Constitutive Endocytosis of GABA_AReceptors by an Association with the Adaptin AP2 Complex Modulates Inhibitory Synaptic Currents in Hippocampal Neurons

Abstract

Type A GABA receptors (GABA A ) mediate the majority of fast synaptic inhibition in the brain and are believed to be predominantly composed of ␣, ␤, and ␥ subunits. Although changes in cell surface GABA A receptor number have been postulated to be of importance in modulating inhibitory synaptic transmission, little is currently known on the mechanism used by neurons to modify surface receptor levels at inhibitory synapses. To address this issue, we have studied the cell surface expression and maintenance of GABA A receptors. Here we show that constitutive internalization of GABA A receptors in hippocampal neurons and recombinant receptors expressed in A293 cells is mediated by clathrin-dependent endocytosis. Furthermore, we identify an interaction between the GABA A receptor ␤ and ␥ subunits with the adaptin complex AP2, which is critical for the recruitment of integral membrane proteins into clathrin-coated pits. GABA A receptors also colocalize with AP2 in cultured hippocampal neurons. Finally, blocking clathrin-dependant endocytosis with a peptide that disrupts the association between amphiphysin and dynamin causes a large sustained increase in the amplitude of miniature IPSCs in cultured hippocampal neurons. These results suggest that GABA A receptors cycle between the synaptic membrane and intracellular sites, and their association with AP2 followed by recruitment into clathrin-coated pits represents an important mechanism in the postsynaptic modulation of inhibitory synaptic transmission.

Hypothesis review: are clathrin-mediated endocytosis and clathrin-dependent membrane and protein trafficking core pathophysiological processes in schizophrenia and bipolar disorder?

“…Similarly, N-methyl-D-Aspartate (NMDA) glutamate receptors, which are thought to be functionally impaired in psychosis [84][85] , are endocytosed by clathrin-dependent mechanisms 70 . Other receptors that are implicated in schizophrenia such as AMPA [86][87] and GABA(A) [88][89] are also regulated by clathrin-dependent mechanisms.…”

Beta-amyloid disrupted synaptic vesicle endocytosis in cultured hippocampal neurons

“…P4 blocks the binding of dynamin 1 to amphiphysin and prevents the recruitment of dynamin 1 to the endocytic complex. Biochemical assays showed that this peptide is highly specific and binds with high affinity to a unique site in the dynamin-proline rich domain (Grabs et al 1997;Kittler et al 2000). Treated neurons were then stimulated and used for subcellular fractionation or FM1-43 imaging as described below.…”

“…To obtain more direct evidence of the role of dynamin 1 in the altered synaptic vesicle endocytosis in Aβ-treated neurons, we examined whether inhibition of dynamin 1 would cause a similar membranous build-up of amphiphysinin in cultured hippocampal neurons. To inhibit dynamin 1 activity, we used a dynamin 1 inhibitory peptide (P4) which blocks the binding of dynamin 1 to amphiphysin and prevents the recruitment of dynamin 1 to the endocytic complex (Grabs et al 1997;Kittler et al 2000). When these neurons were treated with 50 μM P4 for 30 minutes prior to stimulation they did not show any change in the amphiphysin membrane-to-cytosol ratio when compared to untreated stimulated neurons (Fig.…”

Huntingtin-associated protein 1 regulates inhibitory synaptic transmission by modulating γ-aminobutyric acid type A receptor membrane trafficking

“…It is evident that GABA A Rs are not static entities in neuronal plasma membranes but undergo rapid movement into and out of these structures (3). Modifications of GABA A R cell surface number underlie changes in inhibitory postsynaptic current amplitude, providing an effective mechanism for regulating the efficacy of synaptic inhibition (3)(4)(5)(6)(7)(8)(9)(10). Under basal conditions, synaptic GABA A Rs are undergoing clathrin-dependent endocytosis (8,10,11).…”

“…Modifications of GABA A R cell surface number underlie changes in inhibitory postsynaptic current amplitude, providing an effective mechanism for regulating the efficacy of synaptic inhibition (3)(4)(5)(6)(7)(8)(9)(10). Under basal conditions, synaptic GABA A Rs are undergoing clathrin-dependent endocytosis (8,10,11). Given this constitutive endocytosis, the cellular fate of internalized GABA A receptors is critical as their recycling or degradation will affect the number of receptors on the cell surface, and hence the efficacy of synaptic inhibition.…”