Involvement of Ca2+ channel synprint site in synaptic vesicle endocytosis

Abstract

The synaptic protein interaction (synprint) site of the voltage-gated Ca(2+) channel (VGCC) alpha1 subunit can interact with proteins involved in exocytosis, and it is therefore thought to be essential for exocytosis of synaptic vesicles. Here we report that the synprint site can also directly bind the mu subunit of AP-2, an adaptor protein for clathrin-mediated endocytosis, in competition with the synaptotagmin 1 (Syt 1) C2B domain. In brain lysates, the AP-2-synprint interaction occurred over a wide range of Ca(2+) concentrations but was inhibited at high Ca(2+) concentrations, in which Syt 1 interacted with synprint site. At the calyx of Held synapse in rat brainstem slices, direct presynaptic loading of the synprint fragment peptide blocked endocytic, but not exocytic, membrane capacitance changes. We propose that the VGCC synprint site is involved in synaptic vesicle endocytosis, rather than exocytosis, in the nerve terminal, via Ca(2+)-dependent interactions with AP-2 and Syt.

Figure 1.

Direct interaction of AP-2μ with VGCC synprint site. A, Affinity column chromatography for screening N-synprint-binding proteins. Brain extract from 7-d-old rats (Input) was loaded onto a GST- or GST–N-synprint-immobilized column. Bound proteins were eluted and analyzed by SDS-PAGE and silver staining. Two bands indicated by arrows were identified as AP-2αA (110 kDa) and AP-2αC (100 kDa), respectively, by mass spectrometry. B, Eluates from the column with GST–N-synprint (718-963 aa, GST-N), GST–P/Q-synprint (715-1015 aa, GST-P/Q), and corresponding loop of GST–L–L_II–III (800-910 aa, GST-L) were analyzed by immunoblot for AP-1 and AP-2. Standard was 15.6% of the input. C, Pull-down assays using GST, GST–N-synprint, or GST–Syt1-C2B (248–421 aa, GST-Syt1), before (−) and after (+) urea denature of brain lysate. AP-2α, AP-2β, and AP-2μ were detected using their specific antibodies. Standard was 30% of the input. D, In vitro binding assays using recombinant proteins (GST, GST-N, GST-P/Q, GST-L, GST-Syt1)-immobilized beads and MBP-fused AP-2μ subunit (123-435 aa). AP-2μ bindings were detected using anti-MBP antibody. Standard was 3% of the input. E, Scatchard plot indicating concentration-dependent bindings of MBP–AP-2μ with GST–N-synprint. K _d was calculated from a linear regression line. Similar results were obtained from two other trials. In the right, data from three experiments were normalized and were fit by Hill plot (Hill coefficient of 0.85). F, Coimmunoprecipitation (IP) of AP-2 with Ca_V2.2 (middle lane in the left) or β4 (middle lane in the right) subunit of native VGCC in heparin-purified samples of brain tissue. VGCC subunits in starting samples are shown in Western blot (WB) in the bottom row. Standard was 2% of the input. GST–N-synprint disrupted the interactions of AP-2 with Ca_V2.2 and β4 subunits, respectively (right lanes).

Figure 2.

