A Noelin-organized extracellular network of proteins required for constitutive and context-dependent anchoring of AMPA-receptors.

Information processing and storage in the brain rely on AMPA-receptors (AMPARs) and their context-dependent dynamics in synapses and extra-synaptic sites. We found that distribution and dynamics of AMPARs in the plasma membrane are controlled by Noelins, a three-member family of conserved secreted proteins expressed throughout the brain in a cell-type-specific manner. Noelin tetramers tightly assemble with the extracellular domains of AMPARs and interconnect them in a network-like configuration with a variety of secreted and membrane-anchored proteins including Neurexin1, Neuritin1, and Seizure 6-like. Knock out of Noelins1-3 profoundly reduced AMPARs in synapses onto excitatory and inhibitory (inter)neurons, decreased their density and clustering in dendrites, and abolished activity-dependent synaptic plasticity. Our results uncover an endogenous mechanism for extracellular anchoring of AMPARs and establish Noelin-organized networks as versatile determinants of constitutive and context-dependent neurotransmission.

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

In 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.

The molecular appearance of native TRPM7 channel complexes identified by high-resolution proteomics.

The transient receptor potential melastatin-subfamily member 7 (TRPM7) is a ubiquitously expressed membrane protein consisting of ion channel and protein kinase domains. TRPM7 plays a fundamental role in the cellular uptake of divalent cations such as Zn2+, Mg2+, and Ca2+, and thus shapes cellular excitability, plasticity, and metabolic activity. The molecular appearance and operation of TRPM7 channels in native tissues have remained unresolved. Here, we investigated the subunit composition of endogenous TRPM7 channels in rodent brain by multi-epitope affinity purification and high-resolution quantitative mass spectrometry (MS) analysis. We found that native TRPM7 channels are high-molecular-weight multi-protein complexes that contain the putative metal transporter proteins CNNM1-4 and a small G-protein ADP-ribosylation factor-like protein 15 (ARL15). Heterologous reconstitution experiments confirmed the formation of TRPM7/CNNM/ARL15 ternary complexes and indicated that complex formation effectively and specifically impacts TRPM7 activity. These results open up new avenues towards a mechanistic understanding of the cellular regulation and function of TRPM7 channels.

An ER Assembly Line of AMPA-Receptors Controls Excitatory Neurotransmission and Its Plasticity.

Excitatory neurotransmission and its activity-dependent plasticity are largely determined by AMPA-receptors (AMPARs), ion channel complexes whose cell physiology is encoded by their interactome. Here, we delineate the assembly of AMPARs in the endoplasmic reticulum (ER) of native neurons as multi-state production line controlled by distinct interactome constituents: ABHD6 together with porcupine stabilizes pore-forming GluA monomers, and the intellectual-disability-related FRRS1l-CPT1c complexes promote GluA oligomerization and co-assembly of GluA tetramers with cornichon and transmembrane AMPA-regulatory proteins (TARP) to render receptor channels ready for ER exit. Disruption of the assembly line by FRRS1l deletion largely reduces AMPARs in the plasma membrane, impairs synapse formation, and abolishes activity-dependent synaptic plasticity, while FRRS1l overexpression has the opposite effect. As a consequence, FRSS1l knockout mice display severe deficits in learning tasks and behavior. Our results provide mechanistic insight into the stepwise biogenesis of AMPARs in native ER membranes and establish FRRS1l as a powerful regulator of synaptic signaling and plasticity.

Neuroplastin and Basigin Are Essential Auxiliary Subunits of Plasma Membrane Ca2+-ATPases and Key Regulators of Ca2+ Clearance.

Plasma membrane Ca2+-ATPases (PMCAs), a family of P-type ATPases, extrude Ca2+ ions from the cytosol to the extracellular space and are considered to be key regulators of Ca2+ 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 Ca2+ transport, as well as submembraneous Ca2+ concentrations under steady-state conditions. Together, these results establish neuroplastin and basigin as obligatory auxiliary subunits of native PMCAs and key regulators of intracellular Ca2+ concentration.

Heteromeric channels formed by TRPC1, TRPC4 and TRPC5 define hippocampal synaptic transmission and working memory.

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 Trpc1/Trpc4/Trpc5-triple-knockout (Trpc1/4/5-/-) 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. In vivo, Trpc1/4/5-/- mice displayed impaired cross-frequency coupling in hippocampal networks and deficits in spatial working memory, while spatial reference memory was unaltered. Trpc1/4/5-/- 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.

Modular composition and dynamics of native GABAB receptors identified by high-resolution proteomics.

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-ß 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.

Mechanism of Cd2+ coordination during slow inactivation in potassium channels.

