Post-translational conjugation of S mall U biquitin-like Mo difier (SUMO) peptides to lysine (K) residues on target proteins alters their interactions. SUMOylation of a target protein can either promote its interaction with other proteins that possess SUMO binding domains, or it can prevent target protein interactions that normally occur in the absence of SUMOylation. One subclass of voltage-gated potassium channels that mediates an A-type current, I A , exists as a ternary complex comprising Kv4 pore-forming subunits, Kv channel interacting proteins (KChIP) and transmembrane dipeptidyl peptidase like proteins (DPPL). SUMOylation could potentially regulate intra- and/or intermolecular interactions within the complex. This study began to test this hypothesis and showed that Kv4.2 channels were SUMOylated in the rat brain and in human embryonic kidney (HEK) cells expressing a GFP-tagged mouse Kv4.2 channel (Kv4.2g). Prediction software identified two putative SUMOylation sites in the Kv4.2 C-terminus at K437 and K579. These sites were conserved across mouse, rat, and human Kv4.2 channels and across mouse Kv4 isoforms. Increasing Kv4.2g SUMOylation at each site by ~30% produced a significant ~22%–50% decrease in I A G max , and a ~70%–95% increase in channel surface expression. Site-directed mutagenesis of Kv4.2g showed that K437 SUMOylation regulated channel surface expression, while K579 SUMOylation controlled I A G max . The K579R mutation mimicked and occluded the SUMOylation-mediated decrease in I A G max , suggesting that SUMOylation at K579 blocked an intra- or inter-protein interaction involving K579. The K437R mutation did not obviously alter channel surface expression or biophysical properties, but it did block the SUMOylation-mediated increase in channel surface expression. Interestingly, enhancing K437 SUMOylation in the K579R mutant roughly doubled channel surface expression, but produced no change in I A G max , suggesting that the newly inserted channels were electrically silent. This is the first report that Kv4.2 channels are SUMOylated and that SUMOylation can independently regulate Kv4.2 surface expression and I A G max in opposing directions. The next step will be to determine if/how SUMOylation affects Kv4 interactions within the ternary complex.

Small Ubiquitin-like Modifier (SUMO) is a ∼10 kDa peptide that can be post-translationally added to a lysine (K) on a target protein to facilitate protein–protein interactions. Recent studies have found that SUMOylation can be regulated in an activity-dependent manner and that ion channel SUMOylation can alter the biophysical properties and surface expression of the channel. Hyperpolarization-activated cyclic nucleotide-gated (HCN) channel surface expression can be regulated in an activity-dependent manner through unknown processes. We hypothesized that SUMOylation might influence the surface expression of HCN2 channels. In this manuscript, we show that HCN2 channels are SUMOylated in the mouse brain. Baseline levels of SUMOylation were also observed for a GFP-tagged HCN2 channel stably expressed in Human embryonic kidney (Hek) cells. Elevating GFP-HCN2 channel SUMOylation above baseline in Hek cells led to an increase in surface expression that augmented the hyperpolarization-activated current (Ih) mediated by these channels. Increased SUMOylation did not alter Ih voltage-dependence or kinetics of activation. There are five predicted intracellular SUMOylation sites on HCN2. Site-directed mutagenesis indicated that more than one K on the GFP-HCN2 channel was SUMOylated. Enhancing SUMOylation at one of the five predicted sites, K669, led to the increase in surface expression and Ih Gmax. The role of SUMOylation at additional sites is currently unknown. The SUMOylation site at K669 is also conserved in HCN1 channels. Aberrant SUMOylation has been linked to neurological diseases that also display alterations in HCN1 and HCN2 channel expression, such as seizures and Parkinson’s disease. This work is the first report that HCN channels can be SUMOylated and that this can regulate surface expression and Ih.

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.

Neurons operate within defined activity limits, and feedback control mechanisms dynamically tune ionic currents to maintain this optimal range. This study describes a novel, rapid feedback mechanism that uses SUMOylation to continuously adjust ionic current densities according to changes in activity. Small ubiquitin-like modifier (SUMO) is a peptide that can be post-translationally conjugated to ion channels to influence their surface expression and biophysical properties. Neuronal activity can regulate the extent of protein SUMOylation. This study on the single, unambiguously identifiable lateral pyloric neuron (LP), a component of the pyloric network in the stomatogastric nervous system of male and female spiny lobsters (Panulirus interruptus), focused on dynamic SUMOylation in the context of activity homeostasis. There were four major findings: First, neuronal activity adjusted the balance between SUMO conjugation and deconjugation to continuously and bidirectionally fine-tune the densities of two opposing conductances: the hyperpolarization activated current (I h) and the transient potassium current (I A). Second, tonic 5 nM dopamine (DA) gated activity-dependent SUMOylation to permit and prevent activity-dependent regulation of I h and I A , respectively. Third, DA-gated, activity-dependent SUMOylation contributed to a feedback mechanism that restored the timing and duration of LP activity during prolonged modulation by 5 M DA, which initially altered these and other activity features. Fourth, DA modulatory and metamoduatory (gating) effects were tailored to simultaneously alter and stabilize neuronal output. Our findings suggest that modulatory tone may select a subset of rapid activitydependent mechanisms from a larger menu to achieve homeostasis under varying conditions.

Nociceptor sensitization following nerve injury or inflammation leads to chronic pain. An increase in the nociceptor hyperpolarization-activated current, I h , is observed in many models of pathological pain. Pharmacological blockade of I h prevents the mechanical and thermal hypersensitivity that occurs during pathological pain. Alterations in the Hyperpolarization-activated Cyclic Nucleotidegated ion channel 2 (HCN2) mediate I h -dependent thermal and mechanical hyperalgesia. Limited knowledge exists regarding the nature of these changes during chronic inflammatory pain. Modifications in HCN2 expression and post-translational SUMOylation have been observed in the Complete Freund's Adjuvant (CFA) model of chronic inflammatory pain. Intra-plantar injection of CFA into the rat hindpaw induces unilateral hyperalgesia that is sustained for up to 14 days following injection. The hindpaw is innervated by primary afferents in lumbar DRG, L4-6. Adjustments in HCN2 expression and SUMOylation have been well-documented for L5 DRG during the first 7 days of CFAinduced inflammation. Here, we examine bilateral L4 and L6 DRG at day 1 and day 3 post-CFA. Using L4 and L6 DRG cryosections, HCN2 expression and SUMOylation were measured with immunohistochemistry and proximity ligation assays, respectively. Our findings indicate that intra-plantar injection of CFA elicited a bilateral increase in HCN2 expression in L4 and L6 DRG at day 1, but not day 3, and enhanced HCN2 SUMOylation in ipsilateral L6 DRG at day 1 and day 3. Changes in HCN2 expression and SUMOylation were transient over this time course. Our study suggests that HCN2 is regulated by multiple mechanisms during CFA-induced inflammation.