Subunit composition, molecular environment, and activation of native TRPC channels encoded by their interactomes.

peer reviewedIn the mammalian brain TRPC channels, a family of Ca2+-permeable cation channels, are involved in a variety of processes from neuronal growth and synapse formation to transmitter release, synaptic transmission and plasticity. The molecular appearance and operation of native TRPC channels, however, remained poorly understood. Here, we used high-resolution proteomics to show that TRPC channels in the rodent brain are macro-molecular complexes of more than 1 MDa in size that result from the co-assembly of the tetrameric channel core with an ensemble of interacting proteins (interactome). The core(s) of TRPC1-, C4-, and C5-containing channels are mostly heteromers with defined stoichiometries for each subtype, whereas TRPC3, C6, and C7 preferentially form homomers. In addition, TRPC1/C4/C5 channels may co-assemble with the metabotropic glutamate receptor mGluR1, thus guaranteeing both specificity and reliability of channel activation via the phospholipase-Ca2+ pathway. Our results unveil the subunit composition of native TRPC channels and resolve the molecular details underlying their activation

AMPA Receptors Commandeer an Ancient Cargo Exporter for Use as an Auxiliary Subunit for Signaling

Fast excitatory neurotransmission in the mammalian central nervous system is mainly mediated by ionotropic glutamate receptors of the AMPA subtype (AMPARs). AMPARs are protein complexes of the pore-lining α-subunits GluA1-4 and auxiliary β-subunits modulating their trafficking and gating. By a proteomic approach, two homologues of the cargo exporter cornichon, CNIH-2 and CNIH-3, have recently been identified as constituents of native AMPARs in mammalian brain. In heterologous reconstitution experiments, CNIH-2 promotes surface expression of GluAs and modulates their biophysical properties. However, its relevance in native AMPAR physiology remains controversial. Here, we have studied the role of CNIH-2 in GluA processing both in heterologous cells and primary rat neurons. Our data demonstrate that CNIH-2 serves an evolutionarily conserved role as a cargo exporter from the endoplasmic reticulum (ER). CNIH-2 cycles continuously between ER and Golgi complex to pick up cargo protein in the ER and then to mediate its preferential export in a coat protein complex (COP) II dependent manner. Interaction with GluA subunits breaks with this ancestral role of CNIH-2 confined to the early secretory pathway. While still taking advantage of being exported preferentially from the ER, GluAs recruit CNIH-2 to the cell surface. Thus, mammalian AMPARs commandeer CNIH-2 for use as a bona fide auxiliary subunit that is able to modify receptor signaling

γ-2 and GSG1L bind with comparable affinities to the tetrameric GluA1 core.

BACKGROUND: The AMPA-type ionotropic glutamate receptor mediates fast excitatory neurotransmission in the brain. A variety of auxiliary subunits regulate its gating properties, assembly, and trafficking, but it is unknown if the binding of these auxiliary subunits to the receptor core is dynamically regulated. Here we investigate the interplay of the two auxiliary subunits γ-2 and GSG1L when binding to the AMPA receptor composed of four GluA1 subunits. METHODS: We use a three-color single-molecule imaging approach in living cells, which allows the direct observation of the receptors and both auxiliary subunits. Colocalization of different colors can be interpreted as interaction of the respective receptor subunits. RESULTS: Depending on the relative expression levels of γ-2 and GSG1L, the occupancy of binding sites shifts from one auxiliary subunit to the other, supporting the idea that they compete for binding to the receptor. Based on a model where each of the four binding sites at the receptor core can be either occupied by γ-2 or GSG1L, our experiments yield apparent dissociation constants for γ-2 and GSG1L in the range of 2.0-2.5/µm2. CONCLUSIONS: The result that both binding affinities are in the same range is a prerequisite for dynamic changes of receptor composition under native conditions

γ-2 and GSG1L bind with comparable affinities to the tetrameric GluA1 core

Abstract Background The AMPA-type ionotropic glutamate receptor mediates fast excitatory neurotransmission in the brain. A variety of auxiliary subunits regulate its gating properties, assembly, and trafficking, but it is unknown if the binding of these auxiliary subunits to the receptor core is dynamically regulated. Here we investigate the interplay of the two auxiliary subunits γ-2 and GSG1L when binding to the AMPA receptor composed of four GluA1 subunits. Methods We use a three-color single-molecule imaging approach in living cells, which allows the direct observation of the receptors and both auxiliary subunits. Colocalization of different colors can be interpreted as interaction of the respective receptor subunits. Results Depending on the relative expression levels of γ-2 and GSG1L, the occupancy of binding sites shifts from one auxiliary subunit to the other, supporting the idea that they compete for binding to the receptor. Based on a model where each of the four binding sites at the receptor core can be either occupied by γ-2 or GSG1L, our experiments yield apparent dissociation constants for γ-2 and GSG1L in the range of 2.0–2.5/µm2. Conclusions The result that both binding affinities are in the same range is a prerequisite for dynamic changes of receptor composition under native conditions

Auxiliary GABAB Receptor Subunits Uncouple G Protein βγ Subunits from Effector Channels to Induce Desensitization

