Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser65)—which lies within its ubiquitin-like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser65. How Ser65-phosphorylated ubiquitin (ubiquitinPhospho-Ser65) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitinPhospho-Ser65 binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser65 by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site-directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitinPhospho-Ser65, thereby promoting Parkin Ser65 phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser65 phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitinPhospho-Ser65 to Parkin disrupts the interaction between the Ubl domain and C-terminal region, thereby increasing the accessibility of Parkin Ser65. Finally, purified Parkin maximally phosphorylated at Ser65 in vitro cannot be further activated by the addition of ubiquitinPhospho-Ser65. Our results thus suggest that a major role of ubiquitinPhospho-Ser65 is to promote PINK1-mediated phosphorylation of Parkin at Ser65, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho-Ser65-binding pocket on the surface of Parkin that is critical for the ubiquitinPhospho-Ser65 interaction. This study provides new mechanistic insights into Parkin activation by ubiquitinPhospho-Ser65, which could aid in the development of Parkin activators that mimic the effect of ubiquitinPhospho-Ser65.

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A human recombinant homo‐oligomeric 5‐HT3 receptor (h5‐HT3A) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole‐cell recording configuration of the patch clamp technique. 5‐HT evoked transient inward currents (EC50= 3.4 μM; Hill coefficient = 1.8) that were blocked by the 5‐HT3 receptor antagonist ondansetron (IC50= 103 pM) and by the non‐selective agents metoclopramide (IC50= 69 nM), cocaine (IC50= 459 nM) and (+)‐tubocurarine (IC50= 2.8 μM). 5‐HT‐induced currents rectified inwardly and reversed in sign (E5‐HT) at a potential of −2.2 mV. N‐Methyl‐D‐glucamine was finitely permeant. Permeability ratios PNa/PCs and PNMDG/PCs were 0.90 and 0.083, respectively. Permeability towards divalent cations was assessed from measurements of E5‐HT in media where Ca2+ and Mg2+ replaced Na+. PCa/PCs and PMg/PCs were calculated to be 1.00 and 0.61, respectively. Single channel chord conductance (γ) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at −40 mV to 742 fS at −100 mV. Reducing [Ca2+]o from 2 to 0.1 mM caused an increase in the whole‐cell current evoked by 5‐HT. A concomitant reduction in [Mg2+]o produced further potentiation. Fluctuation analysis indicates that a voltage‐independent augmentation of γ contributes to this phenomenon. The data indicate that homo‐oligomeric receptors composed of h5‐HT3A subunits form inwardly rectifying cation‐selective ion channels of low conductance that are permeable to Ca2+ and Mg2+.

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