DOC2B Acts as a Calcium Switch and Enhances Vesicle Fusion

Abstract

Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusioncompetent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PM was supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release.

The C2B Domain Is the Primary Ca2+ Sensor in DOC2B: A Structural and Functional Analysis

“…Such slow dissociation of the C2AB from ULV correlates with the time constants of asynchronous neurotransmitter release at the synapse [24] and with the contribution of DOC2B to asynchronous release phase [24,25] ] microdomains or may increase its affinity due to the vicinity of PLs head groups as suggested for other C 2 domains [8,12]. The finding that C2A can affect DOC2B interaction with the membrane is also supported from observations of DOC2B D218,220N , a DOC2B with mutations in C2A, found to be constitutively associated with the PM [1,23]. These previous data suggested that C2A serves as the primary DOC2B Ca 2+ sensor.…”

“…By interacting with membranes, SNARE proteins, and additional factors, DOC2B enhances spontaneous and asynchronous neurotransmitter release [1,6,[23][24][25]. Here, we structurally and biochemically analyzed the individual C 2 domains and the C2AB tandem to shed light on DOC2B's Ca 2 + -binding properties and the conformational and functional effects of Ca 2 + on DOC2B.…”

“…indicator Fura-4 F AM. We have previously used this approach to characterize the EC 50 of DOC2A and DOC2B [1,23]. During the experiment, cells were repeatedly depolarized by a solution containing high potassium (100 mM) and monitored for their Ca 2 + levels and translocation status ( Fig.…”

“…This process, as well as other processes in the synapse, depends on Ca 2+ , which interacts with Ca 2+ -binding domains of specific regulatory proteins, leading to a series of Ca 2+ -dependent processes in the synapse [1][2][3].…”

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

“…They performed most of their experiments using a buffer without calcium (i.e., Nonidet P-40 lysis buffer). Since DOC2b has conserved Ca 2ϩ -binding sites and is thought to be a Ca 2ϩ sensor protein in other cell types such as neuron (32) and chromaffin cells (33), Ca 2ϩ might be important for the physiological properties of DOC2b in ␤-cells. Further work is required to uncover the underling mechanisms by which DOC2b regulates SNARE assembly in response to insulin.…”