SummaryActivation of K+ channels by the G protein βγ subunits is an important signaling mechanism of G-protein-coupled receptors. Typically, receptor-activated K+ currents desensitize in the sustained presence of agonists to avoid excessive effects on cellular activity. The auxiliary GABAB receptor subunit KCTD12 induces fast and pronounced desensitization of the K+ current response. Using proteomic and electrophysiological approaches, we now show that KCTD12-induced desensitization results from a dual interaction with the G protein: constitutive binding stabilizes the heterotrimeric G protein at the receptor, whereas dynamic binding to the receptor-activated Gβγ subunits induces desensitization by uncoupling Gβγ from the effector K+ channel. While receptor-free KCTD12 desensitizes K+ currents activated by other GPCRs in vitro, native KCTD12 is exclusively associated with GABAB receptors. Accordingly, genetic ablation of KCTD12 specifically alters GABAB responses in the brain. Our results show that GABAB receptors are endowed with fast and reversible desensitization by harnessing KCTD12 that intercepts Gβγ signaling

CNIH-2 is rendered a surface membrane protein by assembly with AMPARs.

<p><b>A</b> Total (T), surface (S) and internal (I) populations of CNIH-2 in HeLa cells expressing either CNIH-2 alone (CTRL) or together with GluA1_o or GluA2_i, respectively. S is concentrated 10fold. Note that in the absence of GluAs, CNIH-2 cannot be detected in the surface fraction. However, it is robustly observed in the plasma membrane when co-expressed with GluAs (n = 4). <b>B</b> Total (T), surface (S) and internal (I) populations of CNIH-2 in dissociated hippocampal neurons (DIV 17) transduced with CNIH-2 (+) or GFP (−). S is concentrated 10fold. Both endogenous (−) and over-expressed (+) CNIH-2 can be detected on the cell surface (n = 5). <b>C</b> (Top) Representative current traces recorded in somatic outside-out patches excised from dissociated hippocampal neurons (DIV 16–21) over-expressing either GFP (CTRL, black) or CNIH-2 (CNIH-2, red) upon 1 ms (left panel) and 100 ms applications (right panel) of 10 mM glutamate. (Bottom) Quantification of deactivation and desensitization kinetics. Data are given as mean ± SD. Asterisk denotes a significant difference from control (p<0.01, unpaired Student's t-test; deactivation: n = 10 and 8 for CTRL and CNIH-2, respectively; desensitization: n = 19 and 8 for CTRL and CNIH-2, respectively).</p

CNIH-2 changes glycosylation of GluAs.

<p><b>A</b> Western blot analysis of total and surface populations of GluA2_i extracted from HeLa cells by surface biotinylation in the absence (CTRL) or presence of CNIH-2 (CNIH-2) (n = 4). Extensive glycosylation of surface GluA2_i during maturation increases its apparent molecular weight. Note the smaller increase upon co-expression of CNIH-2. <b>B</b> Enzymatic deglycosylation analysis of GluA2_i surface populations in the presence (+) or absence (−) of CNIH-2. Surface GluA2_i remained either untreated (C) or was incubated with either endoglycosidase H (H) or PNGase F (F). Note that upon CNIH-2 co-expression, the GluA2_i surface population remains sensitive to endoglycosidase H (n = 2).</p

Surface trafficking of GluAs by CNIH-2 is splicing-dependent.

<p><b>A</b> Quantification of GluA surface expression levels by extracellular epitope tagging in HeLa cells expressing the indicated GluA subunits. Data are mean ± SEM normalized to GluA1_o (GluA1_o: n = 24; GluA1_i: n = 9; GluA2_o: n = 12; GluA2_i: n = 12). <b>B</b> Increase in GluA surface expression mediated by CNIH-2 in HeLa cells. Data are mean ± SEM normalized to surface expression of respective GluA subunits without CNIH-2 (GluA1_o: n = 24; GluA1_i: n = 8; GluA2_o: n = 12; GluA2_i: n = 12).</p

CNIH-2 facilitates ER export of AMPARs.

<p><b>A</b> Representative confocal images of OK cells stably expressing CNIH-2. Co-expression of dominant-negative Sar1 H79G prevents ER export of CNIH-2 leading to its redistribution into the ER. <b>B</b> Quantification of GluA1_o surface expression levels by extracellular epitope tagging in the presence of CNIH-2 and either wildtype (WT) Sar1 (white bar) or mutant Sar1 H79G (grey bar). Data are mean increases in surface expression levels by CNIH-2 ± SEM normalized to GluA1_o+Sar1 WT or GluA1_o+Sar1 H79G without CNIH-2, respectively. Asterisk marks a significant increase in surface expression of GluA1_o by co-expression of CNIH-2 (p<0.001, unpaired Student's t-test; n = 12 for both experimental groups). <b>C</b> Quantification of GluA1_o surface expression levels in the presence of CNIH-2 and either wildtype dynamin-1 (white bar) or dominant-negative dynamin-1 K44A (grey bar) inhibiting clathrin-dependent endocytosis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030681#pone.0030681-Damke1" target="_blank">[38]</a>. Data are mean increases in surface expression levels by CNIH-2 ± SEM normalized as in B (n = 6 for both experimental groups).</p