Heparin modulates the single channel kinetics of reconstituted AMPA receptors from rat brain (original) (raw)
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Effects of heparin on the properties of solubilized and reconstituted rat brain AMPA receptors
Neuroscience Letters, 1996
Heparin was found to bind to a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors and to alter their functional properties. AMPA receptors solubilized in 0.4% Triton X-100 bound to a heparin-agarose column and were eluted by 0.4 M NaCl. Soluble heparin inhibited 10 nM [ 3 H]AMPA binding to detergent-solubilized receptors by 75% (IC 50 = 10 mg/ml), but had little effect on binding to membrane-associated receptors. The inhibition of [ 3 H]AMPA binding to detergent-solubilized receptors was not observed when binding was measured in the presence of 0.4 M NaCl, and no effect of heparin was observed on binding of the AMPA receptor antagonist [ 3 H]6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Scatchard analyses of [ 3 H]AMPA binding to solubilized receptors revealed that the inhibition induced by heparin was caused by a decrease in the apparent affinity of a portion of the total binding sites. Studies on AMPA receptors reconstituted in artificial lipid bilayers indicated that 10 mg/ml heparin enhanced cooperativity between channels and prolonged the lifetime of the open channel, but did not affect the amplitude of single channel currents. Thus, heparin may be added to the list of compounds known to modulate AMPA receptor function. These data also raise the possibility that heparin-containing proteoglycans, which are known to be concentrated at synaptic junctions, might be able to bind AMPA receptors and influence their functional characteristics.
Experimental neurology, 2015
Our previous studies revealed that L-type voltage-dependent Ca(2+) channels (Cav1.2 L-VDCCs) are modulated by the neural extracellular matrix backbone, polyanionic glycan hyaluronic acid. Here we used isothermal titration calorimetry and screened a set of peptides derived from the extracellular domains of Cav1.2α1 to identify putative binding sites between the channel and hyaluronic acid or another class of polyanionic glycans, such as heparin/heparan sulfates. None of the tested peptides showed detectable interaction with hyaluronic acid, but two peptides derived from the first pore-forming domain of Cav1.2α1 subunit bound to heparin. At 25°C the binding of the peptide P7 (MGKMHKTCYN) was at ~50μM, and that of the peptide P8 (GHGRQCQNGTVCKPGWDGPKHG) was at ~21μM. The Cav1.2α1 first pore forming segment that contained both peptides maintained a high affinity for heparin (~23μM), integrating their enthalpic and entropic binding contributions. Interaction between heparin and recombina...
AMPA RECEPTOR: A REVIEW Review Article
2012
α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors (AMPARs) are of fundamental importance in the brain. They are responsible for the majority of fast excitatory synaptic transmission, and their overactivation is potently excitotoxic. Recent findings have implicated AMPA receptors in synapse formation and stabilization, and regulation of functional AMPA receptors is the principal mechanism underlying synaptic plasticity. Changes in AMPA receptor activity have been described in the pathology of numerous diseases, such as Alzheimer’s disease, stroke, and epilepsy. Unsurprisingly, the developmental and activity-dependent changes in the functional synaptic expression of these receptors are under tight cellular regulation. The molecular and cellular mechanisms that control the postsynaptic insertion, arrangement, and lifetime of surface-expressed AMPARs are the subject of intense and widespread investigation. For example, there has been an explosion of information about proteins t...
Neuroscience Letters, 2000
In order to investigate the topography of the channel binding site in (^)-a-amino-3-hydroxy-5-methylisoxazole-4propionic acid (AMPA) and N-methyl-d-aspartate (NMDA) types of glutamate receptors, we have compared the blocking actions of mono-and dicationic derivatives of adamantane and phenylcyclohexyl. The potencies of dicationic derivatives to block AMPA receptor channels are about 1000 times higher than those of monocationic ones, whereas NMDA receptors are equally sensitive to both mono-and dicationic derivatives. The dependence of the activity of dicationic compounds on the length of the polymethylene chain between ammonium groups has a pronounced maximum for AMPA receptor channel block. For NMDA receptor channel dicationic compounds with various internitrogen distances produce similar blocking effects. The results show that hydrophobic and nucleophilic components of the binding site are located close to each other in the NMDA receptor channel but are separated by ,10 A Ê in the AMPA receptor channel. q
Regulated delivery of AMPA receptor subunits to the presynaptic membrane
The EMBO Journal, 2003
In recent years, a role for AMPA receptors as modulators of presynaptic functions has emerged. We have investigated the presence of AMPA receptor subunits and the possible dynamic control of their surface exposure at the presynaptic membrane. We demonstrate that the AMPA receptor subunits GluR1 and GluR2 are expressed and organized in functional receptors in axonal growth cones of hippocampal neurons. AMPA receptors are actively internalized upon activation and recruited to the surface upon depolarization. Pretreatment of cultures with botulinum toxin E or tetanus toxin prevents the receptor insertion into the plasma membrane, whereas treatment with a-latrotoxin enhances the surface exposure of GluR2, both in growth cones of cultured neurons and in brain synaptosomes. Puri®cation of small synaptic vesicles through controlled-pore glass chromatography, revealed that both GluR2 and GluR1, but not the GluR2 interacting protein GRIP, copurify with synaptic vesicles. These data indicate that, at steady state, a major pool of AMPA receptor subunits reside in synaptic vesicle membranes and can be recruited to the presynaptic membrane as functional receptors in response to depolarization.
