Altered phosphorylation of connexin 43 contributes to increased gap-junction coup

University Of Colorado Denver, Aurora CO

Investigators

Abstract

DESCRIPTION (provided by applicant): Pilocarpine-induced status epileptics (SE) induce reactive gliosis and activate calcium signaling in astrocytes of the hippocampus in a time-dependent manner (1, 2). Primary studies demonstrate significant decreases in connexin phosphorylation 48 h after SE and in chronically epileptic rats 30 d after SE. This data combined with previous studies showing that paroxysmal depolarization shifts can be initiated by release of glutamate from astrocytes3 suggests that increased open probability of gap- junction hemi channels via dephosphorylating of connexin 43 in hippocampal astrocytes may contribute to the progression of epileptogenesis. The major goal of this proposal is to characterize the role of serine/threonine kinases and phosphatases in seizure-induced dephosphorylating of connexin 43 and specifically, to evaluate the hypothesis that status epileptics acutely activates phosphatases (and may decrease kinases at specific time points) in glyptic astrocytes which dephosphorylates connexin 43 to subsequently increase connexin 43-containing hemi channel open probability and astrocytes gap- junction coupling during epileptogenesis in the hippocampus. Molecular methods will be used to characterize the time course of phosphatase and kinase activation. Alterations in gap-junction coupling will be quantitatively measured in response to equilibrium shifts between kinase and phosphatase activity in hippocampal slices at multiple time points following SE. Kinase and phosphatase activity will be pharmacologically and genetically modulated to determine the primary mechanism responsible for the seizure-induced dephosphorylating of connexin 43 and downstream effects on gap-junction coupling, potassium currents, and glutamate transporter currents prior to and after the onset of spontaneous seizures. Chronic video-EEG monitoring and freeze-fracture replica immunogold labeling (FRIL) will be used to determine whether seizure-induced alterations in phosphorylated connexin 43 membrane expression and potential increases in seizure frequency associated with phosphorylation-mediated hemi channel opening can be prevented with pharmacological or transgenic methods. The proposal will provide valuable translational information about how phosphatase inhibitors or kinase agonists may be exploited to prevent the progression of epileptogenesis and achieve and disease-modification.