A novel function for the ER retention signals in the C-terminus of kainate receptor subunit,GluK5

Classically, endoplasmic reticulum (ER) retention signals in secreted integral membrane proteins impose the requirement to assemble with other cognate subunits to form functional assemblies before they can exit the ER. We report that GluK5 has two ER retention signals in its cytoplasmic C-terminus: an arginine-based signal and a di-leucine motif previously thought to be an endocytic motif. GluK5 assembles with GluK2, but surprisingly GluK2 association does little to block the ER retention signals. We find instead that the ER retention signals are blocked by two proteins involved in intracellular trafficking, SAP97 and CASK. We show that SAP97, in the presence of CASK and the receptor complex, assumes an extended conformation. In the extended conformation, SAP97 makes its SH3 and GuK domains available to bind and sterically mask the ER retention signals in the GluK5 C-terminus. SAP97 and CASK are also necessary for sorting receptor cargoes into the local dendritic secretory pathway in neurons. We show that the ER retention signals of GluK5 play a vital role in sorting the receptor complex in the local dendritic secretory pathway in neurons. These data suggest a new role for ER retention signals in trafficking integral membrane proteins in neurons.SignificanceWe present evidence that the ER retention signals in the kainate receptors containing GluK5 impose a requirement for sorting into local dendritic secretory pathways in neurons, as opposed to traversing the somatic Golgi apparatus. There are two ER retention signals in the C-terminus of GluK5. We show that both are blocked by physical association with SAP97 and CASK. The SH3 and GuK domains of SAP97, in the presence of CASK, bind directly to each ER retention signal and form a complex. These results support an entirely new function for ER retention signals in the C-termini of neuronal receptors, such as NMDA and kainate receptors, and define a mechanism for selective entry of receptors into local secretory pathways.     

Agonist binding to the GluK5 subunit is sufficient for functional surface expression of heteromeric GluK2/GluK5 kainate receptors

Trafficking of ionotropic glutamate receptors to the plasma membrane commonly requires occupation of the agonist binding sites. This quality control check does not typically involve receptor activation, as binding by competitive antagonists or to non-functional channels may also permit surface expression. The tetrameric kainate receptors can be assembled from five different subunits (GluK1–GluK5). While the “low-affinity” GluK1-3 subunits are able to produce functional homomeric receptors, the “high-affinity” GluK4 and GluK5 subunits require co-assembly with GluK1, 2, or 3 for surface expression. These two different types of subunits have distinct functional roles in the receptor. Therefore, we examined the relative importance of occupancy of the agonist site of the GluK2 or GluK5 subunit for surface expression of heteromeric receptors. We created subunits with a mutation within the S2 ligand-binding domain which decreased agonist affinity. Mutations at this site reduced functional surface expression of homomeric GluK2 receptors, but surface expression of these receptors could be increased with either a competitive antagonist or co-assembly with wild-type GluK5. In contrast, mutations in the GluK5 subunit reduced the production of functional heteromeric receptors at the membrane, and could not be rescued with either an antagonist or wild-type GluK2. These findings indicate that ligand binding to only the GluK5 subunit is both necessary and sufficient to allow trafficking of recombinant GluK2/K5 heteromers to the cell membrane, but that occupancy of the GluK2 site alone is not. Our results suggest a distinct role for the GluK5 subunit in regulating surface expression of heteromeric kainate receptors.     

Crystal structures of the glutamate receptor ion channel GluK3 and GluK5 amino-terminal domains

