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First published online September 29, 2004
doi: 10.1242/10.1242/jcs.01388

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Phosphorylation by cAMP-dependent protein kinase is essential for synapsin-induced enhancement of neurotransmitter release in invertebrate neurons

Ferdinando Fiumara¹, Silvia Giovedì², Andrea Menegon³, Chiara Milanese¹, Daniela Merlo^2,*, Pier Giorgio Montarolo^1,, Flavia Valtorta³, Fabio Benfenati² and Mirella Ghirardi¹

¹ Department of Neuroscience, Section of Physiology, University of Torino, Corso Raffaello 30, 10125 Torino, Italy
² Department of Experimental Medicine, Section of Human Physiology, University of Genova, Viale Benedetto XV 3, 16132, Genova, Italy
³ `Vita-Salute' San Raffaele School of Medicine, Via Olgettina 58, 20132, Milano, Italy

View larger version (20K): [in a new window]   Fig. 1. Time-course and stoichiometry of apSyn phosphorylation by distinct protein kinases. Purified bovine synapsin I (5 pmol; closed symbols) or recombinant apSyn (clone 11.1; 50 pmol; open symbols) cleaved from GST and repurified was incubated at 30°C for the indicated time periods with either the catalytic subunit of PKA (A), CaMK II (B), MAPK/Erk2 (C) or PKC (D) in the presence of radioactive ATP. Phosphorylation stoichiometries (mol [32P]/mol synapsin), calculated at various incubation times, are shown as mean±s.e.m. (n=4-5).

View larger version (39K): [in a new window]   Fig. 2. PKA phosphorylates apSyn at a single site in domain A. (A) Radioactive phosphate incorporation into recombinant apSyn 11.1 or bovine synapsin I (5 pmol) as a function of the incubation time demonstrates that the snail synapsin orthologue is an excellent substrate for PKA. The multiple bands observed in the apSyn lanes are probably caused by partial cleavage by thrombin. (B) Mutation of Ser-9 in the conserved PKA consensus sequence present in apSyn domain A into alanine (apSynALA9) or aspartate (apSynASP9) completely abolishes [32P] incorporation after incubation with the PKA catalytic subunit for 20 minutes, indicating that Ser-9 is the only phosphorylation site for PKA. The positions of molecular mass standards are shown on the left in kDa.

View larger version (31K): [in a new window]   Fig. 3. Intracellular injection of apSyn enhances evoked neurotransmitter release from C1 neuron somata cultured under low-release conditions. (A) Phase-contrast image of the soma of a C1 neuron immediately after contact with a 5HT-sensitive sniffer cell (S). Bar, 50 µm. (B) Phase-contrast image of a C1-C3 soma-soma co-culture. Twenty-four hours after C1-C3 pairing, a sniffer cell (S) was micromanipulated to contact the membrane of the C1 soma to detect neurotransmitter release. Calibration as in A. (C) Sample intracellular recordings of the sniffer cell depolarization (upper trace) induced by a train of action potentials (10 Hz for 10 seconds) in the C1 soma (lower trace) under different experimental conditions. The basal membrane potential of the sniffer cell was kept at -80 mV. (D) The mean sniffer depolarization measured after stimulation of C3-paired C1 somata is significantly lower than the mean depolarization induced by stimulation of isolated C1. The amplitude of sniffer depolarization is significantly higher 24 hours after injection of the GST-apSyn fusion protein. No change is induced by injection of GST alone.

View larger version (16K): [in a new window]   Fig. 4. The apSyn-induced enhancement of neurotransmitter release requires phosphorylation of domain A. (A) Sample recordings of sniffer depolarization induced by C1 firing (10 Hz for 10 seconds) representative of the different experimental groups in which the soma of the C1 neuron paired with C3 was loaded with either wild-type apSyn, apSynASP9, apSynALA9 or GST. (B) Bar graph showing that the mean amplitude of sniffer depolarization recorded after firing of C1 neurons loaded with either apSyn or the pseudophosphorylated apSynASP9 mutant was significantly higher than that recorded after the stimulation of control neurons injected with GST alone. In contrast, the mean sniffer depolarization amplitude measured after stimulation of C1 neurons loaded with the non-phosphorylatable apSynALA9 mutant was not different from that recorded after firing of GST-injected neurons.

View larger version (112K): [in a new window]   Fig. 5. ApSyn induces ultrastructural changes in C1 neurons in a phosphorylation-dependent manner. (A-C) Fine structure of contact areas between C1 and C3 neurons of soma-soma pairs in which C1 has been injected with GST (A-A"), apSyn (B-B") or apSynALA9 (C-C"). The boundaries of the meshwork of interdigitating cellular processes between C1 and C3 somata are highlighted in black (arrows). Note the marked increase in the thickness of this area in the sample injected with apSyn (B-B') with respect to either GST- or apSynALA9-loaded samples (A-A' and C-C', respectively). In the apSyn-injected samples the area of contact was characterized by a dense meshwork of neurite-like processes filled with microtubules and clusters of dense core SV profiles typical of the C1 neuron (see arrowhead in B and high magnification inset). Clusters of SVs were also visible in processes extending outside the contact area (B"). These processes were virtually absent in C1-C3 pairs loaded with GST or apSynALA9 (A-A" and C-C"). (D) Bar graph showing the differences in the thickness of the meshwork of embricated processes between C1 and C3 in the three experimental groups. When the C1 neuron was loaded with apSyn, the interdigitation area was significantly thicker than in control conditions (GST injection) or after injection of the non-phosphorylatable mutant apSynALA9. (E) A cluster of dense core SVs typical of the C1 neuron. (F) Large neurosecretory granules observed in the soma of C3. Bar, 1.8 µm for panels A-C; 8 µm for panels A'-C'; 1 µm for panels A"-C"; 0.5 µm for panels E,F and inset in panel B.

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