SUMOylation of Kir7.1 participates in neuropathic pain through regulating its membrane expression in spinal cord neurons

Abstract Aims Potassium (K+) channels have been demonstrated to play a prominent involvement in nociceptive processing. Kir7.1, the newest members of the Kir channel family, has not been extensively studied in the CNS, and its function remains largely unknown. The present study investigated the role of spinal Kir7.1 in the development of pathological pain. Methods and Results Neuropathic pain was induced by spared nerve injury (SNI). The mechanical sensitivity was assessed by von Frey test. Immunofluorescence staining assay revealed that Kir7.1 was predominantly expressed in spinal neurons but not astrocytes or microglia in normal rats. Western blot results showed that SNI markedly decreased the total and membrane expression of Kir7.1 in the spinal dorsal horn accompanied by mechanical hypersensitivity. Blocking Kir7.1 with the specific antagonist ML418 or knockdown kir7.1 by siRNA led to mechanical allodynia. Co‐IP results showed that the spinal kir7.1 channels were decorated by SUMO‐1 but not SUMO‐2/3, and Kir7.1 SUMOylation was upregulated following SNI. Moreover, inhibited SUMOylation by GA (E1 inhibitor) or 2‐D08 (UBC9 inhibitor) can increase the spinal surface Kir7.1 expression. Conclusion SUMOylation of the Kir7.1 in the spinal cord might contribute to the development of SNI‐induced mechanical allodynia by decreasing the Kir7.1 surface expression in rats.

Potassium (K + ) channels play an essential role in establishing the resting membrane potential and are crucial determinants of neuronal activity throughout the nervous system. 6 Dysfunction of K + channels is linked to many kinds of human diseases or disorders related to nervous system, including neuropathic pain. [7][8][9] On the basis of structural and physiological attributes, K + channels are classified mainly into voltage-gated (K v ), two-pore (K 2P ), calcium-activated (K CA ), and inward-rectifying (Kir) channels. 10 Kir7.1 is the most recently described Kir subtype. 11 Recent study has shown that Kir7.1 is expressed pervasive in the brain than previously recognized and have potential importance in regulating neuronal and glial function. 12 Evidence have shown that some K + channels (for example Kv1,13 Kv2, 14 Kv7 15 and TREK2 16 ) are involved in the development and maintenance of neuropathic pain; however, the role of Kir7.1 in neuropathic pain remains largely unexplored. In the present study, we investigated the role of Kir7.1 in SNI-induced mechanical allodynia.
SUMOylation is a post-translational modification in which a member of the small ubiquitin-like modifier (SUMO) family of proteins is conjugated to lysine residues in target proteins. 17 Such a modification can facilitate or prevent inter-and intra-molecular interactions via conformational changes or direct steric hindrance. 18 In mammals, there are three SUMO paralogues (SUMO1-3). 19 The three SUMO proteins can be covalently conjugated to proteins as a single moiety (SUMO-1) or as polymeric SUMO chains (SUMO-2/3). 20 Similar to ubiquitylation, SUMOylation is regulated by a specialized set of activating (E1), conjugating (E2), and ligating (E3) enzymes, and is reversed by specific isopeptidases referred to as sentrin/SUMO-specific proteases (SENPs). 21 Emerging evidence established that SUMOylation of proteins plays key roles in neuronal function. An increasing number of ion channels, including Kainite receptor GluR6, 22 Kv2.1, 23 Kv1. 5,24 and transient receptor potential melastatin ion channel type 4 (TRPM4), 24 transient receptor potential vanilloid 1 (TRPV1) 18 and hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2), 25 have been reported to be conjugated and regulated by SUMO, suggesting that SUMOylation might be a common mechanism for modulating ion channel function. 18,26 Here, we investigated whether Kir7.1 was modified by SUMOylation in SNI rats.

| Animals
Male Sprague-Dawley rats (200-220 g, purchased from the Institute of Experimental Animals of Sun Yat-sen University) were grouphoused with ad libitum access to food and water in a temperature (24°C) and humidity (50%-60%) and light-controlled (12:12-h light/ dark cycle) quiet room. All experimental procedures were approved by the local Animal Care and Use Committee (No. SYXK [yue] 2015-0107) and were performed in accordance with the guidelines of the National Institutes of Health on animal care and the ethical guidelines.

