Ca2+/Calmodulin Causes Rab3A to Dissociate from Synaptic Membranes

Abstract

The GTPase Rab3A has been postulated to cycle on and off synaptic membranes during the course of neurotransmission. Moreover, a Rab guanine nucleotide dissociation inhibitor has been shown to cause Rab3A to dissociate from synaptic membranes in vitro. We demonstrate here that Ca 2؉ /calmodulin also can cause Rab3A to dissociate from synaptic membranes in vitro. Like Rab guanine nucleotide dissociation inhibitor, it forms a 1:1 complex with Rab3A that requires both the lipidated C terminus of Rab3A and the presence of bound guanine nucleotide. In addition, a synthetic peptide corresponding to the Lys The opening of voltage-gated Ca 2ϩ channels in active zones of nerve terminals causes a brief, localized influx of Ca 2ϩ followed by the secretion of neurotransmitters (1-3). The molecular basis of this effect is still unclear, but increased concentrations of intracellular Ca 2ϩ may act at several levels to trigger fast fusion of pre-docked synaptic vesicles with the synaptic plasma membrane, promote endocytosis of the vesicle membranes and subsequent vesicle reformation, and mobilize additional vesicles to release sites (1, 4). Proteins that bind Ca 2ϩ probably mediate many of these actions, and a number of candidate proteins have been identified. They include rabphilin (5, 6); the ␣-, ␤-II-, and ␥ isoforms of protein kinase C (7); and dynamin (8) (Table I) was from LC Laboratories. Stock solutions of peptides were prepared in Me 2 SO and then added to incubation mixtures at final Me 2 SO concentrations of Ͻ5%. GDP, GTP␥S, and unprenylated Rab3A were from Calbiochem. Rab GDI was purified from bovine brain as described (29), except that all buffers used after the ammonium sulfate precipitation step contained 10% glycerol, 0.25 mM phenylmethylsulfonyl fluoride, 2.5 g/ml each of aprotinin and leupeptin, and 1 g/ml pepstatin A. All other purchased chemicals were reagent grade from Sigma, and all procedures were performed at 4°C unless otherwise indicated.Preparation of Synaptosomes-Two different methods were used to prepare synaptosomes from cerebral cortex of nonhuman primates (Macaca nemestrina), obtained from the tissue distribution program of the Regional Primate Research Center at the University of Washington. In method 1, 50 g of cortex was sliced in ice-cold buffer A (320 mM sucrose; * This work was supported by the Howard Hughes Medical Institute and by National Institutes of Health Grant RR00166 to the Regional Primate Research Center at the University of Washington. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C.

Regulation of GTP-binding state in RalA through Ca2+ and calmodulin

“…Native prenylated RalA binds to CaM in a Ca 2+ -dependent manner (Wang et al, 1997). A peptide of 18 amino acid residues, with the sequence SKEKNGKKKRKSLAKRIR, was identified as a putative CaM-binding domain in RalA (Wang et al, 1997), but prenyl group on the molecule is likely required for the interaction (Park et al, 1997). RalA has C-terminus that end in CCIL (Kinsella et al, 1991) where prenylation occurs.…”

“…Crude synaptic vesicle fraction, which contained Rab3A-enriched membranes (LP2 membrane fraction), was prepared through lysis of synaptosomes in hypotonic solution (Park et al, 1997) Dissociation of small-GTPases from synaptic vesicle membrane by Ca 2+ /CaM LP2 membranes (60 µg protein) were preincubated for 10 min at 30 o C in 50 µl of buffer (50 mM Hepes pH 7.4, 0.5 mM MgCl 2 , 1 mM DTT, 2 µg/ml aprotinin, 2 µg/ml leupeptin, and 0.1 mM CaCl 2 ) in the presence of 10 mM EDTA, or 0.1 mM GDP, or GTP, and incubated with 40 µg CaM for 30 min at 30 o C. The reaction mixtures were centrifuged for 30 min at 100,000 g, at 4 o C in a Beckman TLA45 rotor. The supernatants and the pellets in 50 µl of the same buffer were run on a SDS-PAGE and the proteins were transferred to PVDF membranes (Park et al, 1997). RalA was measured by Western blot analysis using anti-RalA antibody.…”

The proteins of synaptic vesicle membranes are affected during ageing of rat brain

“…Synaptosomes were prepared from the brain of the young (2 months) and the old (20 months) SD rat by using Ficoll gradient method (Fisher von Mollard et al, 1991;Park et al, 1997). Crude SV, which contains Rab3A-enriched membranes, was prepared through lysis of synaptosome in hypotonic solution, and it has been also referred to as LP2 membranes (Huttner et al, 1983;Park et al, 1997).…”

“…Crude SV, which contains Rab3A-enriched membranes, was prepared through lysis of synaptosome in hypotonic solution, and it has been also referred to as LP2 membranes (Huttner et al, 1983;Park et al, 1997).…”

“…A considerable portion of Rab3A in brain is cytosolic, and this cytosolic Rab3A is complexed to guanine nucleotide dissociation inhibitor (GDI), a protein capable of dissociating Rab proteins from membranes (Ullrich, 1993). It was reported that Ca 2+ /calmodulin (CaM) could also dissociate Rab3A from SV, and forming a 1:1 complex of Rab3A-CaM (Park et al, 1997). RalA, another Ras-related small GTP-binding protein, was reported to be also an abundant protein in SV (Bielinski et al, 1993).…”