, when intracellular solutions contained 10 μM Ca2+, we found a major decrease in the mechanical threshold of V14, whereas the threshold of V2 was unaffected (Figures 4C–4E). Piezo2 inactivates rapidly, within ∼10 ms (Coste et al., 2010Coste B. Mathur J. Schmidt M. Earley T.J. Ranade S. Petrus M.J. Dubin A.E. Patapoutian A. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels.Science. 2010; 330: 55-60Crossref PubMed Scopus (1435) Google Scholar), to static indentation, presumably reflecting rapid entry of the channel into a non-conducting desensitized state. This allows Piezo2 to perform basic frequency filtering and is believed to be a key factor in the ability of sensory neurons to respond to rapid and/or vibrational stimuli (Lewis et al., 2017Lewis A.H. Cui A.F. McDonald M.F. Grandl J. Transduction of repetitive mechanical stimuli by Piezo1 and Piezo2 ion channels.Cell Rep. 2017; 19: 2572-2585Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Given that Piezo2 is required for the sensation of vibration and touch discrimination (Chesler et al., 2016Chesler A.T. Szczot M. Bharucha-Goebel D. Čeko M. Donkervoort S. Laubacher C. Hayes L.H. Alter K. Zampieri C. Stanley C. et al.The Role of PIEZO2 in human mechanosensation.N. Engl. J. Med. 2016; 375: 1355-1364Crossref PubMed Scopus (185) Google Scholar, Ranade et al., 2014Ranade S.S. Woo S.H. Dubin A.E. Moshourab R.A. Wetzel C. Petrus M. Mathur J. Bégay V. Coste B. Mainquist J. et al.Piezo2 is the major transducer of mechanical forces for touch sensation in mice.Nature. 2014; 516: 121-125Crossref PubMed Scopus (464) Google Scholar), we explored the possibility that splicing might fine tune inactivation. To quantify rates of inactivation of V2 and V14, we stimulated cells with increasing strengths of mechanical force, measured current decay, and calculated the tau of inactivation. Notably, we found that V2 inactivated significantly slower than V14 (Figures 5A–5C). Comparing rates of inactivation of four additional Piezo2 isoforms (expressed by sensory neurons) revealed that the presence of E35 correlated with the increased rate of inactivation (Figure 5C). In summary, our results establish that alternate splicing selectively tunes the permeability, calcium-mediated sensitivity, and rate of inactivation of Piezo2. Neuronal sensory tissues express at least 17 isoforms of Piezo2 (Figure 2E; Table S1). We hypothesized that the functionally distinct Piezo2 isoforms might be differentially enriched in certain sub-types of sensory neurons. To test this hypothesis, we compared transcriptomic data from sensory neurons that developmentally express the Trpv1 ion channel (Trpv1lineage neurons) with neurons that do not (non-Trpv1lineage neurons; Hjerling-Leffler et al., 2007Hjerling-Leffler J. Alqatari M. Ernfors P. Koltzenburg M. Emergence of functional sensory subtypes as defined by transient receptor potential channel expression.J. Neurosci. 2007; 27: 2435-2443Crossref PubMed Scopus (165) Google Scholar, Mishra et al., 2011Mishra S.K. Tisel S.M. Orestes P. Bhangoo S.K. Hoon M.A. TRPV1-lineage neurons are required for thermal sensation.EMBO J. 2011; 30: 582-593Crossref PubMed Scopus (188) Google Scholar). This strategy allowed us to compare the coding sequences of Piezo2 expressed in neurons that are broadly required for nociception, pruriception, and thermoreception (Le Pichon and Chesler, 2014Le Pichon C.E. Chesler A.T. The functional and anatomical dissection of somatosensory subpopulations using mouse genetics.Front. Neuroanat. 2014; 8: 21Crossref PubMed Scopus (139) Google Scholar) with those found in neurons involved in discriminative touch and proprioception. Interestingly, the relative abundance of alternative" @default.
• W2773700017 created "2017-12-22" @default.
• W2773700017 creator A5003091226 @default.
• W2773700017 creator A5010136511 @default.
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• W2773700017 date "2017-12-01" @default.
• W2773700017 modified "2023-10-02" @default.
• W2773700017 title "Cell-Type-Specific Splicing of Piezo2 Regulates Mechanotransduction" @default.
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• W2773700017 doi "https://doi.org/10.1016/j.celrep.2017.11.035" @default.
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