Sarah K. Coleman

KCC2 interacts with the dendritic cytoskeleton to promote spine development.

The neuron-specific K-Cl cotransporter, KCC2, induces a developmental shift to render GABAergic transmission from depolarizing to hyperpolarizing. Now we demonstrate that KCC2, independently of its Cl(-) transport function, is a key factor in the maturation of dendritic spines. This morphogenic role of KCC2 in the development of excitatory synapses is mediated by structural interactions between KCC2 and the spine cytoskeleton. Here, the binding of KCC2 C-terminal domain to the cytoskeleton-associated protein 4.1N may play an important role. A more general conclusion based on our data is that KCC2 acts as a synchronizing factor in the functional development of glutamatergic and GABAergic synapses in cortical neurons and networks.

Subunit Composition of Kv1 Channels in Human CNS

Abstract : The α subunits of Shaker‐related K+ channels (Kv1.X) show characteristic distributions in mammalian brain and restricted coassembly. Despite the functional importance of these voltage‐sensitive K+ channels and involvement in a number of diseases, little progress has been achieved in deciphering the subunit composition of the (α)4(β)4 oligomers occurring in human CNS. Thus, the association of α and β subunits was investigated in cerebral grey and white matter and spinal cord from autopsy samples. Immunoblotting established the presence of Kv1.1, 1.2, and 1.4 in all the tissues, with varying abundance. Sequential immunoprecipitations identified the subunits coassembled. A putative tetramer of Kv1.3/1.4/1.1/1.2 was found in grey matter. Both cerebral white matter and spinal cord contained the heterooligomers Kv1.1/1.4 and Kv1.1/1.2, similar to grey matter, but both lacked Kv1.3 and the Kv1.4/1.2 combination. An apparent Kv1.4 homooligomer was detected in all the samples, whereas only the brain tissue possessed a putative Kv1.2 homomer. In grey matter, Kvβ2.1 was coassociated with the Kv1.1/1.2 combination and Kv1.2 homooligomer. In white matter, Kvβ2.1 was associated with Kv1.2 only, whereas Kvβ1.1 coprecipitated with all the α subunits present. This represents the first description of Kv1 subunit complexes in the human CNS and demonstrates regional variations, indicative of functional specialisation.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Channels Lacking the N-terminal Domain

Ionotropic glutamate receptor (iGluR) subunits contain a ∼400-residue extracellular N-terminal domain (“X domain”), which is sequence-related to bacterial amino acid-binding proteins and to class C G-protein-coupled receptors. The X domain has been implicated in the assembly, transport to the cell surface, allosteric ligand binding, and desensitization in various members of the iGluR family, but its actual role in these events is poorly characterized. We have studied the properties of homomeric α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)-selective GluR-D glutamate receptors carrying N-terminal deletions. Our analysis indicates that, surprisingly, transport to the cell surface, ligand binding properties, agonist-triggered channel activation, rapid desensitization, and allosteric potentiation by cyclothiazide can occur normally in the complete absence of the X domain (residues 22–402). The relatively intact ligand-gated channel function of a homomeric AMPA receptor in the absence of the X domain indirectly suggests more subtle roles for this domain in AMPA receptors, e.g. in the assembly of heteromeric receptors and in synaptic protein interactions.

Selective Binding of Synapse-associated Protein 97 to GluR-A α-Amino-5-hydroxy-3-methyl-4-isoxazole Propionate Receptor Subunit Is Determined by a Novel Sequence Motif

A family of four closely related PDZ domain-containing membrane-associated guanylate kinase homologues (MAGUKs) is involved in the regulation of the amount and functional state of ionotropic glutamate receptors in excitatory synapses. To understand the mechanisms that determine the specificity of these interactions, we examined the structural basis of the highly selective association between the ionotropic GluR subunit GluR-A and synapse-associated protein 97 (SAP97). The C terminus of GluR-A bound to the PDZ domains of SAP97, but not to those of three related MAGUKs, PSD-93, PSD-95, and SAP102. Experiments with single PDZ domains indicated that the strongest contribution was by the second PDZ domain. Unexpectedly, mutation analysis of the GluR-A C terminus revealed that a tripeptide sequence SSG at position −9 to −11 plays an essential role in this binding, in addition to a C-terminal type I PDZ binding motif (leucine at C terminus and threonine at the −2 position). Analysis of the in vitroMAGUK-binding properties of a GluR-D mutant with a one-residue deletion at the C terminus provides further support for the view that an SSG sequence located N-terminally from a type I PDZ binding motif can mediate selective binding to SAP97 and suggest the existence of a novel variation of the PDZ domain-peptide interaction.

