Yael Stern-Bach

Functional Assembly of AMPA and Kainate Receptors Is Mediated by Several Discrete Protein-Protein Interactions

Functional heterogeneity of ionotropic glutamate receptors arises not only from the existence of many subunits and isoforms, but also from combinatorial assembly creating channels with distinct properties. This heteromerization is subtype restricted and thought to be determined exclusively by the proximal extracellular N-terminal domain of the subunits. However, using functional assays for heteromer formation, we show that, besides the N-terminal domain, the membrane sector and the C-terminal part of S2 are critical determinants for the formation of functional channels. Our results are compatible with a model where the N-terminal domain only mediates the initial subunit associations into dimers, whereas for the assembly of the full functional tetramer, compatibility of the other regions is required.

A Point Mutation in the Glutamate Binding Site Blocks Desensitization of AMPA Receptors

Desensitization of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate (AMPA) receptors is thought to shape the synaptic response and act as a neuroprotective mechanism at central synapses, but the molecular mechanism underlying desensitization is poorly understood. We found that replacing the glutamate binding domain S1 of GluR3 (an AMPA receptor) with S1 of GluR6 (a kainate receptor) resulted in a fully active but completely nondesensitizing receptor. Smaller substitutions within S1 identified, besides two additional modulatory regions, a single exchange, L507Y, as is required and sufficient for the block of desensitization. This phenotype was specific for AMPA receptors and required an aromatic residue at this position. L507 lies between two residues (T504 and R509) that form part of the glutamate binding site. The physical proximity of these residues, which are involved in binding and gating, suggests they may form part of the link between these two events.

Stargazin Reduces Desensitization and Slows Deactivation of the AMPA-Type Glutamate Receptors

The AMPA-type glutamate receptors mediate the majority of the fast excitatory synaptic transmission and critically contribute to synaptic plasticity in the brain, hence the existence of numerous trafficking proteins dedicated to regulation of their synaptic delivery and turnover. Stargazin (also termed γ2) is a member of a recently identified protein family termed transmembrane AMPA receptor regulatory proteins (TARPs). TARPs physically associate with AMPA receptors and participate in their surface delivery and anchoring at the postsynaptic membrane. Here, we report that next to its trafficking roles, stargazin may also act as a positive allosteric modulator of AMPA receptor ion channel function. Coexpression of stargazin with AMPA receptor subunits, either in Xenopus oocytes or in human embryonic kidney 293 cells, significantly reduced receptor desensitization in response to glutamate. Receptor deactivation rates were also slowed, and the recovery from desensitization was accelerated. Structurally, based on the data showing a tight correlation between desensitization and the stability of the AMPA receptor intradimer interface, we propose that binding of stargazin may stabilize the receptor conformation. Functionally, our data suggest that AMPA receptors complexed with stargazin (and possibly also with other TARPs) at the postsynaptic membrane are significantly more responsive to synaptically released glutamate compared with AMPA receptors lacking stargazin/TARP interaction. The putative existence of such two states of synaptic AMPA receptors, with and without stargazin/TARP binding, may provide a novel mechanism for regulation of excitatory synaptic strength during development and/or in synaptic plasticity in the adult brain.

CKAMP44: A Brain-Specific Protein Attenuating Short-Term Synaptic Plasticity in the Dentate Gyrus

Dancing with AMPARs A type of transmembrane receptor for glutamate, known as AMPAR, mediates most of the fast excitatory transmission in the mammalian central nervous system. Their function is regulated by the composition of their subunits, posttranslational modifications, and protein-protein interactions. Recently, several proteins that interact with AMPARs have been identified that affect their subcellular localization, synaptic stabilization, and kinetics. Using proteomic analysis, immunohistochemistry, and electrophysiology, von Engelhardt et al. (p. 1518, published online 25 February; see the Perspective by Farrant and Cull-Candy) identified a protein, CKAMP44, which modulates postsynaptic AMPA receptor gating, deactivation, and desentization. A synaptic protein that regulates postsynaptic AMPA receptor responses has been cloned and functionally characterized. CKAMP44, identified here by a proteomic approach, is a brain-specific type I transmembrane protein that associates with AMPA receptors in synaptic spines. CKAMP44 expressed in Xenopus oocytes reduced GluA1- and A2-mediated steady-state currents, but did not affect kainate- or N-methyl- d-aspartate (NMDA) receptor–mediated currents. Mouse hippocampal CA1 pyramidal neurons expressed CKAMP44 at low abundance, and overexpression of CKAMP44 led to stronger and faster AMPA receptor desensitization, slower recovery from desensitization, and a reduction in the paired-pulse ratio of AMPA currents. By contrast, dentate gyrus granule cells exhibited strong CKAMP44 expression, and CKAMP44 knockout increased the paired-pulse ratio of AMPA currents in lateral and medial perforant path–granule cell synapses. CKAMP44 thus modulates short-term plasticity at specific excitatory synapses.

Cloning and functional expression of a tetrabenazine sensitive vesicular monoamine transporter from bovine chromaffin granules

Using oligonucleotide primers derived from the vesicular monoamine transporters sequences, a cDNA predicted to encode the bovine chromaffin granule amine transporter has been cloned (b‐VMAT2). Surprisingly, its structure is more similar to the rat brain transporter (VMAT2), than to the rat adrenal counterpart (VMAT1). Unlike rat VMAT1, bovine VMAT2 appears to be expressed both in the adrenal medulla and the brain, as judged by Northern analysis. After modification/deletion of the seven amino acids at the N‐terminus of the protein it was expressed in a functional form. The order of affinity of the bovine VMAT2 transporter to substrates is: serotonin>dopamine = norepinephrine>epinephrine. Also, the recombinant bovine adrenal transporter is highly sensitive to tetrabenazine, in sharp contrast to the rat adrenal transporter. The findings indicate, therefore, a clear species variation in which structure and function of the bovine adrenal transporter resemble the rat brain protein, while its tissue distribution is distinct from both types of rat proteins. In addition, the predicted protein sequence is identical to the experimentally determined N‐terminus sequence of the purified vesicular amine transporter [Stern‐Bach et al. (1992) Proc. Natl. Acad. Sci. USA 89, 9730‐9733].

