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, 21 (10), 2760-2771

Cell-Type-Specific Splicing of Piezo2 Regulates Mechanotransduction

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Cell-Type-Specific Splicing of Piezo2 Regulates Mechanotransduction

Marcin Szczot et al. Cell Rep.

Abstract

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.

Keywords: Piezo; ion-channel; sensation; splicing; touch.

Figures

Figure 1
Figure 1. Piezo2 is highly expressed in heterogenous classes of peripheral sensory neurons
A, Single label ISH shows that Piezo2 is expressed at high levels in TG neurons (left panel), it is also expressed at lower levels in scattered cells in the lung and bladder (middle and right panels). B, Double-label ISH reveals that Piezo2 (green) is found in neurons that are associated with sensation of low threshold mechanical stimuli, or are proprioceptive (Ntrk2, Ntrk3, Npy2r, and Th), and is present in HTMR-neurons (Mrgprd). However, Piezo2 is virtually absent from cold responsive neurons (Trpm8). Arrowheads indicate singly labeled neurons. C, Quantification of ISH data shows the extent of overlap in expression with Piezo2: 41/42 Ntrk2-neurons (280 Piezo2-cells), 114/126 Ntrk3-neurons (232), 27/45 Npy2r-neurons (242), 36/58 Th-neurons (464), 35/46 Mrgprd-neurons (464), and 1/38 Trpm8-neurons (232) 4–10 sections n=3 mice.
Figure 2
Figure 2
Multiple splice isoforms of mouse Piezo2 are alternatively expressed in neuronal and non-neuronal tissues. Alignment of sequencing reads of Piezo2 transcripts with the coding sequence of Piezo2 (A) reveals that 5 exons are alternatively spliced and there are profound differences in splicing between neuronal and non-neuronal tissues. Sequence reads from TG (B), lung (C), and bladder (D) are displayed as individual line plots, black indicates sequence identity and open areas regions of sequence lacking similarity (for clarity, junctions between exons are highlighted). Grey bars show the approximate percentage of sequence reads. Exons 17–41 of Piezo2 are colored in alternating blocks, and pink blocks for alternatively spliced exons. E, Analysis of reads from TG (Upper panel) showed that 16 variants are expressed, while lung and bladder (Middle panel) express one predominant form of Piezo2, V2. Lower panel is a schematic of the numbering system for splice variants.
Figure 3
Figure 3. Sequences encoded by alternately spliced exons have intracellular locations
A, Alignment of the coding sequence of alternatively spliced exons from multiple species show their high level of sequence similarity; amino-acids identical to those of human Piezo2 are shaded grey. 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., 2015). The proposed positions of sequences encoded by alternatively spliced exons are indicated in blue as well as the approximate positions of amino-acid for V32 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), E18–19 (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 a 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.
Figure 4
Figure 4
Neuronal and non-neuronal splice forms of Piezo2 exhibit different calcium permeability and calcium-induced receptor sensitization. A, Mechanically-evoked responses of HEK293-cells expressing Piezo2 V2 and V14 show that the current of the non-neuronal splice form of Piezo2 differs from the neuronal variant at the indicated voltages, indicating a difference in relative calcium permeability. Cesium and calcium were the intracellular and extracellular cations, respectively. Lower panels schematize the exons usage of variant V2 and V14; these isoforms have opposite alternate splicing structures: grey boxes indicate presence of an exon and open boxes indicates the absence; all other regions in these constructs were identical. B, Quantification of the relative permeability of calcium versus cesium for V2 and V14. Data represent means ±SEM (n=22 for V2 n=20 for V14); significant difference in calcium permeability between variants * (Student’s t-test) p= 0.028. C, Sample traces demonstrating dose dependence of mechanically activated currents in cells expressing different variants in nominally calcium free intracellular solution (black) and high calcium intracellular solution (green). Traces are normalized to dose response fit maximums. D, The average fit of mechanically evoked currents for non-neuronal splice form, V2 (left panel) is not affected by intracellular calcium, while calcium decreases the indentation needed to gate V14 (right panel). Response profiles fits (middle trace represents the mean and outer traces SEM) were assessed by applying mechanical