We examined synaptic protein complexes isolated from a mildly-denaturing extract of freshly dissected mouse brain cortex, using a gentle extraction procedure that maintains the integrity of the protein complexes. Using co-immunoprecipitation, immunoblotting, and mass spectrometry, we successfully detected novel protein-protein interactions in cortical synapses. Although some of the interactions shown here have been reported previously, they provide not only a validation of our procedure, but in some cases are the first evidence for SNARE protein interactions in the absence of GST-tagged proteins as baits or outside of in vitro
systems. Though the use of artificial systems is useful to explore protein interactions, it is imperative to study synaptic protein interactions under native conditions as shown here. Our protocol also provided appropriate matrix and antibody controls to avoid potential non-specific protein-protein interactions that may occur during co-immunoprecipitation [18
], and we further provided mass spectrometric evidence for the interaction of SNARE proteins. Our experiments do not distinguish between diverse cell compartments since we used membranes from both pre- and postsynaptic fractions. However, using a simplified synaptosomal preparation as described by Ling et al. [7
], we were still able to show the same interactions in both mouse cortex and midbrain (data not shown). Thus, the preparation that we used provides reproducible synaptic interactions that are not dependent on a particular micro-environment and ensures a physiological system that precludes non-specific interactions.
Many proteins interact in the synapses to mediate and maintain neuronal transmission, and they control vesicle-related processes by forming complexes. Synapses have different endocytic mechanisms at their disposal, including clathrin-dependent and -independent pathways, in addition to other mechanisms [23
]. Also, components of SNARE complexes mediate membrane fusion in all of the trafficking steps of the secretory pathway. As we showed here, syntaxin-1A, SNAP-25, and VAMP-2 can establish reciprocal interactions in agreement with previous reports [22
], and these interactions have been shown to mediate neurotransmitter vesicle transport and targeting [4
]. SNAREs and dynamin, both involved in membrane fusion, could be linked in a common protein complex to finely regulate synaptic neurotransmission by controlling the vesicle recycling process. Indeed, other molecules and proteins could connect dynamin and SNAREs, forming a macromolecular complex. For example, synaptophysin can be co-immunoprecipitated using VAMP-2 as a bait protein, as we verified with multiple techniques, in agreement with previous reports [2
] and can also bind dynamin in a Ca2+
-dependent fashion [5
], suggesting that a dynamin/synaptophysin/Ca2+
complex may participate in recycling of synaptic vesicles. A definitive link between endocytic machinery and SNAREs is represented by our finding that dynamin-1 can be co-immunoprecipitated using VAMP-2 as a bait protein. This interaction was assessed with both mass spectrometry and immunoblotting. Thus, VAMP-2 and dynamin may exert their functions as components of the same complex. However, this interaction was not seen using dynamin as a bait, and no interactions were detected between dynamin and syntaxin or SNAP25 (data not shown). These observations are consistent with the current model of synaptic neurotransmission, where VAMP-2 is anchored in the vesicular membrane and can thus be re-internalized by endocytotic processes while syntaxin and SNAP-25 reside in the cell membrane and do not take part in the recycling process. This model of synaptic neurotransmission implicates the disassembly of the SNARE complex or a time/location shift before dynamin/VAMP-2 interaction takes place. VAMP-2 and dynamin could interact during the vesicle recycling process that elapses between exo- and endo-cytosis, but their interaction may be transient and not linked to the start of endocytosis. In addition, other mechanisms have been hypothesized [4
] and we cannot exclude that interactions between dynamin and SNAP-25 or syntaxin could be identified by using different experimental conditions.
We also evaluated the possibility that the BKCa
channel could operate as a link between the endocytic machinery and SNAREs. In fact, it has been recently shown that a different potassium channel, the Kir2.3, can be internalized from the plasma membrane via vesicle-mediated endocytosis in a dynamin-dependent manner [13
]. We also confirmed that the BKCa
channel can co-associate with syntaxin-1A, as previously reported [7
]. Above all, our reciprocal co-immunoprecipitations conclusively showed that the BKCa
channel and the GTP-ase, dynamin-1, established a strong interaction, which was disrupted under denaturing conditions and was not driven by nonspecific compartment binding, given that the BKCa
channel did not interact with clathrin under identical conditions. Mass spectrometric analysis of the BKCa
interactome confirmed the presence of dynamin among co-immunoprecipitated proteins; the sensitivity of the mass spectrometer could ensure the absence of dynamin in the corresponding FLAG control band, thus showing specificity of the BKCa
-dynamin interaction in mouse cortex. BKCa
α-subunit may act as a link between calcium-activated potassium channel activity and the cell’s endocytic machinery, given that it can interact with both syntaxin and dynamin. We speculate that the assembly and density of BKCa
channels could be regulated by endocytosis via the interaction of its alpha subunit with dynamin. Additional experiments are required to verify the functional significance of this interaction.
channel association with the SNARE complex is not limited to its interaction with syntaxin-1A. Immunoblots and mass spectrometry also provided novel evidence for BKCa
channel interaction with syntaxin binding protein, munc-18, a protein known to co-associate with syntaxin [19
], as we confirmed, and to play a role in its conformational switch during exocytosis. Recent evidence suggests that both syntaxin-dependent and -independent functions may exist for this protein; indeed, munc-18 may be able to exert its role in two additional ways, by binding to the N-terminus of syntaxin or to the assembled SNARE complex [24
]. The mechanisms of transition between the different modes of binding are poorly understood. In this context, the novel interaction between BKCa
channel and munc-18 that we report here may be transient and take place during one of the diverse modes cited above. Given that we found that the BKCa
channel does not interact with SNAP-25 and VAMP-2 (data not shown), more experimental information is needed to elucidate the molecular mechanism and significance underlying this novel and selective interaction with munc-18.
The sensitivity of mass spectrometry allowed us to identify a number of other protein-protein interactions. In Suppl. Table 2C
, we report additional partners of BKCa
channels: phosphoinositide-3-kinase subunits, sodium/potassium-transporting ATPase subunits, cytoskeletal proteins like actin and tubulin, and kinesin heavy and light chains which are involved in vesicular transport. The above BKCa
partners, with the exception of kinesin, have also been recently identified by Kathiresan et al. [9
] in the mouse cochlea. Previous studies in the brain reported many partners of BKCa
channels, including actin, tubulin, calcium channels, microtubule proteins [6
], and syntaxin [7
]. SNARE proteins (syntaxin and VAMP), dynamin-1, clathrin, actin, and microtubule proteins have also been identified as interacting with N-type calcium channels [11
]. Taken together, these interactions may help to depict a general synaptic protein-protein network with calcium channels, SNAREs, and cytoskeletal proteins as possible links between BKCa
and dynamin (). The hypothesized network would be consistent with our mass spectrometric analysis of synaptic protein partners, which detected kinesin heavy chains, tubulin, microtubule associated proteins, and AP-2 complex subunit proteins as SNAP-25 partners and sodium/potassium-transporting ATPase subunits as VAMP-2 partners.
Provisional representation of a putative synaptic protein network
In summary, we identified novel synaptic protein complexes in mouse cortical membranes using an interaction proteomics approach, which included co-immunoprecipitation followed by immunoblotting or LC-MS/MS; the experimental conditions combined high-specificity with a native system that resembled the synaptic environment. This is the first report to uncover protein interactions between 1) dynamin and the BKCa channel, 2) dynamin and VAMP-2, and 3) the BKCa channel and munc-18 in synapses. These newly identified interactions could elucidate some of the missing links in synaptic protein complexes and contribute to better understanding of synaptic vesicle trafficking, release, and recycling.