DOC2A and DOC2B are sensors for neuronal activity with unique calcium‐dependent and kinetic properties

Abstract

Elevation of the intracellular calcium concentration ([Ca 2+ ] i ) to levels below 1 lM alters synaptic transmission and induces short-term plasticity. To identify calcium sensors involved in this signalling, we investigated soluble C2 domain-containing proteins and found that both DOC2A and DOC2B are modulated by submicromolar calcium levels. Fluorescent-tagged DOC2A and DOC2B translocated to plasma membranes after [Ca 2+ ] i elevation. DOC2B translocation preceded DOC2A translocation in cells co-expressing both isoforms. Half-maximal translocation occurred at 450 and 175 nM [Ca 2+ ] i for DOC2A and DOC2B, respectively. This large difference in calcium sensitivity was accompanied by a modest kinetic difference (halftimes, respectively, 2.6 and 2.0 s). The calcium sensitivity of DOC2 isoforms can be explained by predicted topologies of their C2A domains. Consistently, neutralization of aspartates D218 and D220 in DOC2B changed its calcium affinity. In neurones, both DOC2 isoforms were reversibly recruited to the plasma membrane during trains of action potentials. Consistent with its higher calcium sensitivity, DOC2B translocated at lower depolarization frequencies. Styryl dye uptake experiments in hippocampal neurones suggest that the overexpression of mutated DOC2B alters the synaptic activity. We conclude that both DOC2A and DOC2B are regulated by neuronal activity, and hypothesize that their calcium-dependent translocation may regulate synaptic activity.

K+Channel Facilitation of Exocytosis by Dynamic Interaction with Syntaxin

“…PC12 cells were incubated with 4 M fura-4 acetoxymethyl ester (AM) (Invitrogen, Eugene, OR) for 45 min in the 37°C incubator before being used for experiments (Groffen et al, 2006). To facilitate loading of the AM ester indicators, the mild detergent pluronic F-127 was added to fura-AM in a 1:1 ratio and vortexed thoroughly with 1 ml of DMEM (Invitrogen) immediately before incubation.…”

DOC2B: A Novel Syntaxin-4 Binding Protein Mediating Insulin-Regulated GLUT4 Vesicle Fusion in Adipocytes

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Detection of Genomic Imbalances by Array-Based Comparative Genomic Hybridization in Bulgarian Patients with Autism Spectrum Disorders

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