XBART47300
Cell Rep.
March 29, 2012;
1
(3):
23440.
Assembly stoichiometry of the GluK2/GluK5 kainate receptor complex.
Reiner A
,
Arant RJ
,
Isacoff EY
.
Abstract
Ionotropic glutamate receptors assemble as homo or heterotetramers. One wellstudied 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 fulllength GluK2/GluK5 receptor complex has yet to be determined, as is the case for all nonNMDA glutamate receptors. Here, we address this question, using a singlemolecule 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.
PubMed ID:
22509486
PMC ID:
PMC3324185
Article link:
Cell Rep.
Grant support:
2PN2EY018241
NEI NIH HHS , PN2 EY01824108
NEI NIH HHS , R01 NS03554915
NINDS NIH HHS , U24NS057631
NINDS NIH HHS , PN2 EY018241
NEI NIH HHS , R01 NS035549
NINDS NIH HHS , PN2 EY01824108
NEI NIH HHS , R01 NS03554915
NINDS NIH HHS , PN2 EY018241
NEI NIH HHS , U24 NS057631
NINDS NIH HHS , U24NS057631
NINDS NIH HHS , 2PN2EY018241
NEI NIH HHS , R01 NS035549
NINDS NIH HHS
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Article Images:
[+] show captions

Figure 1
GluK2 homotetramers
(A) mEGFP was fused to the Cterminus 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.


Figure 2
Coexpression of GluK2mEGFP and GluK5mCherry
(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 greenonly, colocalizing and redonly spots for a total of 599 spots. (B) The coexpression experiment with GluK2mEGFP and GluK5mCherry 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 GluK5mEGFP, 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).


Figure 3
Homomeric GluK5 receptors
(A) GluK2 forms homotetramers and traffics to the plasma membrane, whereas GluK5 is retained in the ER. Coexpression of GluK2 and GluK5 gives heterotetramers with 2:2 stoichiometry (see Figure 2). Impairment of the ER retention motifs on GluK5 (GluK5ΔERret) restores surfaceexpression, but these assemblies are nonfunctional. (B) Bleaching step analysis of GluK5ΔERretmEGFP 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.


Figure 4
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.