| Spared nerve injury
Spared nerve injury (SNI) surgery was performed as previously described. 27 Briefly, rats were anesthetized with sodium pentobarbital (50 mg/kg, Sigma-Aldrich) intraperitoneally (ip.). The left sciatic nerve and its three terminal branches (the sural, common peroneal, and tibial nerves) were exposed through an incision at the midthigh level. The common peroneal and tibial nerves were tightly ligated with 5.0 silk and sectioned (removal of a 2 mm length). The sural nerve was kept intact. In the sham group, identical operation was performed for exposure of the sciatic nerve and its branches but without any nerve injury.

| Behavioral tests
Mechanical withdrawal thresholds of the rats were assessed by the up-down method as described previously. 28,29 Briefly, rats were acclimatized to the testing environment for 3 consecutive days (2 h/d).
After habituation for 15 min, a series of von Frey hairs of bending force (0.41, 0.70, 1.20, 2.04, 3.63, 5.50, 8.51, and 15.14 g) were applied to the plantar surface of the hind paw, which is predominantly innervated by the sciatic nerve. The brisk withdrawal or licking of the paw in response to the stimulus was considered as a positive response. The 50% paw withdrawal thresholds (PWT) were calculated.

| Intrathecal catheter implantation
For intrathecal (i.t.) delivery of drugs, rats were implanted with i.t. catheters as described previously. 30 Briefly, after being anesthetized, a sterile polyethylene (PE-10) tube was inserted through L5/ L6 intervertebral space and the tip of the tube was placed at the spinal lumbar enlargement level. Any rats that developed hind limb paralysis or paresis after surgery were excluded.
The drugs were i.t. injected in a volume of 10 μl followed by additional 10 μl of vehicle to flush the catheter.

| Western blot
Western blot was performed following our previous study. 30 After being anesthetized with sodium pentobarbital (50 mg/kg, i.p.), the and then with HRP-conjugated secondary antibody for 1 h at room temperature (RT). The immune complex was visualized using enhanced chemiluminescence (Thermo Scientific) and quantified by NIH ImageJ.

| Immunohistochemistry
Immunochemistry was performed in accordance with the previous description. 31 In short, perfusion was performed through the ascending aorta with 4% paraformaldehyde under deeply anesthetized. The

| Co-Immunoprecipitation assay
Co-Immunoprecipitation (Co-IP) was carried out following our previous study. 29 Briefly, the extracted spinal cord tissues were lysed in IP-RIPA buffer (Beyotime Institute of Biotechnology, China) with protease inhibitors and N-ethylmaleimide (NEM, 20 mM) with rotation at 4°C for 30 min. After centrifugation (10 min at 12,000 rpm), the supernatant was transferred to fresh tubes. The SUMO1 antibody (1:50) or Kir7.1 antibody, which immobilized with resin, were used to collect the immune complexes. The eluted complexes from the resin were analyzed by Western blot using Kir7.1 antibody or SUMO1 antibody after incubation and washes.
The control group received the same volume of scramble siRNA.

| Statistical analysis
All data were presented as means ± standard deviation (SD) and analyzed with SPSS 20.0 software. Western blot and qPCR data were analyzed by two-tailed, independent Student's t-test and two-way ANOVA followed by a Tukey post hoc test. Behavioral results were performed by one-way or two-way ANOVA with repeated measures followed by a Tukey post hoc test. p < 0.05 was considered statistically significant.

| The expression of Kir7.1 in the neuron of spinal cord dorsal horn was downregulated in SNI rats
To evaluate the development of mechanical allodynia, PWTs were recorded at baseline (Day 0) and Days 1, 3, 7, and 10 after SNI surgery in rats. Consistent with earlier findings, we confirmed that unilateral SNI induced a long-lasting decrease in ipsilateral PWTs As shown in Figure 2A, the PWTs were significantly decreased in the ML418-treated group as compared to the naïve or vehicle group.

| SUMO1-mediated SUMOylation of Kir7.1 was significantly upregulated in the spinal cord after SNI
Previous studies have reported that several ion channels can be SUMOylated, suggesting that SUMOylation might be a common mechanism for modulating ion channel function. SUMOylation of Kir7.1 has never been examined. To assess whether Kir7.1 can be regulated by SUMOylation in SNI-induced chronic pain, Co-IP was performed with SUMO1-or SUMO2/3-specific antibody. The results showed that the potential binding of SUMO1 with Kir7.1, but not SUMO2/3, was increased at Day 10 after SNI ( Figure 3A, B), indicating that Kir7.1 could be post-translationally modified by SUMO1 in the rat spinal cord. In addition, immunofluorescence results showed that Kir7.1 co-localized with SUMO1 in the spinal cord ( Figure 3C). These results implied that Kir7.1 SUMOylation may be dynamically regulated by SNI.  Figure 3D). Then, we verified the conservation of this motif in different organisms. It is intriguing that K314 is conserved among mammals (e.g., rat, human, mouse, and bovine) ( Figure 3E). Taken together, we proposed that K314 might be the potential SUMOylated modification site of Kir7.1 in rat.