Ionotropic glutamate receptors (iGluRs) mediate the majority of fast excitatory synaptic neurotransmission in the central nervous system. The selective assembly of iGluRs into AMPA, kainate, and N-methyl-d-aspartic acid (NMDA) receptor subtypes is regulated by their extracellular amino-terminal domains (ATDs). Kainate receptors are further classified into low-affinity receptor families (GluK1-GluK3) and high-affinity receptor families (GluK4-GluK5) based on their affinity for the neurotoxin kainic acid. These two families share a 42% sequence identity for the intact receptor but only a 27% sequence identity at the level of ATD. We have determined for the first time the high-resolution crystal structures of GluK3 and GluK5 ATDs, both of which crystallize as dimers but with a strikingly different dimer assembly at the R1 interface. By contrast, for both GluK3 and GluK5, the R2 domain dimer assembly is similar to those reported previously for other non-NMDA iGluRs. This observation is consistent with the reports that GluK4-GluK5 cannot form functional homomeric ion channels and require obligate coassembly with GluK1-GluK3. Our analysis also reveals that the relative orientation of domains R1 and R2 in individual non-NMDA receptor ATDs varies by up to 10°, in contrast to the 50° difference reported for the NMDA receptor GluN2B subunit. This restricted domain movement in non-NMDA receptor ATDs seems to result both from extensive intramolecular contacts between domain R1 and domain R2 and from their assembly as dimers, which interact at both R1 and R2 domains. Our results provide the first insights into the structure and function of GluK4-GluK5, the least understood family of iGluRs.     

Kainate Receptor Post-translational Modifications Differentially Regulate Association with 4.1N to Control Activity-dependent Receptor Endocytosis

Kainate receptors exhibit a highly compartmentalized distribution within the brain; however, the molecular and cellular mechanisms that coordinate their expression at neuronal sites of action are poorly characterized. Here we report that the GluK1 and GluK2 kainate receptor subunits interact with the spectrin-actin binding scaffolding protein 4.1N through a membrane-proximal domain in the C-terminal tail. We found that this interaction is important for the forward trafficking of GluK2a receptors, their distribution in the neuronal plasma membrane, and regulation of receptor endocytosis. The association between GluK2a receptors and 4.1N was regulated by both palmitoylation and protein kinase C (PKC) phosphorylation of the receptor subunit. Palmitoylation of the GluK2a subunit promoted 4.1N association, and palmitoylation-deficient receptors exhibited reduced neuronal surface expression and compromised endocytosis. Conversely, PKC activation decreased 4.1N interaction with GluK2/3-containing kainate receptors in acute brain slices, an effect that was reversed after inhibition of PKC. Our data and previous studies therefore demonstrate that these two post-translational modifications have opposing effects on 4.1N association with GluK2 kainate and GluA1 AMPA receptors. The convergence of the signaling pathways regulating 4.1N protein association could thus result in the selective removal of AMPA receptors from the plasma membrane while simultaneously promoting the insertion and stabilization of kainate receptors, which may be important for tuning neuronal excitability and synaptic plasticity.     

Kainate induces various domain closures in AMPA and kainate receptors

Ionotropic glutamate receptors are key players in fast excitatory synaptic transmission within the central nervous system. These receptors have been divided into three subfamilies: the N-methyl-d-aspartic acid (NMDA), 2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA) and kainate receptors. Kainate has previously been crystallized with the ligand binding domain (LBD) of AMPA receptors (GluA2 and GluA4) and kainate receptors (GluK1 and GluK2). Here, we report the structures of the kainate receptor GluK3 LBD in complex with kainate and GluK1 LBD in complex with kainate in the absence of glycerol. Kainate introduces a conformational change in GluK3 LBD comparable to that of GluK2, but different from the conformational changes induced in GluA2 and GluK1. Compared to their domain closures in a glutamate bound state, GluA2 and GluK1 become more open and kainate induces a domain closure of 60% and 62%, respectively, relative to glutamate (100%). In GluK2 and GluK3 with kainate, the domain closure is 88% and 83%, respectively. In previously determined structures of GluK1 LBD in complex with kainate, glycerol is present in the binding site where it bridges interlobe residues and thus, might contribute to the large domain opening. However, the structure of GluK1 LBD with kainate in the absence of glycerol confirms that the observed domain closure is not an artifact of crystallization conditions. Comparison of the LBD structures with glutamate and kainate reveals that contacts are lost upon binding of kainate in the three kainate receptors, which is in contrast to the AMPA receptors where similar contacts are seen. It was revealed by patch clamp electrophysiology studies that kainate is a partial agonist at GluK1 with 36% efficacy compared to glutamate, which is in between the published efficacies of kainate at GluK2 and AMPA receptors. The ranking of efficacies seems to correlate with LBD domain closures.     

