Abstract
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.
Highlights
It was observed in blots probed with anti-HCN2, anti-SUMO1, and anti-SUMO2/3 in both nondenaturing and denaturing IP experiments, which suggests both SUMO1 and SUMO2/3 are covalently attached to HCN2 channels in the mouse forebrain
Since this ∼75 kDa band was not observed for the denaturing IP experiments, it most likely represents an unknown SUMOylated protein that non-covalently associates with HCN2 channels in the mouse forebrain
IP experiments with mouse forebrain membrane preparations followed by Western blotting showed that mouse HCN2 channels were post-translationally modified by SUMO1, as well as SUMO2 and/or SUMO3 in vivo
Summary
Small Ubiquitinlike Modifier (SUMO, a.k.a. Sentrin) is a ∼100 amino acid peptide that is covalently added to a lysine (K) residue on a target protein. The immature SUMO protein is cleaved by a sentrin specific protease (SENP), exposing a C-terminal diglycine (Mukhopadhyay and Dasso, 2007). The SUMO E1 activating enzyme transfers the mature SUMO peptide to the E2 conjugating enzyme, Ubc, which will add SUMO to the target protein (Desterro et al, 1997). SUMO can be deconjugated from a target protein by SENP (Hickey et al, 2012). SUMO2 and SUMO3 proteins are ∼97% identical and are not readily distinguishable in most experiments. SUMO1 and SUMO2/3 proteins share ∼46% identity, and their sets of targets greatly overlap. SUMO4 cannot be cleaved into the mature form by SENP, and its function is unclear
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