Alterations in SUMOylation of the hyperpolarization‐activated cyclic nucleotide‐gated ion channel 2 during persistent inflammation

Abstract

Background Unilateral injection of Complete Freund's Adjuvant (CFA) into the intra‐plantar surface of the rodent hindpaw elicits chronic inflammation and hyperalgesia in the ipsilateral hindlimb. Mechanisms contributing to this hyperalgesia may act over multiple time courses and can include changes in ion channel expression and post‐translational SUMOylation. Hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels mediate the hyperpolarization‐activated current, I h . An HCN2‐mediated increase in C‐nociceptor I h contributes to mechanical hyperalgesia in the CFA model of inflammatory pain. Changes in HCN2 post‐translational SUMOylation and protein expression have not been systematically documented for a given dorsal root ganglia (DRG) throughout the time course of inflammation. Methods This study examined HCN2 protein expression and post‐translational SUMOylation in a rat model of CFA‐induced hindpaw inflammation. L5 DRG cryosections were used in immunohistochemistry experiments and proximity ligation assays to investigate HCN2 expression and SUMOylation, respectively, on days 1 and 3 post‐CFA. Results Unilateral CFA injection elicited a significant bilateral increase in HCN2 staining intensity in small diameter DRG neurons on day 1 post‐CFA, and a significant bilateral increase in the number of small neurons expressing HCN2 but not staining intensity on day 3 post‐CFA. HCN2 channels were hyper‐SUMOylated in small diameter neurons of ipsilateral relative to contralateral DRG on days 1 and 3 post‐CFA. Conclusions Unilateral CFA injection elicits unilateral mechanical hyperalgesia, a bilateral increase in HCN2 expression and a unilateral increase in post‐translational SUMOylation. This suggests that enhanced HCN2 expression in L5 DRG is not sufficient for mechanical hyperalgesia in the early stages of inflammation and that hyper‐SUMOylation of HCN2 channels may also be necessary. Significance Nociceptor HCN2 channels mediate an increase in I h that is necessary for mechanical hyperalgesia in a CFA model of chronic pain, but the mechanisms producing the increase in nociceptor I h have not been resolved. The data presented here suggest that the increase in I h during the early stages of inflammation may be mediated by an increase in HCN2 protein expression and post‐translational SUMOylation.

Changes in peripheral HCN2 channels during persistent inflammation

“…In these cases, the decrease in expression may represent compensation for prolonged nociceptor hyperexcitability, or redistribution of the channels from soma to axon. In other models of inflammatory pain, both I h and HCN2 protein expression increased [ 21 , 28 , 29 , 49–51 , 54 , 55 ]. In most instances, it was not clear if altered expression represented an adjustment to transcription, translation, and/or post-translational modifications that modify channel turnover.…”

“…HCN2 expression is enhanced in multiple subcellular compartments leading to mechanical hyperalgesia [ 21 , 28 , 49 , 51 ]. A detailed immunohistochemical examination of somatic HCN2 protein expression in lumbar 5 (L5) DRG neurons that innervate the hindlimb showed that HCN2 immunoreactivity significantly increased on day 1 post-CFA in small and medium neurons relative to control animals that did not receive CFA injections [ 55 ]. HCN2 staining intensity then returned to baseline by day 3, but the number of neurons expressing HCN2 significantly increased [ 55 ].…”

“…A detailed immunohistochemical examination of somatic HCN2 protein expression in lumbar 5 (L5) DRG neurons that innervate the hindlimb showed that HCN2 immunoreactivity significantly increased on day 1 post-CFA in small and medium neurons relative to control animals that did not receive CFA injections [ 55 ]. HCN2 staining intensity then returned to baseline by day 3, but the number of neurons expressing HCN2 significantly increased [ 55 ]. By days 5–7 post-CFA, HCN2 staining intensity was once again significantly increased, and the number of neurons expressing HCN2 remained elevated relative to controls [ 28 ].…”

“…Inflammation causes a global increase in the SUMOylation of DRG proteins, and experimentally enhanced SUMOylation in sensory neurons produced pathological pain [ 72 ]. In a rat model of CFA-induced inflammatory pain, HCN2 SUMOylation was increased in small DRG neurons on days 1 and 3 post-CFA (later times not examined) [ 55 ]. A target protein’s phosphorylation status often determines its ability to be SUMOylated [ 69 , 73 ].…”

“…SUMOylation of HCN2 channels is dynamically regulated as inflammation progresses. SUMO2/3 conjugation to HCN2 increased at day 1 post-CFA, and SUMO1 conjugation to HCN2 increased at day 3 post-CFA [ 55 ]. These data imply that inflammatory mediators regulate HCN2 interaction with components of the SUMOylation machinery that show target and SUMO-isoform specificity [ 69 ].…”