force (indentation of the plasma membrane) in the absence of intracellular calcium (black) and in the presence of 10 μM intracellular calcium (green). Note leftward shift in mechanical threshold of V14 in the presence of intracellular calcium (V2 calcium free n=8, V2 high calcium n=5, V14 calcium free n=8, V2 high calcium n=6) E, Quantification of the calcium induced shift in half maximal force showed that V2 is insensitive to intracellular calcium, while V14 is sensitized. Data are means ±SEM (V2 calcium free n=8, V2 high calcium n=5, V14 calcium free n=8, V2 high calcium n=6); significant difference in response sensitivity for V14 between no calcium and 10 μM calcium * (Student’s t-test) p= 0.0064, and for V2 it is not significant, p= 0.92.
Figure 5
Figure 5
Neuronal and non-neuronal splice forms of Piezo2 exhibit different rates of inactivation to mechanical stimulation. The neuronal variant V14 inactivates to mechanical stimulation significantly faster than the non-neuronal, V2 isoform of Piezo2. A, Mechanically evoked membrane currents for HEK293-cells transfected with the non-neuronal, V2 (left) and the neuronal, V14 (right) variants. Increasing mechanical stimulation (upper panel) elicits greater current (lower panel). B, Schematized exons usage of variants (see Figure 4 for details). C, Quantification of inactivation kinetics for different Piezo2 variants shows that V14 has significantly faster rates of inactivation than V2, V18, and V23. Data are means ±SEM V2 (n=12), V8 (n=16), V14 (n=12), V16 (n=9), V18 (n=12), and V23 (n=13) significant difference in rates of inactivation between variants * (one-way ANOVA with Dunnett’s multiple comparison test) V14 vs V2 p=0.009, V14 vs V8 p>0.99, V14 vs V16 p>0.99, V14 vs V18 p=0.0029, and V14 vs V23 p=0.0204.
Figure 6
Figure 6. Subsets of sensory neurons selectively splice exon 35 and Merkel cells express restricted classes of Piezo2 splice variants
E35 is selectively spliced in Piezo2 transcripts expressed by LTMRs and proprioceptive neurons. A, Analysis of NextGen sequence reads reveals that E35 is expressed at high levels in transcripts from non-Trpv1lineage neurons and at a lower level in transcripts from Trpv1lineage neurons. Exon reads were normalized to individual length of exons and to the overall Piezo2 reads in non-Trpv1lineage neuron and Trpv1lineage neuron datasets. B, RT-PCR of cDNA from non-Trpv1lineage and Trpv1lineage neurons confirmed that there is significantly greater expression of E35 in non-Trpv1lineage neurons than Trpv1lineage neurons. Significant difference (Student’s t-test) in expression between cell-types *, were p<0.05 (means ±SEM, n=3 samples). C, Double-label ISH reveals that E35 (green) is expressed in many Ntrk3-positive neurons (purple; upper panel), but there is no co-expression of E35 with Mrgprd (purple; lower panel). D, Comparison of the density of staining of positive profiles of Ntrk3 cells (purple) relative to those for E35 (green) shows there is high correspondence in the expression profiles of these two molecules. In contrast, there is very poor correspondence in the profiles stained for Mrgprd (purple) and E35 (green). Sequencing of Piezo2 from purified Merkel cell preparations reveals that they predominantly express V5. E, plot of reads from Piezo2 transcripts aligned to the coding sequence of Piezo2 (exons 17–41). F, structures of V5 and V2 splice variants of Piezo2. Note that E10 is also variably spliced in Piezo2 mRNA from Merkel cells. G, Schematic of the location of Merkel cells illustrating their close association to hair-follicles; Merkel cells are indicated in green and surround the root of a hair-follicle. Double label ISH of tissue from Atoh1-EGFP mice show that E35 (left) and E33 (right), are expressed with GFP (green). Individual Merkel cells and the position of a hair follicle are outlined.
Figure 7
Figure 7. Human Piezo2 is alternatively spliced
Sequencing human Piezo2 transcripts reveals that six exons are alternatively spliced and that there are major differences in the splicing of Piezo2 in DRG and lung. A, E16–41 are colored in alternating grey and open blocks with the junctions of alternatively spliced exons highlighted in pink. Sequence reads from DRG (B), and from lung (C) are displayed as plots, black dots indicate sequence identity and open areas sequence lacking similarity. In DRG sensory neurons E19, E33, E35, and E40 are variably spliced while E18 is present in most transcripts. In contrast, in lung, transcripts E18, E19, E35, and E40 are present rarely. See Figure S3 and Table S3 for details.

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