Ligand binding is a critical requirement for plasma membrane expression of heteromeric kainate receptors

Intracellular trafficking of ionotropic glutamate receptors is controlled by multiple discrete determinants in receptor subunits. Most such determinants have been localized to the cytoplasmic carboxyl-terminal domain, but other domains in the subunit proteins can play roles in modulating receptor surface expression. Here we demonstrate that formation of an intact glutamate binding site also acts as an additional quality-control check for surface expression of homomeric and heteromeric kainate receptors. A key ligand-binding residue in the KA2 subunit, threonine 675, was mutated to either alanine or glutamate, which eliminated affinity for the receptor ligands kainate and glutamate. We found that plasma membrane expression of heteromeric GluR6/KA2(T675A) or GluR6/KA2(T675E) kainate receptors was markedly reduced compared with wild-type GluR6/KA2 receptors in transfected HEK 293 and COS-7 cells and in cultured neurons. Surface expression of homomeric KA2 receptors lacking a retention/retrieval determinant (KA2-R/A) was also reduced upon mutation of Thr-675 and elimination of the ligand binding site. KA2 Thr-675 mutant subunits were able to co-assemble with GluR5 and GluR6 subunits and were degraded at the same rate as wild-type KA2 subunit protein. These results suggest that glutamate binding and associated conformational changes are prerequisites for forward trafficking of intracellular kainate receptors following multimeric assembly.     

P2X7 receptor cell surface expression and cytolytic pore formation are regulated by a distal C-terminal region

The importance of the cytosolic C-terminal region of the P2X7 receptor (P2X7R) is unquestioned, yet little is known about the functional domains of this region and how they may contribute to the numerous properties ascribed to this receptor. A structure-function analysis of truncated and single-residue-mutated P2X7 receptors was performed in HEK-293 cells and Xenopus oocytes. Cells expressing receptors truncated at residue 581 (of 595) have negligible ethidium ion uptake, whereas those expressing the P2X7R truncated at position 582 give wild type ethidium ion uptake suggesting that pore formation requires over 95% of the C-terminal tail. Channel function was evident even in receptors that were truncated at position 380 indicating that only a small portion of the cytosolic region is required for channel activity. Surprisingly, truncations in the region between residues 551 and 581 resulted in non-functional receptors with no detectable cell surface expression in HEK-293 cells. A more detailed analysis revealed that mutations of single residues within this region could also abolish receptor function and cell surface expression, suggesting that this region may participate in regulating the surface expression of the pore-forming P2X7R.     

Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles

RNA editing at the Q/R site near the apex of the pore loop of AMPA and kainate receptors controls a diverse array of channel properties, including ion selectivity and unitary conductance and susceptibility to inhibition by polyamines and cis-unsaturated fatty acids, as well as subunit assembly into tetramers and regulation by auxiliary subunits. How these different aspects of channel function are all determined by a single amino acid substitution remains poorly understood; however, several lines of evidence suggest that interaction between the pore helix (M2) and adjacent segments of the transmembrane inner (M3) and outer (M1) helices may be involved. In the present study, we have used double mutant cycle analysis to test for energetic coupling between the Q/R site residue and amino acid side chains along the M3 helix. Our results demonstrate interaction with several M3 locations and particularly strong coupling to substitution for L614 at the level of the central cavity. In this location, replacement with smaller side chains completely and selectively reverses the effect of fatty acids on gating of edited channels, converting strong inhibition of wild-type GluK2(R) to nearly 10-fold potentiation of GluK2(R) L614A.     

