Fig. 5

EndoA2 increases Piezo2-mediated mechanically activated (MA) currents. a Representative images showing Piezo2 membrane and cytoplasm staining in DRG neurons from EndoA2^fl/fl, EndoA2^fl/fl × Nefh-Cre and rescue mice. Scale bar = 15 μm. b Profile plots were used to define membrane staining. A ratio of membrane to cytoplasm was calculated, and those cells with a ratio greater than 1.5 times were defined as Piezo2 membrane positive. c The proportion of Piezo2 membrane positive neurons in NF200⁺ cultured DRG neurons from EndoA2^fl/fl, EndoA2^fl/fl × Nefh-Cre and rescue mice. n = 3 repeats. Eighty, 57 and 74 NF200⁺ neurons were analyzed in the EndoA2^fl/fl, EndoA2^fl/fl × Nefh-Cre and rescue groups, respectively. d, e MA inward currents recorded in HEK293 Piezo1 knockout (P1KO) cells transiently transfected with Piezo2 + mock and Piezo2 + EndoA2. Cells were subjected to a series of mechanical steps of 1-μm movements of a stimulation pipette (inset illustration, arrow) in the whole-cell patch configuration at a holding potential of − 80 mV. f The maximal amplitude of MA inward currents elicited at a holding potential of − 80 mV in Piezo2 + mock- and Piezo2 + EndoA2-transfected HEK293 P1KO cells. n = 8. g The representative traces of RA, IA, and SA MA inward currents expressed in DRG neurons are characterized by distinct inactivation kinetics. Neurons were subjected to a series of mechanical steps in 1-μm increments at a holding potential of − 80 mV. Frequency histograms indicating the proportion of larger DRG neurons (> 30 μm) from EndoA2^fl/fl, EndoA2^fl/fl × Nefh-Cre and rescue mice that respond to mechanical stimulation, with MA currents characterized by their inactivation kinetics. The proportion of neurons from 7 separate experiments (h) (n = 7 to 13 neurons per condition and per experiment) or the proportion from all neurons pooled from all 7 experiments (i). n = 71, 68 and 65 neurons in the EndoA2^fl/fl, EndoA2^fl/fl × Nefh-Cre and rescue groups, respectively. One-way ANOVA followed by Tukey’s multiple comparisons test (c, h); Two-tailed independent Student’s t test (f). ^**P < 0.01, ^***P < 0.001, ns non-significant. The error bars indicate the SEMs. EndoA2 endophilin A2, NF200 neurofilament-200, P1KO Piezo1 knockout, RA rapidly adapting, IA intermediately adapting, SA slowly adapting