Neural Cell Adhesion Molecule-associated Polysialic Acid Potentiates α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid Receptor Currents
2004; Elsevier BV; Volume: 279; Issue: 46 Linguagem: Inglês
10.1074/jbc.m407138200
ISSN1083-351X
AutoresThirumalini Vaithianathan, Katja Matthias, Ben A. Bahr, Melitta Schachner, Vishnu Suppiramaniam, Alexander Dityatev, Christian Steinhäuser,
Tópico(s)Receptor Mechanisms and Signaling
ResumoThe highly negatively charged polysialic acid (PSA) is a carbohydrate predominantly carried by the neural cell adhesion molecule (NCAM) in mammals. NCAM and, in particular, PSA play important roles in cellular and synaptic plasticity. Here we investigated whether PSA modulates the activity of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors (AMPA-Rs). Single channel recordings of affinity-purified AMPA-Rs reconstituted in lipid bilayers revealed that bacterially derived PSA, called colominic acid, prolonged the open channel time of AMPA-R-mediated currents by severalfold and altered the bursting pattern of the receptor channels but did not modify AMPA-R single channel conductance. This effect was reversible, concentration-dependent, and specific, since monomers of sialic acid and another negatively charged carbohydrate, chondroitin sulfate, did not potentiate single channel AMPA-R currents. Recombinant PSA-NCAM also potentiated currents mediated by reconstituted AMPA-Rs. In pyramidal neurons acutely isolated from the CA1 region of the early postnatal hippocampus, l-glutamate or AMPA (applied in the presence of antagonists blocking voltage-gated Na+ and K+ currents and N-methyl-d-aspartate and metabotropic glutamate receptors) induced inward currents, which were significantly increased by co-application of colominic acid. Chondroitin sulfate did not affect AMPA-R-mediated currents in CA1 neurons. The effect of colominic acid was age-dependent, since in pyramidal neurons from adult hippocampus, colominic acid failed to potentiate glutamate responses. Thus, our study demonstrates age-dependent potentiation of AMPA receptors by PSA via a mechanism probably involving direct PSA-AMPA-R interactions. This mechanism might amplify AMPA-R-mediated signaling in immature cells, thereby affecting their development. The highly negatively charged polysialic acid (PSA) is a carbohydrate predominantly carried by the neural cell adhesion molecule (NCAM) in mammals. NCAM and, in particular, PSA play important roles in cellular and synaptic plasticity. Here we investigated whether PSA modulates the activity of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) subtype of glutamate receptors (AMPA-Rs). Single channel recordings of affinity-purified AMPA-Rs reconstituted in lipid bilayers revealed that bacterially derived PSA, called colominic acid, prolonged the open channel time of AMPA-R-mediated currents by severalfold and altered the bursting pattern of the receptor channels but did not modify AMPA-R single channel conductance. This effect was reversible, concentration-dependent, and specific, since monomers of sialic acid and another negatively charged carbohydrate, chondroitin sulfate, did not potentiate single channel AMPA-R currents. Recombinant PSA-NCAM also potentiated currents mediated by reconstituted AMPA-Rs. In pyramidal neurons acutely isolated from the CA1 region of the early postnatal hippocampus, l-glutamate or AMPA (applied in the presence of antagonists blocking voltage-gated Na+ and K+ currents and N-methyl-d-aspartate and metabotropic glutamate receptors) induced inward currents, which were significantly increased by co-application of colominic acid. Chondroitin sulfate did not affect AMPA-R-mediated currents in CA1 neurons. The effect of colominic acid was age-dependent, since in pyramidal neurons from adult hippocampus, colominic acid failed to potentiate glutamate responses. Thus, our study demonstrates age-dependent potentiation of AMPA receptors by PSA via a mechanism probably involving direct PSA-AMPA-R interactions. This mechanism might amplify AMPA-R-mediated signaling in immature cells, thereby affecting their development. Glutamate receptors mediate excitatory synaptic transmission in the vertebrate central nervous system (1Barnard E.A. Trends Biochem. Sci. 1992; 17: 368-374Abstract Full Text PDF PubMed Scopus (146) Google Scholar, 2Sprengel R. Seeburg P.H. FEBS Lett. 1993; 325: 90-94Crossref PubMed Scopus (23) Google Scholar). According to their pharmacological properties and gene sequence homology, three subfamilies of ionotropic glutamate receptors have been distinguished: N-methyl-d-aspartate, kainate, and α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) 1The abbreviations used are: AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; AMPA-R, AMPA receptor; NCAM, neural cell adhesion molecule; PC, phosphatidylcholine; TTX, tetrodotoxin; ECF, pseudoextracellular