Assembly and Trafficking of Homomeric and Heteromeric Kainate Receptors with Impaired Ligand Binding Sites

Kainate receptors (KARs) are a subfamily of ionotropic glutamate receptors (iGluRs) mediating excitatory synaptic transmission. Cell surface expressed KARs modulate the excitability of neuronal networks. The transfer of iGluRs from the endoplasmic reticulum (ER) to the cell surface requires occupation of the agonist binding sites. Here we used molecular modelling to produce a range of ligand binding domain (LBD) point mutants of GluK1–3 KAR subunits with and without altered agonist efficacy to further investigate the role of glutamate binding in surface trafficking and activation of homomeric and heteromeric KARs using endoglycosidase digestion, cell surface biotinylation and imaging of changes in intracellular Ca²⁺ concentration [Ca²⁺]_i. Mutations of conserved amino acid residues in the LBD that disrupt agonist binding to GluK1–3 (GluK1-T675V, GluK2-A487L, GluK2-T659V and GluK3-T661V) reduced both the total expression levels and cell surface delivery of all of these mutant subunits compared to the corresponding wild type in transiently transfected human embryonic kidney 293 (HEK293) cells. In contrast, the exchange of non-conserved residues in the LBD that convert antagonist selectivity of GluK1–3 (GluK1-T503A, GluK2-A487T, GluK3-T489A, GluK1-N705S/S706N, GluK2-S689N/N690S, GluK3-N691S) did not alter the biosynthesis and trafficking of subunit proteins. Co-assembly of mutant GluK2 with an impaired LBD and wild type GluK5 subunits enables the cell surface expression of both subunits. However, [Ca²⁺]_i imaging indicates that the occupancy of both GluK2 and GluK5 LBDs is required for the full activation of GluK2/GluK5 heteromeric KAR channels.

     

Conserved ectodomain cysteines are essential for rat P2X7 receptor trafficking

The P2X7 receptor (P2X7R) is a member of the ATP-gated ion channel family that exhibits distinct electrophysiological and pharmacological properties. This includes low sensitivity to ATP, lack of desensitization, a sustained current growth during prolonged receptor stimulation accompanied with development of permeability to large organic cations, and the coupling of receptor activation to cell blebbing and death. The uniquely long C-terminus of P2X7R accounts for many of these receptor-specific functions. The aim of this study was to understand the role of conserved ectodomain cysteine residues in P2X7R function. Single- and double-point threonine mutants of C119-C168, C129-C152, C135-C162, C216-C226, and C260-C269 cysteine pairs were expressed in HEK293 cells and studied using whole-cell current recording. All mutants other than C119T-P2X7R responded to initial and subsequent application of 300-μM BzATP and ATP with small amplitude monophasic currents or were practically nonfunctional. The mutagenesis-induced loss of function was due to decreased cell-surface receptor expression, as revealed by assessing levels of biotinylated mutants. Coexpression of all double mutants with the wild-type receptor had a transient or, in the case of C119T/C168T double mutant, sustained inhibitory effect on receptor trafficking. The C119T-P2X7R mutant was expressed on the plasma membrane and was fully functional with a slight decrease in the sensitivity for BzATP, indicating that interaction of liberated Cys168 with another residue rescues the trafficking of receptor. Thus, in contrast to other P2XRs, all disulfide bonds of P2X7R are individually essential for the proper receptor trafficking.     

