Towards a Mechanism of TARP Action at AMPA Receptors

Abstract

Rapid excitatory synaptic transmission is carried out primarily by glutamate-gated AMPA receptors. In the last few years, a number of AMPA receptor auxiliary proteins have been identified which assemble with the AMPAR at synapses to aid in the trafficking and alter the gating of channel. The prototypical auxiliary protein is stargazin, or γ2, which is a member of the transmembrane AMPA receptor regulatory protein (TARP) family. Stargazin slows desensitization and deactivation, increases the frequency of opening to larger conductances and increases the relative efficacy of partial agonists such as kainate. At present, the structural mechanism by which TARPs enhance AMPAR gating is unclear but there are two distinct possibilities. First, TARPs may stabilize the ligand binding domain in more closed conformations, promoting the activation of the channel. And second, TARPs may increase the probability of opening the channel by increasing the efficiency of coupling between changes at the ligand binding domain and channel segments. To distinguish between these alternatives, we used previously characterized ligand binding domain mutations which de-stabilize the closed-bound states of the channel and asked whether stargazin could rescue gating in these mutants. using rapid perfusion in outside-out patches in both desensitizing and non-desensitizing conditions we found that stargazin rescued deficits in gating as measured by the ratio of glutamate to quisqualate responses and single channel conductance, suggesting that TARPs work by stabilizing closed-cleft states of the ligand binding domain. We further tested this hypothesis using FRET to directly measure conformation changes of the ligand binding domain in AMPAR alone or with stargazin. Preliminary FRET experiments are consistent with our electrophysiology data and support the hypothesis that TARPs enhance AMPAR gating by stabilizing closed bound states of the ligand binding domain.