Abstract
Different families of auxiliary subunits regulate the function and trafficking of native α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in the central nervous system. While a facilitatory role of auxiliary subunits in ER export and forward trafficking of newly synthesized AMPA receptors is firmly established, it is unclear whether auxiliary subunits also control endosomal receptor turnover in dendrites. Here, we manipulated the composition of AMPA receptor complexes in cultured hippocampal neurons by overexpression of two auxiliary subunits, transmembrane AMPAR regulatory protein (TARP) γ-8 or cysteine knot AMPAR-modulating protein (CKAMP) 44a, and monitored dendritic receptor cycling in live-cell imaging experiments. Receptor surface delivery was assayed using a modified AMPA receptor subunit carrying the pH-dependent fluorophore superecliptic pHluorin (SEP-GluA1), which regains its fluorescence during receptor exocytosis, when transiting from the acidic lumen of transport organelles to the neutral extracellular medium. Strikingly, we observed a dramatic reduction in the spontaneous fusion rate of AMPA receptor-containing organelles in neurons overexpressing either type of auxiliary subunit. An analysis of intracellular receptor distribution also revealed a decreased receptor pool in dendritic recycling endosomes, suggesting that incorporation of TARPγ-8 or CKAMP44a in receptor complexes generally diminishes cycling through the endosomal compartment. To directly analyze dendritic receptor turnover, we also generated a new reporter by N-terminal fusion of a self-labeling HaloTag to an AMPA receptor subunit (HaloTag-GluA1), which allows for selective, irreversible staining of surface receptors. Pulse chase-experiments with HaloTag-GluA1 indeed demonstrated that overexpression of TARPγ-8 or CKAMP44a reduces the constitutive internalization rate of surface receptors at extrasynaptic but not synaptic sites. Thus, our data point to a yet unrecognized regulatory function of TARPγ-8 and CKAMP44a, by which these structurally unrelated auxiliary subunits delay local recycling and increase surface lifetime of extrasynaptic AMPA receptors.
Highlights
Α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) constitute the primary class of ligand-gated cation channel found at central glutamatergic synapses
We present evidence for a critical involvement of the auxiliary subunits TARPγ-8 and CKAMP44a in controlling constitutive endosomal cycling of AMPARs in the dendrites of hippocampal neurons
We show that an increased abundance of either of these auxiliary subunits results in a reduced turnover of AMPARs, prolonging the surface lifetime of receptors by delaying receptor uptake (Figure 6D)
Summary
Α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptors (AMPARs) constitute the primary class of ligand-gated cation channel found at central glutamatergic synapses. Altering the expression of CKAMP44 in neurons results in corresponding changes in AMPAR surface density (Khodosevich et al, 2014), which suggests that CKAMPs control AMPAR forward trafficking like TARPs. That said, different CKAMP isoforms have been shown to differentially support AMPAR surface expression, which possibly reflects additional influences of AMPAR subunit composition and cell type on trafficking (von Engelhardt et al, 2010; Khodosevich et al, 2014; Chen et al, 2018). The association of AMPARs with specific auxiliary subunit types determines local turnover and receptor lifetime on the dendritic surfaces
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