Abstract/Summary

SUMOylation is an important post-translational modification process involving covalent attachment of SUMO (Small Ubiquitin-like MOdifier) protein to target proteins. Here, we investigated the potential for SUMO-1 protein to modulate the function of the CaV2.2 (N-type) voltage-gated calcium channel (VGCC), a protein vital for presynaptic neurotransmitter release. Co-expression of SUMO-1, but not the conjugation-deficient mutant SUMO-1ΔGG, increased heterologously-expressed CaV2.2 Ca2+ current density, an effect potentiated by the conjugating enzyme Ubc9. Expression of sentrin-specific protease (SENP)-1 or Ubc9 alone, had no effect on recombinant CaV2.2 channels. Co-expression of SUMO-1 and Ubc9 caused an increase in whole-cell maximal conductance (Gmax) and a hyperpolarizing shift in the midpoint of activation (V1/2). Mutation of all five CaV2.2 lysine residues to arginine within the five highest probability (>65 %) SUMOylation consensus motifs (SCMs) (construct CaV2.2-Δ5KR), produced a loss-of-function mutant. Mutagenesis of selected individual lysine residues identified K394, but not K951, as a key residue for SUMO-1-mediated increase in CaV2.2 Ca2+ current density. In synaptically-coupled superior cervical ganglion (SCG) neurons, SUMO-1 protein was distributed throughout the cell body, axons and dendrites and presumptive presynaptic terminals, whilst SUMO-1ΔGG protein was largely confined to the cell body, in particular, the nucleus. SUMO-1 expression caused increases in paired excitatory postsynaptic potential (EPSP) ratio at short (20− 120 ms) inter-stimuli intervals in comparison to SUMO-1ΔGG, consistent with an increase in residual presynaptic Ca2+ current and an increase in release probability of synaptic vesicles. Together, these data provide evidence for CaV2.2 VGCCs as novel targets for SUMOylation pathways.