GABAA receptor-associated phosphoinositide 3-kinase is required for insulin-induced recruitment of postsynaptic GABAA receptors
Introduction
Most of the fast inhibitory synaptic transmission within the central nervous system is mediated by type A γ-aminobutyric acid (GABAA) receptors (Mehta and Ticku, 1999). These receptors are vital in modulating neural activity and maintaining electrical balance. Structurally, GABAA receptors are thought to be heteropentameric complexes (Tretter et al., 1997) made up of various subunits from different subfamilies (Macdonald and Olsen, 1994). So far, at least six α, three β, three γ, one δ, one ε, one π, and one θ subunits have been cloned from the mammalian nervous system (Barnard et al., 1998). In the mammalian brain, the most prevalent GABAA receptor subunit combination is believed to be α1β2γ2 (McKernan and Whiting, 1996).
An emerging theme in the area of synaptic transmission is that of the dynamic nature of postsynaptic ionotropic receptors within the brain, whereby rapid changes in the number of these receptors at postsynaptic domains can modulate the efficacy of receptor-mediated synaptic transmission (Wan et al., 1997b, Nusser et al., 1998, Man et al., 2000, Lu et al., 2001, Malinow, 2003, Collingridge et al., 2004). As such, understanding of the molecular mechanisms underlying the rapid trafficking of these ligand-gated receptors at postsynaptic membranes has been a subject of intensive research.
We have previously shown that insulin produces a rapid recruitment of postsynaptic GABAA receptors and hence potentiates the receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) (Wan et al., 1997b). Recent work from our lab further demonstrated that this recruitment is mediated by a facilitated plasma membrane insertion of GABAA receptors that is dependent on Akt phosphorylation of GABAA receptor β subunits (Wang et al., 2003). However, the detailed mechanisms by which insulin activates Akt and leads to facilitated receptor insertion remain to be determined.
It is well established that the activation of phosphoinositide 3-kinase (PI3-K) is a critical step in stimulating Akt kinase activity in many intracellular signaling pathways. PI3-Ks constitute a family of lipid kinases that is involved in mitogenic signaling, cell survival, cytoskeletal rearrangements, and vesicular trafficking (Cantley, 2002). In neuronal cells, there is a recruitment to, and activation of, Akt at postsynaptic domains of GABAergic synapses following insulin stimulation (Wang et al., 2003), but the underlying mechanisms remain to be specified. Since several subunits of GABAA receptors contain tyrosine residues that can be phosphorylated both in vitro and in vivo (Moss et al., 1995, Valenzuela et al., 1995, Wan et al., 1997a), we hypothesize that insulin stimulation leads to tyrosine phosphorylation of one or more of the GABAA receptor subunits and creates SH2-binding domains for PI3-K, promoting a complex formation between the PI3-K and GABAA receptors, and that this association plays an essential role in insulin-mediated GABAA receptor insertion into postsynaptic domains and hence potentiation of the receptor-mediated synaptic transmission. The present study tests this hypothesis.
Section snippets
Methods
All animals used in the study were housed, cared for, and used experimentally in accordance with the Guide for the Care and Use of Experimental Animals, Volumes I and II, published by the Canadian Council on Animal Care.
Results
Previous work from our laboratory has shown that insulin treatment can increase plasma membrane insertion of postsynaptic GABAA receptors and hence potentiate the receptor-mediated IPSCs in cultured hippocampal neurons (Wan et al., 1997b, Wang et al., 2003). In order to determine whether or not PI3-K plays a role in the insulin-induced cell-surface amplification of GABAA receptors, we examined the effects of wortmannin, a specific PI3-K inhibitor at nanomolar concentrations (Arcaro and Wymann,
Discussion
In the present work, we have established a role for PI3-K in the modulation of GABAA receptors by insulin. We observed an increase in the amplitude and frequency of GABAA receptor-mediated currents following insulin stimulation of hippocampal neuronal cultures that was dependent on PI3-K activation (Fig. 2). Activation of PI3-K has been shown to modulate the function of other neural ion channels. For example, insulin-like growth factor-1 (IGF-1)-mediated increases in calcium channel currents
Acknowledgments
This work was supported by grants from the Canadian Institutes of Health Research (CIHR), Howard Hughes Medical Institute (HHMI), and the Heart and Stroke Foundation of British Columbia and Yukon (HSFBC&Y) to Y.T.W. Y.T.W. is a CIHR Investigator, an HHMI International Scholar and a Michael Smith Foundation for Medical Research Senior Scholar, and is also the holder of HSFBC&Y Chair in Stroke Research at the University of British Columbia and the Vancouver Hospital and Health Sciences Centre. We
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