Block of Kainate Receptor Desensitization Uncovers a Key Trafficking Checkpoint

A prominent feature of ionotropic glutamate receptors from the AMPA and kainate subtypes is their profound desensitization in response to glutamate-a process thought to protect the neuron from overexcitation. In AMPA receptors, it is well established that desensitization results from rearrangements of the interface formed between agonist-binding domains of adjacent subunits; however, it is unclear how this mechanism applies to kainate receptors. Here we show that stabilization of the binding domain dimer by the generation of intermolecular disulfide bonds apparently blocked desensitization of the kainate receptor GluR6. This result establishes a common desensitization mechanism in both AMPA and kainate receptors. Surprisingly, however, surface expression of these nondesensitizing mutants was drastically reduced and did not depend on channel activity. Therefore, in addition to its role at the synapse, we now propose an intracellular role for desensitization in controlling maturation and trafficking of glutamate receptors.

Learning from stargazin : the mouse, the phenotype and the unexpected

The stargazin gene (also referred to as Cacng2) has been identified by forward genetics in a spontaneous mouse mutant with ataxic gait, upward head-elevating movements (hence the name stargazer for the mouse) and episodes of spike-wave discharges. Stargazin is related to the gamma-1 subunit of skeletal muscle voltage-dependent calcium channel (VDCC), and a deficit in its role as auxiliary VDCC subunit was proposed to underlie the epileptic phenotype of the mouse; yet, a conclusive demonstration of stargazin function in VDCC regulation is still lacking. In contrast, stargazin and its three closely related isoforms gamma-3, gamma-4 and gamma-8 were shown to function as auxiliary subunits for a very different ion channel - the AMPA-type glutamate receptor - prominently regulating early intracellular transport, synaptic targeting and anchoring, and ion channel functions of this major excitatory receptor in the brain.

Two regions in the N-terminal domain of ionotropic glutamate receptor 3 form the subunit oligomerization interfaces that control subtype-specific receptor assembly.

The N-terminal domain (NTD) of α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) and kainate glutamate receptors plays an important role in controlling subtype specific receptor assembly. To identify NTD subdomains involved in this process we generated AMPA glutamate receptor 3 (GluR3) mutants having intra-NTD substitutions with the corresponding regions of the kainate receptor GluR6 and tested their ability to form functional heteromers with wild-type subunits. The chimeric design was based on the homology of the NTD to the NTD of the metabotropic GluR1, shown to form two globular lobes and to assemble in dimers. Accordingly, the NTD was divided into four regions, termed here N1–N4, of which N1 and N3 correspond to the regions forming lobe-1 and N2 and N4 to those forming lobe-2. Substituting N1 or N3 impaired functional heteromerization but allowed protein-protein interactions. Conversely, exchanging N2 or N4 preserved functional heteromerization, although it significantly decreased homomeric activity, indicating a role in subunit folding. Moreover, a deletion in GluR3 corresponding to the hotfoot mouse mutation of the glutamate receptor δ2, covering part of N2, N3, and N4, impaired both homomeric and heteromeric oligomerization, thus explaining the null-like mouse phenotype. Finally, computer modeling suggested that the dimer interface, largely formed by N1, is highly hydrophobic in GluR3, whereas in GluR6 it contains electrostatic interactions, hence offering an explanation for the subtype assembly specificity conferred by this region. N3, however, is positioned perpendicular to the dimer interface and therefore may be involved in secondary interactions between dimers in the assembled tetrameric receptor.

Auxiliary subunits of the CKAMP family differentially modulate AMPA receptor properties

AMPA receptor (AMPAR) function is modulated by auxiliary subunits. Here, we report on three AMPAR interacting proteins—namely CKAMP39, CKAMP52 and CKAMP59—that, together with the previously characterized CKAMP44, constitute a novel family of auxiliary subunits distinct from other families of AMPAR interacting proteins. The new members of the CKAMP family display distinct regional and developmental expression profiles in the mouse brain. Notably, despite their structural similarities they exert diverse modulation on AMPAR gating by influencing deactivation, desensitization and recovery from desensitization, as well as glutamate and cyclothiazide potency to AMPARs. This study indicates that AMPAR function is very precisely controlled by the cell-type specific expression of the CKAMP family members.

Molecular Mechanism of AMPA Receptor Modulation by TARP/Stargazin

AMPA receptors (AMPARs) mediate the majority of fast excitatory transmission in the brain and critically contribute to synaptic plasticity and pathology. AMPAR trafficking and gating are tightly controlled by auxiliary transmembrane AMPAR regulatory proteins (TARPs). Here, using systematic domain swaps with the TARP-insensitive kainate receptor GluK2, we show that AMPAR interaction with the prototypical TARP stargazin/γ2 primarily involves the AMPAR membrane domains M1 and M4 of neighboring subunits, initiated or stabilized by the AMPAR C-tail, and that these interactions are sufficient to enable full receptor modulation. Moreover, employing TARP chimeras disclosed a key role in this process also for the TARP transmembrane domains TM3 and TM4 and extracellular loop 2. Mechanistically, our data support a two-step action in which binding of TARP to the AMPAR membrane domains destabilizes the channel closed state, thereby enabling an efficient opening upon agonist binding, which then stabilizes the open state via subsequent interactions.