Skip to main content
. 2010 Jan 20;30(3):1064–1072. doi: 10.1523/JNEUROSCI.3909-09.2010

Figure 4.

Figure 4.

Variable stoichiometry of stargazin on AMPA receptors. A, Oocytes were injected with GluA1 cRNA (2 ng) and various amounts (from left to right: 0, 2.5, 1.25, 0.625, 0.313, 0.156, and 0.078 ng) of stargazin tagged with HA epitope at the extracellular domain and with four monomeric AcGFP units (GFP×4) at its cytoplasmic domain (HA-STG-GFP×4). Single bands of HA-stargazin-GFP×4 and GluA1 were detected on SDS-PAGE (10%) at around 150 kDa, as assessed using an anti-HA antibody, and at around 100 kDa, as assessed using an anti-GluA1 antibody. B, Up-regulation of the expression of HA-tagged stargazin (STG) containing four GFP units (HA-STG-GFP×4) shifted the molecular weight of the GluA1 complex detected using an anti-GluA1 antibody. We detected four distinct bands, in addition to a band corresponding to GluA1 alone, which suggests that a maximum of four stargazin molecules can bind to one AMPA receptor dimer-of-dimers structure. In addition, the molecular weight of the GluA1/stargazin complex was shifted linearly upon increase of stargazin expression, which suggests that all four stargazin-binding sites on the AMPA receptor were independent. C, Kainate-evoked (20 μm) currents from oocytes that were coinjected with GluA1 (2 ng) and various amounts of stargazin tagged with four GFP units (STG-GFP×4) were measured using TEVC recording. Data represent the means ± SEM (n = 5–7); asterisks indicate p < 0.05 using Tukey's test with ANOVA, a significant difference compared with GluA1 alone. NQ, non-quantitative because current amplitudes were too high (>12 μA). D, HA-stargazin tagged with four monomeric GFP units did not form oligomers in an expression-level-dependent manner (from left to right, 4, 2, 1, 0.5, 0.25, 0.125, 0.0625, and 0.031 ng) on BN-PAGE (4–15%). E, HA-tagged stargazin and HA-stargazin tagged with four monomeric GFP units did not form a dimer and were expressed as monomers on BN-PAGE (4–15%). Ratio of HA-STG (0.8 ng)/HA-STG-GFP×4 (1.6 ng), from left: 1/0, 7/1, 5/1, 3/1, 1/1, 1/3, 1/5, 1/7, and 0/1. WB, Western blotting.