Isoform-Specific Early Trafficking of AMPA Receptor Flip and Flop Variants

Flip and flop splice variants of AMPA receptor subunits are expressed in distinct but partly overlapping patterns and impart different desensitization kinetics to cognate receptor channels. In the absence of specific antibodies, isoform-specific differences in trafficking or localization of native flip and flop subunits remain uncharacterized. We report that in several transfected cell lines, transport of homomeric glutamate receptor (GluR)-Dflop receptors is largely blocked at the endoplasmic reticulum (ER) exit, whereas GluR-Dflip undergoes complex glycosylation and reaches the plasma membrane at >10× higher levels than GluR-Dflop, as determined by immunofluorescence, patch-clamp recordings and biochemical assays. The transport difference between flip and flop is independent of activity, is primarily determined by amino acid residue 780 (Leu in flop, Val in flip), and is manifested even in the secretion of the soluble ligand-binding domain, suggesting it is independent of oligomerization. Coexpression with stargazin or with the flip isoform rescues the surface expression of GluR-Dflop near to the level exhibited by GluR-Dflip. Our results demonstrate that the extracellular flip/flop region, via interactions with ER luminal splice form-specific protein(s), plays a hitherto unappreciated and important role in AMPA-receptor trafficking.

Agonist Occupancy Is Essential for Forward Trafficking of AMPA Receptors

Regulated trafficking of AMPA receptors to cell surface and to synapses is an important determinant of neuronal excitability. In the present study, we have addressed the role of agonist binding and desensitization in the early trafficking of glutamate receptor-D (GluR-D) AMPA receptors. Analysis of point-mutated GluR-D receptors, via electrophysiology and immunofluorescence, revealed that agonist-binding activity is essential for efficient delivery to cell surface in transfected cell lines and in neurons. Cotransfection with stargazin could fully rescue the surface expression of nonbinding mutant receptors in cell lines, indicating that stargazin is able to interact with and promote exit of AMPA receptors from endoplasmic reticulum (ER) independently of agonist binding. Secretion of separately expressed ligand-binding domain constructs showed a similar dependency of agonist binding to that observed with full-length GluR-D, supporting the idea that glutamate-induced closure of the binding site cleft is registered by ER quality control as a necessary priming step for transport competence. In contrast to agonist binding, the ability of the receptor to undergo desensitization had only a minor influence on trafficking. Our results are consistent with the hypothesis that AMPA receptors are synthesized as intrinsically unstable molecules, which require glutamate binding for structural stability and for transport-competence.

Up-regulation of the Homophilic Adhesion Molecule Sidekick-1 in Podocytes Contributes to Glomerulosclerosis

Focal segmental glomerulosclerosis (FSGS) is a leading cause of nephrotic syndrome and end-stage renal disease worldwide. Although the mechanisms underlying this important disease are poorly understood, the glomerular podocyte clearly plays a central role in disease pathogenesis. In the current work, we demonstrate that the homophilic adhesion molecule sidekick-1 (sdk-1) is up-regulated in podocytes in FSGS both in rodent models and in human kidney biopsy samples. Transgenic mice that have podocyte-specific overexpression of sdk-1 develop gradually progressive heavy proteinuria and severe FSGS. We also show that sdk-1 associates with the slit diaphragm linker protein MAGI-1, which is already known to interact with several critical podocyte proteins including synaptopodin, α-actinin-4, nephrin, JAM4, and β-catenin. This interaction is mediated through a direct interaction between the carboxyl terminus of sdk-1 and specific PDZ domains of MAGI-1. In vitro expression of sdk-1 enables a dramatic recruitment of MAGI-1 to the cell membrane. Furthermore, a truncated version of sdk-1 that is unable to bind to MAGI-1 does not induce podocyte dysfunction when overexpressed. We conclude that the up-regulation of sdk-1 in podocytes is an important pathogenic factor in FSGS and that the mechanism involves disruption of the actin cytoskeleton possibly via alterations in MAGI-1 function.