The common binding region of VGCC synprint site, shared by AP-2μ and Syt 1 C2B domain. A, Narrowing down the binding region of N-synprint for AP-2μ, Syt 1, and Stx 1 by pull-down assay using GST–N-synprint fragments (left). Their bindings to MBP-fused AP-2μ (standard was 2.2% of the input), Syt 1 C2B (248-421 aa; standard was 2% of the input), or Stx 1 (1-288 aa; standard was 5% of the input) were analyzed using anti-MBP antibody. B, Narrowing down the binding region of AP-2μ for binding to N-synprint and Syt1 C2B. GST or GST-fusion proteins were incubated with cell lysates obtained from COS 7 cells transfected with EGFP-fused AP-2μ fragments (left). Binding was evaluated by immunoblotting using anti-EGFP antibody (right). Standard was 33% of the input. C, Reduced binding of two-points mutants (Y344A, K354A) of AP-2μ to N-synprint (GST-N), P/Q-synprint (GST-P/Q), and Syt 1 C2B (GST-Syt 1). Myc tag-fused wild-type (WT) and mutant (mut) AP-2μ subunits overexpressed in COS7 cells were pulled down, and their binding was detected using anti-Myc tag antibody. Standard was 30% of the input. D, Competitions between AP-2μ and Syt for binding to N-synprint. Left, MBP–AP-2μ (50 nm) was pulled down by GST–N-synprint alone (None) or in the presence of MBP–Syt (1-421 aa) at 500 nm (10×) or at 1 μm (20×). Standard was 5% of the input. Right, MBP–Syt 1 (400 nm) was pulled down by GST–N-synprint alone (None) or in the presence of MBP–AP-2μ at 4 μm (10×) or at 8 μm (20×). Standard was 1% of the input. E, Competitions between AP-2μ and Stx for binding to N-synprint. Left, MBP–AP-2μ alone (50 nm; None) or in the presence of MBP–Stx 1 at 500 nm (10×) or at 1 μm (20×). Standard was 10% of the input. Right, MBP–Stx 1 alone (50 nm; None) or in the presence of MBP–AP-2μ at 500 nm (10×) or at 1 μm (20×), Standard was 10% of the input. Experiments were performed in nominally Ca²⁺-free media.

Figure 3.

Ca²⁺ concentration-dependent binding of VGCC synprint site to AP-2μ and Syt. A, Rat brain extracts were dissolved in Ca²⁺ buffers of various free Ca²⁺ concentrations and mixed with GST–N-synprint-bound beads. Top, Binding of AP-2μ and Syt were detected using anti-AP-2α (top row) or anti-Syt 1 (middle row) antibody, respectively. Standard was 14% of the input. Bottom row shows CBB stainings of bead-bound GST–N-synprint proteins. Bottom, Densitometric quantifications of binding intensities (ordinate in an arbitrary scale) at different Ca²⁺ concentrations (abscissa) for AP-2μ (filled circles) and Syt 1 (open circles). Vertical bars indicate SEMs of three experiments. B, Effects of Ba²⁺ and Sr²⁺ on bindings of N-synprint to AP-2μ (top row) and Syt 1 (bottom row). Standard was 14% of the input. C, Recombinant AP-2μ was incubated with N-synprint in 1 mm (1000) or 0 mm Ca²⁺ (0) in the presence (right) or absence (left) of brain extract. Binding of AP-2μ to N-synprint was detected using anti-MBP antibody. Standard was 20% of the input.

Figure 4.

Effects of intraterminal loadings of N-synprint fragment on vesicle exocytosis and endocytosis at the calyx of Held. A, Sample records of I _pCa and C _m changes induced by a 1 ms depolarizing command voltage pulse (V _com; from −80 to 0 mV), in the presence of GST–N-synprint (5 μm; red) or GST–L–L_II–III (5 μm, black) in the presynaptic pipette. Middle column, In the presence of GST–N-synprint fragment, command pulse amplitude was reduced (by 5–10 mV) to match the I _pCa amplitude to that in GST–L–L_II–III (superimposed). Arrows indicate the onset of command pulse in A and C. Gray lines indicate the level of I _pCa amplitude and ΔC _m in control. B, The mean amplitude of I _pCa (left) and ΔC _m (right) in the terminals loaded with GST–L–L_II–III (n = 10; black) or GST–N-synprint (n = 18; red). Terminals loaded with GST–N-synprint showed significantly larger I _pCa (*p < 0.02) and ΔC _m (**p < 0.002) than GST–L–L_II–III-loaded terminals. When I _pCa in the terminal loaded with GST–N-synprint was matched to that with GST–L–L_II–III (Weak depol.), ΔC _m was similar to that in the terminal loaded with GST–L–L_II–III (n = 7, p > 0.4). C, Presynaptic C _m change, induced by the 1 ms depolarizing pulse, shown in slow timescale. Averaged C _m traces, in the presence of GST–N-synprint (red; n = 6) or GST–L–L_II–III (black; n = 6) in the recording patch pipette, were superimposed, after normalizing at 200–250 ms from the pulse onset.