Journal of Neurochemistry, 2005
The effects of divalent cations on Ca 2+ -impermeable containing (GluR2 subunit) MPA receptors of hippocampal pyramidal neurones isolated from rat brain was studied using patch-clamping. Ca 2+ , Mg 2+ , Mn 2+ , Co 2+ , Ni 2+ and Zn 2+ inhibited currents induced by kainate and glutamate. Inhibition was fast, reversible and voltage independent. The rank order of activities was Ni 2+ > Zn 2+ > Co 2+ > Ca 2+ > Mn 2+ > Mg 2+ . Cyclothiazide (0.1 mM) significantly reduced inhibition by divalent cations and 6, 7 dinitroquinoxaline-2.3-dione (DNQX). However, high concentrations of Ni 2+ and DNQX inhibited AMPA receptors even in the presence of cyclothiazide. The inhibitory effect of divalent cations as well as DNQX was counteracted by an increase in agonist concentration. In the presence of divalent cations the EC 50 values of kainate and glutamate were increased, but the maximal response was not changed. An increase in agonist concentration induced a parallel shift in the concentration-inhibition curve for a divalent cation. These data suggest a competitive-like type of inhibition. However, an increase in agonist concentration reduced the inhibitory action of Ni 2+ less than that of DNQX. This gave evidence against direct competition between divalent cations and AMPA receptor agonists. A 'complex-competition' hypothesis was proposed to explain the inhibitory action of divalent cations; it is suggested that divalent cations form ion-agonist complexes, which compete with free agonist for agonistbinding sites on AMPA receptors.
Characterization of the functional role of the N-glycans in the AMPA receptor ligand-binding domain
Journal of Neurochemistry, 2003
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 ligandbinding 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.
Neurochemistry International, 1986
This study describes the specificity, time-course and characteristics of the solubilization of class I-A forms of AChE by heparin, from the endplate regions of rat diaphragm muscle. Heparin fractions which differed in size charge, anticoagulant activity and capacity to bind type I collagen, were probed in their ability to extract ACHE. No differences were found among all the fractions tested. Affinity chromatography on heparin-agarose of class I-and class II-A forms of esterase showed that both classes were able to bind to the column with the same relative affinity. Our results establish the use of heparin, as a solubilizing agent for the class I-A. The existence of a heparin-binding domain in class I-and class II-A forms of ACHE, opens the possibility, that heparan sulfate proteoglycans could be involved in the anchorage of both types of esterase to synaptic regions. Finally, our results suggest that class I and class II-A do not correspond to intrinsically distinct molecules, but rather to identical molecules engaged in different interactions in the tissue.
Journal of Neurochemistry, 2002
Quantitative s-[3H]amino-3-hydroxy-5-methyl-tors of fast excitatory synaptic transmission in mammaisoxazole-4-propionic acid ([3H]AMPA) binding autoradiog-han brain. AMPA receptors have been found colocalraphy was performed on frozen-thawed sections from rat ized with NMDA (Bekkers and Stevens, 1989), and brain after preincubation at 0 or 35°Cfor 1 h. Preincubation glutamate metabotropic receptors (Baude et al., 1993), at 35°Cinstead of 0°Cresulted in a selective decrease of and modulation of AMPA receptors is generally as-[3H]AMPAbinding assayed at a low concentration of [3H}-sumed to be responsible for producing long-lasting AMPA (50 nM) and an enhancement of binding at a high concentration (500 nM). The decrease in [3H]AMPA bind-changes in excitatory synaptic transmission as a result of NMDA and/or glutamate metabotropic receptor acing after preincubation at 35°Cwas accompanied with the loss of the lighter organelles of P3 (microsomal) fractions.
Voltage‐dependent block of native AMPA receptor channels by dicationic compounds
British Journal of Pharmacology, 2000
The kinetics of open channel block of GluR2‐containing and GluR2‐lacking AMPA receptors (AMPAR) by dicationic compounds (IEM‐1460, IEM‐1754, and IEM‐1925) have been studied in rat hippocampal neurones using whole‐cell patch clamp recording and concentration‐jump techniques. Neurones were isolated from hippocampal slices by vibrodissociation. The dicationic compounds were approximately 100–200 times more potent as blockers of GluR2‐lacking AMPAR than as blockers of GluR2‐containing AMPAR. The subunit specificity of channel block is determined by the blocking rate constant of a dicationic compound, whereas differences in unblocking rate constants account for differences in potency. Hyperpolarization may decrease the block produced by IEM‐1460 and IEM‐1754 block due to the voltage‐dependence of the unblocking rate constants for these compounds. This suggests that dicationic compounds permeate the AMPAR channel at negative membrane potentials. The effect was particularly apparent for Gl...