Ligand-binding Domain Determines Endoplasmic Reticulum Exit of AMPA Receptors

AMPA receptors (AMPARs) are tetrameric ion channels that mediate rapid glutamate signaling in neurons and many non-neuronal cell types. Endoplasmic reticulum (ER) quality control mechanisms permit only correctly folded functional receptors to be delivered to the cell surface. We analyzed the biosynthetic maturation and transport of all 12 GluA1–4 subunit splice variants as homomeric receptors and observed robust isoform-dependent differences in ER exit competence and surface expression. In contrast to inefficient ER exit of both GluA3 splice forms and the flop variants of GluA1 and GluA4, prominent plasma membrane expression was observed for the other AMPAR isoforms. Surprisingly, deletion of the entire N-terminal domain did not alter the transport phenotype, nor did the different cytosolic C-terminal tail splice variants. Detailed analysis of mutant receptors led to the identification of distinct residues in the ligand-binding domain as primary determinants for isoform-specific maturation. Considered together with the essential role of bound agonist, our findings reveal the ligand-binding domain as the critical quality control target in AMPAR biogenesis.

Ethanol increases desensitization of recombinant GluR-D AMPA receptor and TARP combinations

Glutamate receptors are important target molecules of the acute effect of ethanol. We studied ethanol sensitivity of homomeric GluR-D receptors expressed in human embryonic kidney 293 cells and examined whether recently discovered transmembrane alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory proteins (TARPs) affect ethanol sensitivity. Coexpression of the TARPs, stargazin, and gamma4 increased the time constant (tau-value) of current decay in the presence of agonist, thus slowing the onset of desensitization and increasing the steady-state current. Ethanol produced less inhibition of the peak current than the steady-state current for all types of the GluR-D receptors. In addition, ethanol concentration-dependently accelerated the rate of desensitization, measured as the tau-value of fast decay of peak current. This effect was enhanced with coexpression of TARPs. The recovery from desensitization was slowed down by coexpression of gamma4 but ethanol did not affect this process in any GluR-D combination. The results support the idea that increased desensitization is an important mechanism in the ethanol inhibition of AMPA receptors and indicate that coexpression of TARPs can alter this effect of ethanol.

Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain

The ligand‐binding domains of AMPA receptor subunits carry two conserved N‐glycosylation sites. In order to gain insight into the functional role of the corresponding N‐glycans, we examined how the elimination of glycosylation at these sites (N407 and N414) affects the ligand‐binding characteristics, structural stability, cell‐surface expression, and channel properties of homomeric GluR‐D (GluR4) receptor and its soluble ligand‐binding domain (S1S2). GluR‐D S1S2 protein expressed as a secreted protein in insect cells was found to be glycosylated at N407 and N414. No major differences in the ligand‐binding properties were observed between the ‘wild‐type’ S1S2 and non‐glycosylated N407D/N414Q double mutant, or between S1S2 proteins expressed in the presence or absence of tunicamycin, an inhibitor of N‐glycosylation. Purified glycosylated and non‐glycosylated S1S2 proteins also showed similar thermostabilities as determined by CD spectroscopy. Full‐length homomeric GluR‐D receptor with N407D/N414Q mutation was expressed on the surface of HEK293 cells like the wild‐type GluR‐D. In outside‐out patches, GluR‐D and the N407D/N414Q mutant produced similar rapidly desensitizing current responses to glutamate and AMPA. We therefore report that the two conserved ligand‐binding domain glycans do not play any major role in receptor–ligand interactions, do not impart a stabilizing effect on the ligand‐binding domain, and are not critical for the formation and surface localization of homomeric GluR‐D AMPA receptors in HEK293 cells.