GABAB receptors, the most abundant inhibitory G protein-coupled receptors in the mammalian brain, display pronounced diversity in functional properties, cellular signaling and subcellular distribution. We used high-resolution functional proteomics to identify the building blocks of these receptors in the rodent brain. Our analyses revealed that native GABAB receptors are macromolecular complexes with defined architecture, but marked diversity in subunit composition: the receptor core is assembled from GABAB1a/b, GABAB2, four KCTD proteins and a distinct set of G-protein subunits, whereas the receptor's periphery is mostly formed by transmembrane proteins of different classes. In particular, the periphery-forming constituents include signaling effectors, such as Cav2 and HCN channels, and the proteins AJAP1 and amyloid-β A4, both of which tightly associate with the sushi domains of GABAB1a. Our results unravel the molecular diversity of GABAB receptors and their postnatal assembly dynamics and provide a roadmap for studying the cellular signaling of this inhibitory neurotransmitter receptor.

Small ubiquitin modifier 1 (SUMO1) is shown to regulate K2P1 background channels in the plasma membrane (PM) of live mammalian cells. Confocal microscopy reveals native SUMO1, SAE1, and Ubc9 (the enzymes that activate and conjugate SUMO1) at PM where SUMO1 and expressed human K2P1 are demonstrated to colocalize. Silent K2P1 channels in excised PM patches are activated by SUMO isopeptidase (SENP1) and resilenced by SUMO1. K2P1-Lys274 is crucial: when mutated to Gln, Arg, Glu, Asp, Cys, or Ala, the channels are constitutively active and insensitive to SUMO1 and SENP1. Tandem mass spectrometry confirms conjugation of SUMO1 to the ε-amino group of Lys274 in vitro. FRET microscopy shows that assembly of K2P1 and SUMO1 requires Lys274. Singleparticle TIRF microscopy shows that wild-type channels in PM have two K2P1 subunits and assemble with two SUMO1 monomers. Although channels engineered with one Lys274 site carry just one SUMO1 they are activated and silenced by SENP1 and SUMO1 like wild-type channels.T he activities of many cellular proteins are regulated by covalent conjugation of small ubiquitin modifier (SUMO) proteins on ε-amino groups of specific lysine residues. Such proteins are sumoylated and desumoylated via conserved enzymes that form or hydrolyze isopeptide bonds to these target lysines. This pathway is well-recognized to modulate nuclear import and export, DNA repair, and transcription factor activity (1). Unexpectedly, we found the SUMO pathway to regulate the activity of human K2P1 potassium channels expressed at the PM of Xenopus laevis oocytes and COS-7 African Green Monkey fibroblasts (2). At baseline, K2P1 channels in PM were electrically silent due to the indigenous SUMO machinery. Channel activation required exposure to an isopeptidase (SENP1) that removes SUMO or mutation of a specific lysine residue of K2P1, Lys274. Regulatory events were shown to be reversible by "cramming" of channels in PM patches into cells producing SENP1 or SUMO1. Mutation of Lys274 prevented SUMO conjugation producing active channels insensitive to suppression by SUMO1 or activation by SENP1.Many biochemical and structural studies have examined the interaction of SUMO with transcription factors. In contrast, the mechanisms underlying SUMO regulation at the PM of cells has been controversial in the literature. Even as additional PM substrates for sumoylation were reported [including, Kv2.1 and Kv1.5 voltage-gated potassium channels (3, 4), GluR6 receptors (5), and TRPM4 channels (6)], our description of SUMO action on K2P1 has been questioned (7,8). Here, we confirm the regulation of human K2P1 channels in PM by sumoylation and characterize the SUMO-channel interaction in detail using CHO cells.Results K2P channels are dedicated pathways for background flux of potassium ions that set cellular resting potentials and mediate electrical excitability subject to a broad array of regulatory influences (9). Identified by their unique primary structure of two poreforming domains in each subunit (10) and their operation as potas...

Canonical transient receptor potential (TRPC) channels influence various neuronal functions. Using quantitative high-resolution mass spectrometry, we demonstrate that TRPC1, TRPC4, and TRPC5 assemble into heteromultimers with each other, but not with other TRP family members in the mouse brain and hippocampus. In hippocampal neurons from -triple-knockout () mice, lacking any TRPC1-, TRPC4-, or TRPC5-containing channels, action potential-triggered excitatory postsynaptic currents (EPSCs) were significantly reduced, whereas frequency, amplitude, and kinetics of quantal miniature EPSC signaling remained unchanged. Likewise, evoked postsynaptic responses in hippocampal slice recordings and transient potentiation after tetanic stimulation were decreased. , mice displayed impaired cross-frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. animals also exhibited deficiencies in adapting to a new challenge in a relearning task. Our results indicate the contribution of heteromultimeric channels from TRPC1, TRPC4, and TRPC5 subunits to the regulation of mechanisms underlying spatial working memory and flexible relearning by facilitating proper synaptic transmission in hippocampal neurons.

Highlights d Assembly of native AMPARs occurs in discrete steps defined by ER-resident interactors d ABHD6 nurses GluA monomers; FRRS1l/CPT1c complexes drive multimer-formation of GluAs d FRRS1l is a potent regulator of synapse maturation and synaptic plasticity d FRRS1l knockout phenocopies the severe intellectual disability of human patients

Plasma membrane Ca-ATPases (PMCAs), a family of P-type ATPases, extrude Ca ions from the cytosol to the extracellular space and are considered to be key regulators of Ca signaling. Here we show by functional proteomics that native PMCAs are heteromeric complexes that are assembled from two pore-forming PMCA1-4 subunits and two of the single-span membrane proteins, either neuroplastin or basigin. Contribution of the two Ig domain-containing proteins varies among different types of cells and along postnatal development. Complex formation of neuroplastin or basigin with PMCAs1-4 occurs in the endoplasmic reticulum and is obligatory for stability of the PMCA proteins and for delivery of PMCA complexes to the surface membrane. Knockout and (over)-expression of both neuroplastin and basigin profoundly affect the time course of PMCA-mediated Ca transport, as well as submembraneous Ca concentrations under steady-state conditions. Together, these results establish neuroplastin and basigin as obligatory auxiliary subunits of native PMCAs and key regulators of intracellular Ca concentration.