Cleavage of Notch1 by granzyme B disables its transcriptional activity

AE1 (anion exchanger 1) and protein 4.2 associate in a protein complex bridging the erythrocyte membrane and cytoskeleton; disruption of the complex results in unstable erythrocytes and HS (hereditary spherocytosis). Three HS mutations (E40K, G130R and P327R) in cdAE1 (the cytoplasmic domain of AE1) occur with deficiencies of protein 4.2. The interaction of wild-type AE1, AE1HS mutants, mdEA1 (the membrane domain of AE1), kAE1 (the kidney isoform of AE1) and AE1SAO (Southeast Asian ovalocytosis AE1) with protein 4.2 was examined in transfected HEK (human embryonic kidney)-293 cells. The HS mutants had wild-type expression levels and plasma-membrane localization. Protein 4.2 expression was not dependent on AE1. Protein 4.2 was localized throughout the cytoplasm and co-localized at the plasma membrane with the HS mutants mdAE1 and kAE1, but at the ER (endoplasmic reticulum) with AE1SAO. Pull-down assays revealed diminished levels of protein 4.2 associated with the HS mutants relative to AE1. The mdAE1 did not bind protein 4.2, whereas kAE1 and AE1SAO bound wild-type amounts of protein 4.2. A protein 4.2 fatty acylation mutant, G2A/C173A, had decreased plasma-membrane localization compared with wild-type protein 4.2, and co-expression with AE1 enhanced its plasma-membrane localization. Subcellular fractionation showed the majority of wild-type and G2A/C173A protein 4.2 was associated with the cytoskeleton of HEK-293 cells. The present study shows that cytoplasmic HS mutants cause impaired binding of protein 4.2 to AE1, leaving protein 4.2 susceptible to loss during erythrocyte development.     

Intramembrane Charge Movement Associated with Endogenous K+ Channel Activity in HEK-293 Cells

1. The use of molecular biology in combination with electrophysiology in the HEK-293 cell line has given fascinating insights into neuronal ion channel function. Nevertheless, to fully understand the properties of channels exogenously expressed in these cells, a detailed evaluation of endogenous channels is indispensable. 2. Previous studies have shown the expression of endogenous voltage-gated K+, Ca2+, and Cl- channels and this predicts that changes in membrane potential will cause intramembrane charge movement, though this gating charge translocation remain undefined. Here, we confirm this prediction by performing patch-clamp experiments to record ionic and gating currents. Our data show that HEK-293 cells express at least two types of K+-selective endogenous channels which sustain the majority of the ionic current, and exclude a significant contribution from Ca2+ and Cl- channels to the whole-cell current. 3. Gating currents were unambiguously resolved after ionic current blockade enabling this first report of intramembrane charge movement in HEK-293 cells arising entirely from endogenous K+ channel activity, and providing valuable information concerning the activation mechanism of voltage-gated K+ channels in these cells.     

Profilin II regulates the exocytosis of kainate glutamate receptors

The trafficking of ionotropic glutamate receptors to and from synaptic sites is regulated by proteins that interact with their cytoplasmic C-terminal domain. Profilin IIa (PfnIIa), an actin-binding protein expressed in the brain and recruited to synapses in an activity-dependent manner, was shown previously to interact with the C-terminal domain of the GluK2b subunit splice variant of kainate receptors (KARs). Here, we characterize this interaction and examine the role of PfnIIa in the regulation of KAR trafficking. PfnIIa directly and specifically binds to the C-terminal domain of GluK2b through a diproline motif. Expression of PfnIIa in transfected COS-7 cells and in cultured hippocampal neurons from PfnII-deficient mice decreases the level of extracellular of homomeric GluK2b as well as heteromeric GluK2a/GluK2b KARs. Our data suggest a novel mechanism by which PfnIIa exerts a dual role on the trafficking of KARs, by a generic inhibition of clathrin-mediated endocytosis through its interaction with dynamin-1, and by controlling KARs exocytosis through a direct and specific interaction with GluK2b.     

N-glycosylation determines ionic permeability and desensitization of the TRPV1 capsaicin receptor

