The overall goals of this research program during a more than a 30-year period of continual funding are to define structure-function relationships of vitamin K-dependent coagulation proteins, both in vitro and in vivo, with specific attention paid to interactions of the g-carboxyglutamic acid (Gla) domains (GD) with metal ions, membranes, and receptors. We have addressed these issues by use of protein chemistry, enzymology, structural biology, molecular/cell biology, and gene targeting strategies. During the recent past, this grant has centered on GD-mimetic neuroactive peptides, the conantokins, especially their structure-function relationships with regard to cation binding, N-methyl-D-aspartate receptor (NMDAR) binding, and their biological properties of allosteric inhibition of ion channel opening and consequent regulation of Ca2+ homeostasis in neuronal cells and in other cell types transfected with NMDAR subunit combinations. In this renewal application, efforts will be focused on relating the structures of these small neuroactive GD mimetic peptides to their specificities for functional interactions with different subunit combinations of the neuronal NMDAR, which are temporally and spatially variable in the brain, and with their cell signaling properties. To exploit the relationships between the chemical (aim 1) and cell biology (aim 2) results with the in vivo use of conantokin-based drugs, we will use a model of occlusive stroke in rats (aim 3), which results in downstream hypoxia of brain cells and consequent dysregulation of the NMDAR ion channel, leading to abnormal levels of calcium in cells, with deleterious consequences to the host organism (e.g., neuronal apoptosis resulting in cell death after ischemic stroke). Three highly interrelated specific aims are proposed: 1) to delineate the components of the extracellular regions of NR1 and NR2 subunits required for interaction with conantokins and to identify structural elements within the conantokins that dictate NMDAR selectivity, testing the hypothesis that conantokins can be engineered to enable their NMDAR subunit- selectivity. 2) to study the role of conantokins in modulating the NMDAR-dependent activation of ERK1/2 in cell signaling, in both NMDAR subunit-transfected HEK293 cells and in primary neurons, testing the hypothesis that the downstream neuroprotective effects of NR2B-specific conantokins are achieved by modulation of steps of the the ERK1/2 pathway. 3) to employ an in vivo model of occlusive stroke in rats to investigate the effects of native and variant conantokins on downstream hypoxia-induced brain cell apoptosis, examining the hypothesis that NMDAR subunit specificity of the conantokins can be employed to attenuate the apoptotic processes that occur. The overall hypothesis of this proposal is that conantokins that differ with respect to their structures display varying potency and efficacy at different NMDAR subunit combinations, an important consideration for understanding the molecular bases for many neurological diseases, e.g., ischemic stroke-related cell death.
Abnormal calcium ion flow into neuronal cells through activated N-methyl-D-aspartate receptor (NMDAR) channels is wholly or partially responsible for a variety of neuropathies, including neuronal apoptotis-driven cell death consequent to ischemic stroke. In such cases, therapeutic inhibition of calcium flow by the NMDAR is desirable. Direct NMDAR ion channel blockade drugs exist, but psychomimetic side-effects associated with their application limit the use of these agents. Vitamin K-dependent gamma-carboxyglutamate (Gla)- containing natural peptides from cone snails represent a potentially significant therapeutic class of NMDAR inhibitors that are allosteric to the ion channel and inhibit calcium flow by processes not associated with direct channel blocking. This proposal is concerned with the complex mechanisms of action of the conantokins in vitro and in vivo and relating these properties to their neuroprotective effects in a model of occlusive stroke.
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