Abstracts

Rationale: The antiepileptic drug levetiracetam (KEPPRA; LEV) binds to a unique site in brain, the synaptic vesicle protein SV2A, a member, by sequence homology, of the MFS class of transporter proteins. Despite evidence that LEV acts at least partly via its binding to SV2A, very little is known about the molecular and cellular functions of SV2A, and there is no current understanding of what the effects of LEV binding to SV2A might be. In order to advance our understanding of SV2A, we undertook a project to image the protein using advanced methods of tomographic analysis of Electron microscopy (EM) images of presynaptic terminals. Mice were treated with either LEV or saline, and slices were made from their hippocampii, for EM imaging of SV2A in the presence and absence of bound LEV, at the presynaptic termini of hippocampal neurons.Methods: One hour after ip LEV administration (170mg/kg), C57BL/6 mice were subjected to fixative perfusion (PAF 4%). EM micrograph tilt series was taken on Ultrathin cryosections of mice hippocampii highly enriched in SV2A. The region of interest was selected and sectioned based on immunohistochemical analysis of SV2A expression. A mouse monoclonal antibody was used for primary detection, and 5 nm colloidal gold, was used as a secondary marker. Three-dimensional reconstructions of the tilt series was performed using Protein Tomography™, which is a novel method for three-dimensional visualization of individual molecules in biological samples. Results: Two major conformation classes of SV2A have been identified; the first conformation is a compact funnel shaped structure with a pore-like opening towards the cytoplasmic side. The other major conformation is a more open V-shaped structure, with no visible pore-structure, but with a cleft-like opening towards the intravesicular space. The observed large conformational difference between the inward-facing and outward-facing conformations is evidence of a high degree of flexibility in the protein supporting a valve like action needed for molecular transport. The data presented in this study gives the first direct structural evidence for a large conformational flexibility of SV2A.The inward-facing conformation and outward-facing conformation of SV2A is found in both LEV treated and untreated samples. Conclusions: In this study, Protein Tomography™ has been used to visualize membrane bound SV2A in tissue samples, both in free form and in LEV associated form. The results of the study showed that SV2A exist in two major conformations. The two major conformations are in good agreement with inward- and outward-facing conformations of the proposed alternating access model (Kaback et al., Proc Natl Acad Sci U S A. 2007, 104(2), 491-4.) with a high level of flexibility in the protein, corresponding to a movement of the two proposed six-helix bundles constrained by a long loop on the cytoplasmic side of the protein. To our knowledge this study has provided the first direct structural data on the large conformational flexibility of SV2A, supportive of the assumed transporter function.