| DISCUSS ION
In the present study, we showed that Kir7.1 expression in the spi-

| Kir7.1 is expressed in the spinal cord neurons
Kir channels, which conduct atypical inward (rather than outward) K + currents at depolarized membrane potentials, play key roles in ion homeostasis and neuronal excitability. There are seven subfamilies of Kir (Kir1-7) discovered so far and each of which has multiple subfamily members. 33 As one of the newest members of the Kir channel family, In the CNS, it has been reported recently that Kir7.1 is widely expressed in the cortex, cerebellum, hippocampus, hypothalamus, pons, and striatum, 12 while its distribution in the spinal cord is still unclear.
In the present study, we observed the expression of Kir7.1 in the spinal cord. Immunofluorescence results showed that Kir7.1 is expressed in the spinal cord, and predominantly co-localized with NeuN, but not with GFAP or Iba1, indicating that Kir7.1 was expressed in the spinal neurons but not astrocytes or microglias in normal rats. We provided a piece of novel evidence for the wide distribution of Kir7.1 in CNS.

| Kir7.1 contributes to the development of SNIinduced chronic pain
Accumulating evidence has highlighted that K + channels may play a crucial role in nociceptive processing through its dominant contributions in regulating neuron excitability. 6 Studies have suggested that changes in K + channel expression may be important in the pathophysiology of peripheral sensitization and neuropathic pain. 10,38 The present work explored the role of Kir7.1 in the SNI-induced mechanical allodynia.
Western blotting assays showed that both the total and membrane expression of Kir7.1 in the spinal dorsal horn was significantly decreased by SNI surgery compared with the sham rats, indicating that the spinal Kir7.1 may be involved in the development of SNI-induced chronic pain. Indeed, behavioral testing found that intraperitoneal injection of the Kir7.1 selective blocker ML418 induced mechanical allodynia behavior in naive rats, which was similar to the behavior in rats after SNI treatment. Moreover, we depleted Kir7.1 from WT rats by intrathecal injection of siRNA also induced the mechanical allodynia. These data suggest that Kir7.1 serves as an inhibitor for the genesis of neuropathic pain and downregulation of Kir7.1 in spinal neurons is essential for mechanical hypersensitivity induced by peripheral nerve injury.
It is Kir channel that is open in its steady state to create the resting membrane potential that most cells obey. 11 The Kir7.1 channels provide a steady background K + current to help set resting membrane potentials 11 and appear to be essential for depolarization of the cells in which it is expressed. 39 We assumed that the downregulation of kir7.1 might lead to the decrease in Kir7.1 current, which affects the resting potential and repolarization process of the membrane, thus increases the excitability and leads to pain hypersensitivity.

| Kir7.1 SUMOylation is involved in the mechanism of SNI-induced chronic pain
SUMOylation is a covalently reversible binding process between small ubiquitin-like modifying proteins (SUMO1, 2, or 3) and the specific substrate. A variety of neuronal proteins have been identified as SUMO substrates, and disrupting the SUMO modification of these proteins results in defects in several kinds of neuronal function, including neuronal excitability. 19  The regulation by SUMO has been shown to control membrane trafficking. SUMOylation retains VEGFR2 in the Golgi, blocking it trafficking to cell surface, reduces its surface expression, and attenuating VEGFR2-dependent signaling in mice. 49 Upon kainite or glutamateinduced stimulation, GluR6 is SUMOylated and subsequently internalized, lead to a downregulation in GluR6 expression and a decrease in KAR-EPSC amplitude. 22 We assumed that SNI-induced decrease in Kir7.1 surface expression might be due to SUMO-modification controlling membrane trafficking and/or endocytosis of the channels.
The detailed mechanism requires further studies.
Besides regulating the surface expression, SUMO pathway also operates directly at the plasma membrane and to control ion channel function. 41,[43][44][45][46]50 With the existing data generally suggesting that enhanced SUMOylation of K + channel subunits inhibit outward currents and increase cell excitability, [42][43][44][45][46] we speculated that the SUMOylation of Kir7.1 have the potential to increase the excitability of neurons by altering K + current through the channel and modulating either the action potential duration or resting membrane potential. Future studies are needed to confirm it.

| CON CLUS ION
In summary, the present study provided the first evidence that Kir7.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.

D I SCLOS U R E
This study has never been published elsewhere.