March 29, 2012;
Assembly stoichiometry of the GluK2/GluK5 kainate receptor complex.
Ionotropic glutamate receptors assemble as homo- or heterotetramers. One well-studied heteromeric complex is formed by the kainate receptor subunits GluK2 and GluK5. Retention motifs prevent trafficking of GluK5 homomers to the plasma membrane, but coassembly with GluK2 yields functional heteromeric receptors. Additional control over GluK2/GluK5 assembly seems to be exerted by the aminoterminal domains, which preferentially assemble into heterodimers as isolated domains. However,the stoichiometry of the full-length GluK2/GluK5 receptor complex has yet to be determined, as is the case for all non-NMDA glutamate receptors. Here, we address this question, using a single-molecule imaging technique that enables direct counting of the number of each GluK subunit type in homomeric and heteromeric receptors in the plasma membranes of live cells. We show that GluK2 and GluK5 assemble with 2:2 stoichiometry. This is an important step toward understanding the assembly mechanism, architecture, and functional consequences of heteromer formation in ionotropic glutamate receptors.
2PN2EY018241 NEI NIH HHS
, PN2 EY018241-08 NEI NIH HHS
, R01 NS035549-15 NINDS NIH HHS
, U24NS057631 NINDS NIH HHS
[+] show captions
(A) mEGFP was fused to the C-terminus of GluK2, expressed in Xenopus oocytes and imaged by TIRF spectroscopy. Circles mark single, stationary receptors that satisfy the criteria for analysis. Scale bar: 2 µm. (B) Fluorescence intensity trace of a representative spot bleaching in four steps indicated by arrows. (C) Number of bleaching steps observed for a total of 438 spots. The error bars represent the counting uncertainty. The red line gives the binominal distribution expected for a tetramer, based on a probability of 0.80 for an individual mEGFP to be fluorescent.
Co-expression of GluK2-mEGFP and GluK5-mCherry
(A) Images from a representative movie. The circles indicate stationary spots that were observed in both the green mEGFP and the red mCherry channel. The bar graph shows the fractions of green-only, colocalizing and red-only spots for a total of 599 spots. (B) The co-expression experiment with GluK2-mEGFP and GluK5-mCherry allows to count the bleaching step distribution of GluK2 subunits colocalizing with GluK5. Left: One example trace with two mEGFP bleaching steps is shown. Right: Bleaching step analysis of 124 colocalizing spots. The red line gives the binominal distribution expected for two subunits with a probability of 0.80 for a single mEGFP to be fluorescent. The error bars represent the counting uncertainty. (C) Experiment with GluK2 and GluK5-mEGFP, which allows to count the number of GluK5 subunits per complex. Left: One example trace is shown. Right: The number of bleaching steps from 932 receptors agrees well with the binominal distribution expected for two subunits (red line, probability of 0.80 for a single mEGFP to be fluorescent).
Homomeric GluK5 receptors
(A) GluK2 forms homotetramers and traffics to the plasma membrane, whereas GluK5 is retained in the ER. Co-expression of GluK2 and GluK5 gives heterotetramers with 2:2 stoichiometry (see Figure 2). Impairment of the ER retention motifs on GluK5 (GluK5ΔERret) restores surface-expression, but these assemblies are non-functional. (B) Bleaching step analysis of GluK5ΔERret-mEGFP based on 658 spots. The red line gives the binominal distribution expected for four subunits with a probability of 0.80 for a single mEGFP to be fluorescent. In this experiment the maturation probability might be closer to 0.83. The error bars represent the counting uncertainty.
Possible stoichiometries for the assembly of GluK2 and GluK5
The dimeric states are hypothetical. We only observe surface expression of (GluK2)4 homotetramers along with 2:2 GluK2/GluK5 heteromers. The drawing of the tetrameric assemblies does not denote the actual topology of the complexes.