The balance of glycosylation and deglycosylation of ion channels can markedly influence their function and regulation. However, the functional importance of glycosylation of the TRPV1 receptor, a key sensor of pain-sensing nerves, is not well understood, and whether TRPV1 is glycosylated in neurons is unclear. We report that TRPV1 is N-glycosylated and that N-glycosylation is a major determinant of capsaicin-evoked desensitization and ionic permeability. Both N-glycosylated and unglycosylated TRPV1 was detected in extracts of peripheral sensory nerves by Western blotting. TRPV1 expressed in HEK-293 cells exhibited various degrees of glycosylation. A mutant of asparagine 604 (N604T) was not glycosylated but did not alter plasma membrane expression of TRPV1. Capsaicin-evoked increases in intracellular calcium ([Ca(2+)](i)) were sustained in wild-type TRPV1 HEK-293 cells but were rapidly desensitized in N604T TRPV1 cells. There was marked cell-to-cell variability in capsaicin responses and desensitization between individual cells expressing wild-type TRPV1 but highly uniform responses in cells expressing N604T TRPV1, consistent with variable levels of glycosylation of the wild-type channel. These differences were also apparent when wild-type or N604T TRPV1-GFP fusion proteins were expressed in neurons from trpv1(-/-) mice. Capsaicin evoked a marked, concentration-dependent increase in uptake of the large cationic dye YO-PRO-1 in cells expressing wild-type TRPV1, indicative of loss of ion selectivity, that was completely absent in cells expressing N604T TRPV1. Thus, TRPV1 is variably N-glycosylated and glycosylation is a key determinant of capsaicin regulation of TRPV1 desensitization and permeability. Our findings suggest that physiological or pathological alterations in TRPV1 glycosylation would affect TRPV1 function and pain transmission.     

Complex formation with the Type B gamma-aminobutyric acid receptor affects the expression and signal transduction of the extracellular calcium-sensing receptor. Studies with HEK-293 cells and neurons

We co-immunoprecipitated the Ca(2+)-sensing receptor (CaR) and type B gamma-aminobutyric acid receptor (GABA-B-R) from human embryonic kidney (HEK)-293 cells expressing these receptors and from brain lysates where both receptors are present. CaRs extensively co-localized with the two subunits of the GABA-B-R (R1 and R2) in HEK-293 cell membranes and intracellular organelles. Coexpressing CaRs and GABA-B-R1s in HEK-293 cells suppressed the total cellular and cell surface expression of CaRs and inhibited phospholipase C activation in response to high extracellular [Ca(2+)] ([Ca(2+)](e)). In contrast, coexpressing CaRs and GABA-B-R2s enhanced CaR expression and signaling responses to raising [Ca(2+)](e). The latter effects of the GABA-B-R2 on the CaR were blunted by coexpressing the GABA-B-R1. Coexpressing the CaR with GABA-B-R1 or R2 enhanced the total cellular and cell surface expression of the GABA-B-R1 or R2, respectively. Studies with truncated CaRs indicated that the N-terminal extracellular domain of the CaR participated in the interaction of the CaR with the GABA-B-R1 and R2. In cultured mouse hippocampal neurons, CaRs co-localized with the GABA-B-R1 and R2. CaRs and GABA-B-R1s also co-immunoprecipitated from brain lysates. The expression of the CaR was increased in lysates from GABA-B-R1 knock-out mouse brains and in cultured hippocampal neurons with their GABA-B-R1 genes deleted in vitro. Thus, CaRs and GABA-B-R subunits can form heteromeric complexes in cells, and their interactions affect cell surface expression and signaling of CaR, which may contribute to extracellular Ca(2+)-dependent receptor activation in target tissues.     

PKC SUMOylation inhibits the binding of 14–3–3τ to GluK2

Phosphorylation and SUMOylation of the kainate receptor (KAR) subunit GluK2 have been shown to regulate KAR surface expression, trafficking and synaptic plasticity. In addition, our previous study has shown that a phosphorylation-dependent interaction of 14–3–3τ and GluK2a-containing receptors contributes to the slow decay kinetics of native KAR-EPSCs. However, it is unknown whether SUMOylation participates in the regulation of the interaction between 14–3–3τ and GluK2a-containing receptors. Here we report that SUMOylation of PKC, but not GluK2, represses the binding of 14–3–3τ to GluK2a via decreasing the phosphorylation level of GluK2a. These results suggest that PKC SUMOylation is an important regulator of the 14–3–3 and GluK2a protein complex and may contribute to regulate the decay kinetics of KAR-EPSCs.