Figure 7. TMEM163 Is a Modulator of ATP-Evoked Behavior and P2XR Activity In Vivo.
(A) ATP-injected responses were evaluated for at least 4 min after injection of 3 μmol ATP into mouse hind paw. The mean cumulative durations of freezing behavior of wild-type (WT) and knockout (KO) mice were each significantly greater in response to ATP than to saline. On the other hand, the TMEM163 KO mice froze significantly less than WT mice in response to ATP, indicating that TMEM163 modulates this pain-related behavior (n = 10 for ATP-injected mice and 3 for saline-injected mice).
(B) The mRNA expression of TMEM163 and P2XRs in mouse dorsal root ganglion (DRG) detected by fluorescence in situ hybridization (FISH). Low-magnification images (top) showed a heterogeneous expression of TMEM163, P2X3R, and P2X4R across DRG neurons. Middle: double-labeling FISH for TMEM163 (green) and P2X3R or P2X4R (red) showed TMEM163 mRNA was expressed in both P2X3R- and P2X4R-positive cells.
Scale bars, 50 μm (top panels); 10 μm (middle and bottom panels).
(C and D) ATP-evoked whole-cell responses were measured in primary DRG neurons from adult WT and TMEM163 KO mice under whole-cell configuration (Vh = −70 mV) with an inter-stimulus interval longer than 3 min for full recovery. (C) Representative traces and quantitation of peak amplitudes of 100 μM ATP-evoked currents (n = 11). Artifacts from 2 mV step for 150 ms for access resistance measurement were omitted from traces shown. (D) Representative traces and dose-response curves of peak amplitudes normalized at 1 mM (n = 5 each). The estimated EC50 values were 5.92 ± 1.17 μM for WT and 31.0 ± 15.1 μM for TMEM163 KO, and these EC50 values were significantly different (p = 0.006). The Hill co-efficient values were unaltered (1.52 ± 0.15 for WT and 1.93 ± 0.37 for TMEM163 KO).
Data are mean ± SEM; one-way ANOVA with Bonferroni’s post-test (A), unpaired t test (C and D); *p < 0.05, ***p < 0.001.