In K+ channels, rearrangements of the pore outer vestibule have been associated with C-type inactivation gating. Paradoxically, the crystal structure of Open/C-type inactivated KcsA suggests these movements to be modest in magnitude. In this study, we show that under physiological conditions, the KcsA outer vestibule undergoes relatively large dynamic rearrangements upon inactivation. External Cd2+ enhances the rate of C-type inactivation in an cysteine mutant (Y82C) via metal-bridge formation. This effect is not present in a non-inactivating mutant (E71A/Y82C). Tandem dimer and tandem tetramer constructs of equivalent cysteine mutants in KcsA and Shaker K+ channels demonstrate that these Cd2+ metal bridges are formed only between adjacent subunits. This is well supported by molecular dynamics simulations. Based on the crystal structure of Cd2+ -bound Y82C-KcsA in the closed state, together with electron paramagnetic resonance distance measurements in the KcsA outer vestibule, we suggest that subunits must dynamically come in close proximity as the channels undergo inactivation.

One SUMO is sufficient to silence the dimeric potassium channel K2P1.

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 epsilon-amino group of Lys274 in vitro. FRET microscopy shows that assembly of K2P1 and SUMO1 requires Lys274. Single-particle 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.

A designer ligand specific for Kv1.3 channels from a scorpion neurotoxin-based library.

Venomous animals immobilize prey using protein toxins that act on ion channels and other targets of biological importance. Broad use of toxins for biomedical research, diagnosis, and therapy has been limited by inadequate target discrimination, for example, among ion channel subtypes. Here, a synthetic toxin is produced by a new strategy to be specific for human Kv1.3 channels, critical regulators of immune T cells. A phage display library of 11,200 de novo proteins is designed using the alpha-KTx scaffold of 31 scorpion toxin sequences known or predicted to bind to potassium channels. Mokatoxin-1 (moka1) is isolated by affinity selection on purified target. Moka1 blocks Kv1.3 at nanomolar levels that do not inhibit Kv1.1, Kv1.2, or KCa1.1. As a result, moka1 suppresses CD3/28-induced cytokine secretion by T cells without cross-reactive gastrointestinal hyperactivity. The 3D structure of moka1 rationalizes its specificity and validates the engineering approach, revealing a unique interaction surface supported on an alpha-KTx scaffold. This scaffold-based/target-biased strategy overcomes many obstacles to production of selective toxins.

A structural model for K2P potassium channels based on 23 pairs of interacting sites and continuum electrostatics.

K(2P)Ø, the two-pore domain potassium background channel that determines cardiac rhythm in Drosophila melanogaster, and its homologues that establish excitable membrane activity in mammals are of unknown structure. K(2P) subunits have two pore domains flanked by transmembrane (TM) spans: TM1-P1-TM2-TM3-P2-TM4. To establish spatial relationships in K(2P)Ø, we identified pairs of sites that display electrostatic compensation. Channels silenced by the addition of a charge in pore loop 1 (P1) or P2 were restored to function by countercharges at specific second sites. A three-dimensional homology model was determined using the crystal structure of K(V)1.2, effects of K(2P)Ø mutations to establish alignment, and compensatory charge-charge pairs. The model was refined and validated by continuum electrostatic free energy calculations and covalent linkage of introduced cysteines. K(2P) channels use two subunits arranged so that the P1 and P2 loops contribute to one pore, identical P loops face each other diagonally across the pore, and the channel complex has bilateral symmetry with a fourfold symmetric selectivity filter.

Arg(362) and Tyr(365) of the botulinum neurotoxin type a light chain are involved in transition state stabilization.

The botulinum neurotoxin type A (BoNT/A) light chain (LC) acts as zinc endopeptidase. The X-ray structure of the toxin demonstrated that Zn(2+) is coordinated by His(222) and His(226) of the Zn(2+) binding motif HisGluXXHis and Glu(261), whereas Glu(223) coordinates the water molecule required for hydrolysis as the fourth ligand. Recent analysis of a cocrystal of the BoNT/B LC and its substrate synaptobrevin 2 suggested that Arg(362) and Tyr(365) of the homologous BoNT/A may be directly involved in catalysis. Their role and that of Glu(350) which is also found in the vicinity to the active site were analyzed by site-directed mutagenesis. Various replacements of Arg(362) and substitution of Tyr(365) with Phe resulted in 79- and 34-fold lower k(cat)/K(m) values, respectively. These changes were provoked by decreased catalytic rates (k(cat)) and not by alterations of ground state substrate binding as evidenced by largely unchanged K(d) and K(m) values. None of these mutations affected the overall secondary structure or zinc content of the LC. These findings suggest that the guanidino group of Arg(362) and the hydroxyl group of Tyr(365) together accomplish transition state stabilization as was proposed for thermolysin, being the prototypical member of the gluzincin superfamily of metalloproteases. Mutation of Glu(350) dramatically diminished the hydrolytic activity which must partly be attributed to an altered active site fine structure as demonstrated by an increased sensitivity toward heat-induced denaturing and a lower Zn(2+) binding affinity. Glu(350) apparently occupies a central position in the active site and presumably positions His(222) and Arg(362).