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. 2015 Jan 30;25(3):318–334. doi: 10.1038/cr.2015.12

Figure 3.

Figure 3

FXYD2 negatively regulates NKA activity and depolarizes the membrane potential. (A) Co-expression of FXYD2 with α1 and β1 subunits of NKA in COS7 cells reduced NKA activity. Treatment with FSTL1 increased NKA activity in COS7 cells transfected with the plasmids expressing α1 and β1 subunits, and partially reversed the reduction of NKA activity in COS7 cells expressing FXYD2 together with the α1 and β1 subunits. n = 5, **P < 0.01 vs vector with vehicle and ##P < 0.01 vs indicated. (B) Immunoblotting and immunostaining showed that FXYD2 protein was absent in the DRG of Fxyd2−/− mice, whereas the expression of the α1 subunit of NKA was not apparently changed. Scale bar, 50 μm. (C) NKA activity in the DRG of Fxyd2−/− mice was higher than that of Fxyd2+/+ mice. FSTL1 activated NKA in the DRG of Fxyd2+/+ mice and also increased NKA activity in Fxyd2−/− DRGs. n = 3, *P < 0.05 vs Fxyd2+/+with vehicle and #P < 0.05 vs Fxyd2−/− with vehicle. (D) The traces recorded from IB4-positive DRG neurons by current-clamp were averaged and compared between Fxyd2+/+ (n = 36) and Fxyd2−/− mice (n = 50). (E) Quantitative analysis showed that the IB4-positive small DRG neurons of Fxyd2−/− mice were hyperpolarized (n = 63 for Fxyd2+/+ and n = 54 for Fxyd2−/− mice, ***P < 0.001). (F) A long duration (200 ms) current was injected to induce sustained firing of small DRG neurons. The number of IB4-positive small DRG neurons with a low firing frequency of AP (0-3 APs per stimulation) was increased slightly in Fxyd2−/− mice, whereas the number of IB4-positive small DRG neurons with high firing frequency (4-7 APs per stimulation) was reduced.