Synaptotagmin I is a molecular target for lead.

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 specific 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 findings, we conclude that synaptotagmin I may be an important, physiologically relevant target of lead.

Calcium triggers an intramolecular association of the C2 domains in synaptotagmin.

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) approximately 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.

207Pb-1H two-dimensional NMR spectroscopy: a useful new tool for probing lead(II) coordination chemistry.

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).