Ampa Receptor Transport

Due to the highly dynamic nature of AMPA receptors as a mechanism of synaptic plasticity64-66, there exists a wealth of studies which focus on their exo-and endocytosis at mature synapses (see Chapters 22-24). A number of studies suggest that similar mechanisms governing the activity-dependent insertion of AMPA receptors may also be occurring during development8,67,68.

AMPA receptors have long been shown to interact directly with NSF and a and P-SNAPs69. These proteins form part of the exocytic fusion complex and are integral to vesicle fusion with the plasma membrane. The interaction between AMPA receptors and NSF has been shown to be required for the increase in the number of AMPA receptors at synapses in response to activity70,71. In addition, AMPA receptors are endocytosed by a dynamin-dependent mechanism44. These studies all suggest that exo/endocytosis is integral to the local trafficking of AMPA receptors at mature synapses. However, can one assume that AMPA receptor trafficking prior to synapse formation also involves cycling with the plasma membrane, as seen for NMDA receptors?

AMPA receptors, like NMDA receptors, are also seen as nonsynaptic clusters in dendrites of young cortical and hippocampal neurons9,53. These clusters have also been described to be mobile9 (see Figure 14.2; Colorplate 9). However, it appears that AMPA receptor trafficking may be governed by somewhat different mechanisms. The number of clusters that are seen to move at any one time is greatly reduced as compared to NMDA receptors and the velocity of their movement is also less9. This may be attributable to the fact that AMPA receptors are transported by the the motor protein KIF5A72 as compared with KIF17 for NMDA receptors41. Interestingly, Hirokawa and colleagues demonstrated that interaction of GluR2-associated protein (GRIP1) with KIF5A preferentially directs the AMPA receptor complex into dendrites rather than axons72.

Occasionally, clusters containing both AMPA and NMDA receptors are transported together and these appear to move with the kinetics of NMDA receptor transport packets9. These may represent vesicles recently derived from the Golgi apparatus prior to sorting at the plasma membrane. During development AMPA receptors are also seen at the surface of neurons at nonsynaptic sites38,39,73,74. It appears that AMPA receptors are found in intracellular and extracellular pools in neuronal dendrites74. This suggests that while AMPA receptors may be transported separately from NMDA receptors, perhaps employing a different motor protein, both receptor types may share the mechanism of cycling with the plasma membrane.

As with NMDA receptors, AMPA receptors are able to diffuse in the plasma membrane. Electrophysiological recordings38, immunocytological methods74, and single-particle tracking experiments73 point to a diffuse presence of extrasynaptic glutamate receptors in the plasma membrane. Electrophysiological recordings combined with iontophoretic application of glutamate did not find large clusters of AMPA in extrasynaptic membrane38. Single-particle tracking experiments clearly demonstrate active transport of surface receptors and trapping of receptors at synapses, suggesting that interactions with a submembrane scaffold may be a mechanism for recruiting receptors that are already in the plasma membrane39,40,73. It remains to be determined which exact route AMPA receptors take to arrive at the synaptic plasma membrane.

Ampa Receptor

Figure 14.2. AMPA Receptor Transport and Recruitment. (A) Time-lapse images of AMPA receptor clusters in vitro. Clusters of GFP-tagged AMPA receptor subunit GluR1 are mobile (white arrow) in the dendrites of cortical neurons cultured for 4 days. Yellow arrow (see Colorplate 9) shows original location; time in minutes and seconds; scale bar = 6 jm. (B) Insertion of pH-dependent GFP-tagged GluR1 (pHluorin-GluR1) at new sites of synapse formation. Contact of a P-neurexin expressing PC12 cell (red) induces the accumulation of CFP-PSD-95 (blue). Scale bar = 10 jm. (C) The appearance of pHluorin-GluR1 at the PSD-95 cluster (arrowheads) is induced by the bath application of glutamate and glycine in brief pulses. Time in minutes and seconds. Reproduced with permission from ref. 88. Copyright (2005) National Academy of Sciences, U.S.A.

Figure 14.2. AMPA Receptor Transport and Recruitment. (A) Time-lapse images of AMPA receptor clusters in vitro. Clusters of GFP-tagged AMPA receptor subunit GluR1 are mobile (white arrow) in the dendrites of cortical neurons cultured for 4 days. Yellow arrow (see Colorplate 9) shows original location; time in minutes and seconds; scale bar = 6 jm. (B) Insertion of pH-dependent GFP-tagged GluR1 (pHluorin-GluR1) at new sites of synapse formation. Contact of a P-neurexin expressing PC12 cell (red) induces the accumulation of CFP-PSD-95 (blue). Scale bar = 10 jm. (C) The appearance of pHluorin-GluR1 at the PSD-95 cluster (arrowheads) is induced by the bath application of glutamate and glycine in brief pulses. Time in minutes and seconds. Reproduced with permission from ref. 88. Copyright (2005) National Academy of Sciences, U.S.A.

There are four different subunits of AMPA receptors, GluR1-4. A great number of studies have focused on their differential recruitment to synapses and this topic is discussed in more detail in Chapter 24. However, pertinent at this point is the observation that GluR4-only AMPA receptors are the first AMPA receptors to be incorporated at new synapses. During the development of neuronal circuits in a number of regions of the nervous system, synapses first only show NMDA receptors and are thus termed "silent"8,67. In response to spontaneous synaptic activity, GluR4-containing AMPA receptors are inserted at "silent" synapses75. Subsequently, these receptors are exchanged for GluR2-containing AMPA receptors, a process that requires little synaptic activity75. The consequences and reasons for such switching of AMPAR receptor subtype delivery remain largely obscure; however it has been hypothesized that long-term maintenance of a potentiated synapse requires the exchange of AMPA receptors for GluR2-containing AMPA receptors70.

Prior to incorporation at new synaptic sites, AMPA receptors associate with a MAGUK protein early during the biosynthetic pathway. SAP-97 binds to AMPA receptors while the receptors are still in the ER or Golgi76. Since SAP-97 does not associate with synaptic AMPA receptors, we can assume that there is a switch in MAGUK protein association that serves to stabilize AMPA receptors at synapses. Recently, it has been hypothesized that a transmembrane protein that also associates with AMPA receptors plays a far greater role in AMPA receptor trafficking and targeting than previously surmised. Stargazin and the related transmembrane AMPA regulatory proteins (TARPs) were identified due to a naturally occurring mutation in the Stargazin gene in Stargazer mice. These mice show dystonia and often look upward, hence their name. This phenotype probably arises due to the fact that the granule cells in the cerebellum have no AMPA receptors at the plasma membrane77. TARPs, as this family of proteins is now known78, have been shown to direct trafficking of AMPA receptors to the cell surface and to synapses via distinct mechanisms77. TARPs interact with AMPA receptors through PSD-95 and this interaction controls the number of AMPA receptors at synapses79. However, it appears that a direct interaction between AMPA receptors and these four transmembrane-domain proteins in their extracellular domains can modulate AMPA receptor gating80. Furthermore, quantitative immunoprecipitations from mouse brain suggest that the majority of AMPA receptors are present in an exclusive complex with TARPs, with less than a total of 1% of AMPA receptors bound to MAGUK proteins such as SAP 102, SAP97, PSD-95, PICK1, and GRIP81. This recent study brings a large amount of controversy into the field and much work is needed to determine the overriding molecular mechanism by which AMPA receptors are transported through the biosynthetic pathway to the plasma membrane and then to synapses.

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