Deubiquitinating Enzyme UCH-L1 Regulates Neuronal Ca2+ Signaling

Abstract

Ubiquitination influences a myriad of biological processes, such as the trafficking or degradation of ubiquitin-tagged target proteins. This posttranslational modification can be reversed by deubiquitinating enzymes (DUBs) that counterbalance the action of E3 ubiquitin ligases. We investigated the impact of PR-619, a membrane-permeable and broad-spectrum DUB inhibitor, on the entry of Ca²⁺ through native voltage-gated Ca²⁺ channels (VGCCs) of cultured embryonic cortical neurons. Fura-2-based Ca²⁺ imaging experiments showed that PR-619 reduced the cytosolic Ca²⁺ rises induced by depolarization by affecting mainly dihydropyridine-sensitive (L-type) VGCCs. This inhibition was sensitive to dynamin inhibitor Myr-Dip and lysosomal agents chloroquine and bafilomycin-A. PR-619 also reduced the amount of Ca_v1.2 proteins. A pharmacological approach was set out to better delineate the identity of the DUB responsible for this inhibitory action of PR-619. Since UCH-L1 and USP19 are two highly expressed neuronal DUBs, we investigated the effects of selective UCH-L1 (IMP1710, GK13S) and USP19 (ADC141) inhibitors. IMP1710 and GK13S depressed the Ca²⁺ uptake through L-type VGCCs, whereas ADC141 and the UCH-L3 inhibitor TCID had no effects. In addition, UCH-L1 inhibition impaired the neuronal Ca²⁺ storage capacities of neurons and reduced the Ca_v1.2 protein levels. Thus, UCH-L1 influences the neuronal uptake and storage of Ca²⁺, which is likely to have important physiological implications. Altogether, these results posit UCH-L1, the main DUB of the brain, as an important regulator of neuronal Ca²⁺ homeostasis and add to our understanding of its cellular functions.

Keywords: DUBs; brain; calcium homeostasis; ubiquitination; voltage-gated calcium channels.