FXYD2, a γ subunit of Na⁺, K⁺-ATPase, maintains persistent mechanical allodynia induced by inflammation

Abstract

Na⁺, K⁺-ATPase (NKA) is required to generate the resting membrane potential in neurons. Nociceptive afferent neurons express not only the α and β subunits of NKA but also the γ subunit FXYD2. However, the neural function of FXYD2 is unknown. The present study shows that FXYD2 in nociceptive neurons is necessary for maintaining the mechanical allodynia induced by peripheral inflammation. FXYD2 interacted with α1NKA and negatively regulated the NKA activity, depolarizing the membrane potential of nociceptive neurons. Mechanical allodynia initiated in FXYD2-deficient mice was abolished 4 days after inflammation, whereas it persisted for at least 3 weeks in wild-type mice. Importantly, the FXYD2/α1NKA interaction gradually increased after inflammation and peaked on day 4 post inflammation, resulting in reduction of NKA activity, depolarization of neuron membrane and facilitation of excitatory afferent neurotransmission. Thus, the increased FXYD2 activity may be a fundamental mechanism underlying the persistent hypersensitivity to pain induced by inflammation.

Figure 1

FXYD2 is expressed in subsets of small DRG neurons. (A) Real-time PCR showed that the highest level of FXYD2 mRNA (Fxyd2) in all tested tissues was observed in the DRG, and very low levels were observed in the spinal cord and brain of adult mice. In situ hybridization showed that FXYD2 mRNA was mainly present in small DRG neurons. Scale bar, 50 μm. (B) Quantitative analysis showed that almost all of FXYD2 mRNA-containing neurons were small DRG neurons with cross-neuronal area less than 600 μm². (C) Co-localization of FXYD2 with neuronal markers by in situ hybridization combined with immunostaining or double fluorescent in situ hybridization. Most of FXYD2 mRNA-positive DRG neurons were peripherin-positive neurons and only a few FXYD2-expressing neurons were NF200-positive large neurons. About half of FXYD2 mRNA-positive neurons bound to IB4 and were non-peptidergic neurons. Some FXYD2-expressing neurons contained mRNA encoding Mrgprd or Mrgprb4. FXYD2 mRNA was also present in TH mRNA-positive neurons. Some CGRP-positive peptidergic neurons also contained FXYD2 mRNA. Scale bar, 50 μm. (D) Diagram showing the profile of FXYD2-expressing neurons in subsets of DRG neurons.

Figure 2

FXYD2 is transported to the dorsal spinal cord and interacts with α1NKA. (A) Immunoblotting (IB) showed that FXYD2 was present in both the DRG and the dorsal horn of spinal cord. This image represents five experiments with similar results. (B) Immunoblotting showed that the level of FXYD2 in the ipsilateral dorsal horn of spinal cord was decreased after dorsal root transection. Four experiments showed similar results. (C) Double fluorescent in situ hybridization showed that Fxyd2 mRNA and α1 subunit mRNA (Atp1a1) were co-localized in small DRG neurons. Scale bar, 50 μm. (D) Co-immunoprecipitation (IP) showed FXYD2 among proteins precipitated with the α1 subunit antibody from mouse DRGs. Similar results were observed in three experiments. (E) In COS7 cells, overexpressed FXYD2 was co-immunoprecipitated with the α1 subunit of NKA. This image is representative of three experiments.

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.

Figure 4

FXYD2 is required for maintaining inflammatory nociceptive responses. (A) The drop latency of Fxyd2^−/− mice was similar to that of Fxyd2^+/+ mice in rotarod test (n = 14 for Fxyd2^+/+ and n = 10 for Fxyd2^−/− mice). (B) The Hargreaves test showed that the thermal latency of Fxyd2^−/− mice was not altered compared to Fxyd2^+/+ mice (n = 10 for both Fxyd2^+/+ and Fxyd2^−/− mice). (C) The hotplate test showed that thermal latency at 50, 52, and 55 °C was unchanged between Fxyd2^+/+ and Fxyd2^−/− mice (n = 11 for Fxyd2^+/+ and n = 12 for Fxyd2^−/− mice). (D) The mechanical threshold of Fxyd2^−/− mice was slightly increased compared to that of Fxyd2^+/+ mice (n = 14 for Fxyd2^+/+ and n = 16 for Fxyd2^−/− mice). (E) The Fxyd2^−/− mice did not show any apparent changes in the number of flinches in either the first or the second phase of the formalin test (n = 11 for both Fxyd2^+/+ and Fxyd2^−/− mice). (F) The radiant heat test showed that recovery from the thermal hyperalgesia was facilitated in Fxyd2^−/− mice 2 days after CFA injection (n = 10 for both Fxyd2^+/+ and Fxyd2^−/− mice). (G) The von Frey test showed that the mechanical allodynia was maintained only in Fxyd2^+/+ mice and not in Fxyd2^−/− mice 2 days after CFA injection (n = 9 for both Fxyd2^+/+ and Fxyd2^−/− mice, ^***P < 0.001).

