Lead poisoning can cause a wide range of symptoms with particularly severe clinical effects on the CNS. Lead can increase spontaneous neurotransmitter release but decrease evoked neurotransmitter release. These effects may be caused by an interaction of lead with speci®c molecular targets involved in neurotransmitter release. We demonstrate here that the normally calcium-dependent binding characteristics of the synaptic vesicle protein synaptotagmin I are altered by lead. Nanomolar concentrations of lead induce the interaction of synaptotagmin I with phospholipid liposomes. The C2A domain of synaptotagmin I is required for lead-mediated phospholipid binding. Lead protects both recombinant and endogenous rat brain synaptotagmin I from proteolytic cleavage in a manner similar to calcium. However, lead is unable to promote the interaction of either recombinant or endogenous synaptotagmin I and syntaxin. Finally, nanomolar concentrations of lead are able to directly compete with and inhibit the ability of micromolar concentrations of calcium to induce the interaction of synaptotagmin I and syntaxin. Based on these ®ndings, we conclude that synaptotagmin I may be an important, physiologically relevant target of lead.

Despite the fact that lead poisoning is the most common disease of environmental origin in the United States, the spectroscopic properties of aqueous Pb(II) coordination compounds have not been extensively investigated. Spectroscopic techniques that can be used to probe the fundamental coordination chemistry of Pb(II) will aid in both the development of water-soluble ligands that bind lead both tightly and selectively and the characterization of potential biological targets. Here, we report the preparation and characterization of a series of Pb(II) complexes of amido- derivatives of EDTA. The 207Pb chemical shift observed in these complexes (2441, 2189, and 1764 ppm for [Pb(EDTA)]2-, Pb(EDTA-N2), and [Pb(EDTA-N4)]2+, respectively) provides an extremely sensitive measure of the local environment and the charge on each complex. These shifts help to map out the lead chemical shift range that can be expected for biologically relevant sites. In addition, we report the first two-dimensional 207Pb-1H heteronuclear multiple-quantum correlation (HMQC) nuclear magnetic resonance spectra and demonstrate that this experiment can provide useful information about the lead coordination environment in aqueous Pb(II) complexes. Because this technique allows 207Pb-1H couplings through three bonds to be identified readily, 207Pb-1H NMR spectroscopy should prove useful for the investigation of Pb(II) in more complex systems (e.g., biological and environmental samples).

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Synaptotagmin I is a critical component of the synaptic machinery that senses calcium influx and triggers synaptic vesicle fusion and neurotransmitter release. Fluorescence resonance energy transfer studies conducted on synaptotagmin demonstrate that calcium concentrations required for fusion induce a conformational change (EC 50 Ϸ 3 mM) that brings the two calcium-binding C2 domains in synaptotagmin closer together. Analytical ultracentrifugation studies reveal that synaptotagmin is monomeric under these conditions, indicating that this calcium-triggered association between the C2 domains is intramolecular, rather than intermolecular. These results suggest a mechanism for synaptotagmin function at the presynaptic plasma membrane that involves the self-association of C2 domains. C alcium mediates neurological signal transduction by triggering the release of neurotransmitter from synaptic vesicles into the synaptic cleft. Substantial progress toward understanding how calcium triggers this response has been made over the last decade, following the identification of a number of proteins involved in docking and fusion of synaptic vesicles (1). Strong evidence points to synaptotagmin I [''synaptotagmin'' (syt)] (2) as the protein that senses calcium influx (3). On binding of Ca 2ϩ , synaptotagmin binds to the SNARE (soluble N-ethylmaleimidesensitive factor attachment protein receptor) complex (4, 5) on the synaptic cleft and triggers fusion of synaptic vesicles. However, the mechanism by which calcium regulates the fusogenic activity of synaptotagmin has remained elusive.Synaptotagmin I is anchored to the wall of synaptic vesicles via an N-terminal transmembrane domain and contains two cytosolic calcium-binding C2 domains (C2A and C2B; see Fig. 1 A). § The C2 domains in synaptotagmin I are each highly homologous with the C2 regulatory region of protein kinase C (2), the protein in which the C2 domain was originally identified. C2 domains have subsequently been reported in over 100 proteins in signaling pathways (http:͞͞www.expasy.ch͞cgi-bin͞prosite-searchac?PS50004) ranging from phospholipid binding and calcium signaling to ubiquitination (6, 7). Despite the pervasive nature of this domain, many fewer structural studies have been reported for proteins containing C2 domains (8-17) than for calciumbinding proteins that contain EF-hand domains (e.g., calmodulin) (18)(19)(20). Those structures that have been reported reveal striking differences between the two classes of calcium proteins: whereas EF-hand proteins are entirely ␣-helical, typically contain a single calcium ion per EF-hand, and have calcium sites that are buried, C2 domains contain a prominent ␤-barrel and bind multiple calcium ions in a highly exposed surface site.Synaptotagmin is an unusual calcium sensor even compared with other C2 proteins in that synaptotagmin must respond quickly and reversibly to both low and high influxes of calcium: low calcium concentrations (5-10 M) trigger phospholipid binding (2, 21-23) and higher calcium concentrations (Ͼ200 M...