Figure 5

FXYD2 is required for maintaining the inflammation-induced membrane depolarization and excitability. (A) Co-IP showed that the interaction between FXYD2 and the α1 subunit of NKA increased gradually after peripheral inflammation and peaked on day 4 after injection. (B) Quantitation showed that the interaction between FXYD2 and the α1 subunit of NKA increased gradually and peaked on day 4 (n = 4, ^***P < 0.001). The increased interaction lasted for 14 days, although the average interaction level on day 14 was lower than that on day 4 (n = 3, ^*P < 0.05). (C) The NKA activity gradually decreased in the DRGs of Fxyd2^+/+ mice treated with CFA for 2 and 4 days (n = 3 for CFA day 2 and n = 6 for CFA day 4, ^***P < 0.001 vs Fxyd2^+/+mice without CFA treatment). However, 4 days after CFA treatment, the NKA activity in the DRGs of Fxyd2^−/− mice was not significantly different from that in the DRGs of Fxyd2^+/+ mice without CFA treatment. The reduction of NKA activity in the DRGs of Fxyd2^+/+ mice was reversed by deletion of Fxyd2 (n = 5, ^***P < 0.001 vs Fxyd2^+/+mice without CFA treatment and ^###P < 0.001 vs indicated). (D) Whole-cell patch clamp recording at current-clamp mode showed that the membrane potential of small DRG neurons cultured from Fxyd2^+/+mice was depolarized after CFA injection (n = 52 for control, n = 55 for CFA 2 days and n = 65 for CFA 4 days, ^*P < 0.05 and ^***P < 0.001 vs Fxyd2^+/+mice without CFA treatment). The membrane potential of small DRG neurons cultured from Fxyd2^−/− mice was also depolarized 2 days after CFA injection (n = 54 for control and n = 49 for CFA 2 days, ^###P < 0.001 vs indicated). However, 4 days after CFA injection, the depolarized membrane potential was reversed in small DRG neurons of Fxyd2^−/− mice (n = 67 for CFA 4 days; N.S., non-significant difference). The data were shown as the change of membrane potential and statistic analysis was performed for the membrane potential between the indicated groups. (E) Bath-applied ouabain (1 mM) depolarized the membrane potential of small DRG neurons of both Fxyd2^+/+ and Fxyd2^−/− mice 4 days after CFA injection (n = 13 for Fxyd2^+/+ mice and n = 14 for Fxyd2^−/− mice, ^***P < 0.001 vs Fxyd2^+/+ mice 4 days after CFA injection, before ouabain treatment and ^###P < 0.001 vs indicated). The data were shown as the change of membrane potential and statistic analysis was performed for the membrane potential between the indicated groups. (F) The current (70 pA, 200 ms) was injected to induce sustained firing of IB4-positive small DRG neurons. Representative traces showed that the firing frequency of AP was increased in IB4-positive small DRG neurons from Fxyd2^+/+ mice 2 and 4 days after CFA injection, whereas the firing rate of AP was decreased in the neurons from Fxyd2^−/− mice 4 days after CFA injection. The number of IB4-positive small DRG neurons with a low firing frequency of AP (0-3 APs per stimulation) was decreased gradually in Fxyd2^+/+ mice after CFA injection, but was increased in Fxyd2^−/− mice 4 days after CFA injection (n = 42 for control, n = 39 for CFA 2 days and n = 50 for CFA 4 days in Fxyd2^+/+ mice; n = 59 for control, n = 46 for CFA 2 days and n = 57 for CFA 4 days in Fxyd2^−/− mice).

Figure 6

FXYD2 is required for facilitating afferent synaptic transmission during inflammation. (A) Whole-cell recording from lamina II neurons in the spinal slice showed that the sEPSC frequency in Fxyd2^+/+ mice was increased 2 and 4 days after CFA injection, and it was reduced to the basal level 14 days after CFA injection (n = 21 for control, n = 11 for CFA 2 days, n = 10 for CFA 4 days and n = 15 for CFA 14 days). In Fxyd2^−/− mice, the increase of sEPSC frequency appeared 2 days after CFA injection, but was not detected 4 days after CFA injection (n = 11 for control, n = 12 for CFA 2 days, n = 14 for CFA 4 days and n = 20 for CFA 14 days). (B) The mean frequency of sEPSCs in Fxyd2^−/− mice was reduced to 58% of that in Fxyd2^+/+ mice. Two days after CFA injection, the increase in sEPSC frequency in Fxyd2^−/− mice was less pronounced than that in Fxyd2^+/++ mice. The increase of sEPSC frequency was absent in Fxyd2^−/− mice 4 days after CFA injection. The changes of sEPSC amplitude in Fxyd2^−/− mice were similar to those in Fxyd2^+/+ mice. The increase in sEPSC frequency was not detected in either Fxyd2^+/+ or Fxyd2^−/− mice 14 days after inflammation. ^*P < 0.05, ^**P < 0.01 and ^***P < 0.001 vs Fxyd2^+/+ mice, and ^#P < 0.01 and ^##P < 0.01 vs indicated. (C) Proposed model for two regulatory mechanisms of α1NKA at the axonal terminals of nociceptive afferent fibers. First, membrane depolarization triggers Ca²⁺-dependent exocytosis of synaptic vesicles and FSTL1 vesicles. Secreted FSTL1 activates the presynaptic α1NKA to hyperpolarize the membrane (Vm) and reduce Ca²⁺ influx, enabling an inhibitory regulation of the excitatory synaptic transmission. Second, FXYD2 interacts with α1NKA and reduces α1NKA activity. Such an effect of FXYD2 can be facilitated by peripheral inflammation, resulting in membrane depolarization and the facilitation of synaptic transmission.