fluid; ICF, pseudointracellular fluid; MOPS, 4-morpholinepropanesulfonic acid; pF, picofarads; pS, picosiemens; PSA, polysialic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; 4-AP, 4-aminopyridine; APV, DL-2-amino-5-phosphonopentanoic acid.1The abbreviations used are: AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; AMPA-R, AMPA receptor; NCAM, neural cell adhesion molecule; PC, phosphatidylcholine; TTX, tetrodotoxin; ECF, pseudoextracellular fluid; ICF, pseudointracellular fluid; MOPS, 4-morpholinepropanesulfonic acid; pF, picofarads; pS, picosiemens; PSA, polysialic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; 4-AP, 4-aminopyridine; APV, DL-2-amino-5-phosphonopentanoic acid. receptors (3Borges K. Dingledine R. Prog. Brain Res. 1998; 116: 153-170Crossref PubMed Google Scholar, 4Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar). AMPA receptors (AMPA-Rs) are composed of four subunits, GluR1 to -4, and exhibit diverse properties depending on subunit composition, RNA splicing and editing (4Dingledine R. Borges K. Bowie D. Traynelis S.F. Pharmacol. Rev. 1999; 51: 7-61PubMed Google Scholar, 5Sommer B. Keinanen K. Verdoorn T.A. Wisden W. Burnashev N. Herb A. Kohler M. Takagi T. Sakmann B. Seeburg P.H. Science. 1990; 249: 1580-1585Crossref PubMed Scopus (972) Google Scholar, 6Hollmann M. Heinemann S. Annu. Rev. Neurosci. 1994; 17: 31-108Crossref PubMed Scopus (3638) Google Scholar, 7Bettler B. Mulle C. Neuropharmacology. 1995; 34: 123-139Crossref PubMed Scopus (419) Google Scholar), and glycosylation (8Lis H. Sharon N. Eur. J. Biochem. 1993; 218: 1-27Crossref PubMed Scopus (790) Google Scholar, 9Kawamoto S. Hattori S. Sakimura K. Mishina M. Okuda K. J. Neurochem. 1995; 64: 1258-1266Crossref PubMed Scopus (31) Google Scholar, 10Standley S. Tocco G. Wagle N. Baudry M. J. Neurochem. 1998; 70: 2434-2445Crossref PubMed Scopus (43) Google Scholar, 11Standley S. Baudry M. Cell. Mol. Life Sci. 2000; 57: 1508-1516Crossref PubMed Scopus (37) Google Scholar). AMPA-Rs have received considerable attention due to their involvement in activity-dependent synaptic plasticity. During this process, insertion of new AMPA-Rs and their posttranslational modifications may go hand-in-hand with structural modifications of synapses (12Luscher C. Nicoll R.A. Malenka R.C. Muller D. Nat. Neurosci. 2000; 3: 545-550Crossref PubMed Scopus (522) Google Scholar). Here, we examined a possible link between AMPA-Rs and an interesting carbohydrate, polysialic acid, which is also involved in synaptic plasticity (13Kiss J.Z. Rougon G. Curr. Opin. Neurobiol. 1997; 7: 640-646Crossref PubMed Scopus (206) Google Scholar). PSA is a highly negatively charged homomeric polymer of sialic acid that can form an unusual α2,8-linkage in chains that can be up to 200 residues long. This polymer is predominantly carried by the neural cell adhesion molecule (NCAM) and modulates its functions during cell migration and axonal outgrowth (see Ref. 13Kiss J.Z. Rougon G. Curr. Opin. Neurobiol. 1997; 7: 640-646Crossref PubMed Scopus (206) Google Scholar for a review). Furthermore, delivery of PSA-NCAM to the cell surface in neurons and endocrine cells is activity-dependent (14Kiss J.Z. Wang C. Olive S. Rougon G. Lang J. Baetens D. Harry D. Pralong W.F. EMBO J. 1994; 13: 5284-5292Crossref PubMed Scopus (136) Google Scholar, 15Muller D. Wang C. Skibo G. Toni G. Cremer H. Calaora V. Rougon G. Kiss J.Z. Neuron. 1996; 17: 413-422Abstract Full Text Full Text PDF PubMed Scopus (525) Google Scholar). Both NCAM protein backbone and PSA are expressed pre- and postsynaptically in a subset of spine synapses, as visualized by immunoelectron microscopy (16Schuster T. Krug M. Stalder M. Hackel N. Gerardy-Schahn R. Schachner M. J. Neurobiol. 2001; 49: 142-158Crossref PubMed Scopus (81) Google Scholar). The extracellular domain of PSA-NCAM can be cleaved by the tissue plasminogen activator-plasmin system at the cell surface so that it becomes a soluble molecule (17Endo A. Nagai N. Urano T. Ihara H. Takada Y. Hashimoto K. Takada A. Neurosci. Lett. 1998; 246: 37-40Crossref PubMed Scopus (33) Google Scholar, 18Hoffman K.B. Larson J. Bahr B.A. Lynch G. Brain Res. 1998; 811: 152-155Crossref PubMed Scopus (37) Google Scholar). The concentration of soluble NCAM is increased 10-fold after induction of long term potentiation in the dentate gyrus (19Fazeli M.S. Breen K. Errington M.L. Bliss T.V. Neurosci. Lett. 1994; 169: 77-80Crossref PubMed Scopus (112) Google Scholar). Several other observations provide strong evidence that PSA is required for long term potentiation and depression in the CA1 region of the hippocampus (20Becker C.G. Artola A. Gerardy-Schahn R. Becker T. Welzl H. Schachner M. J. Neurosi. Res. 1996; 45: 143-152Crossref PubMed Scopus (274) Google Scholar, 21Schachner M. Curr. Opin. Cell Biol. 1997; 9: 627-634Crossref PubMed Scopus (249) Google Scholar, 22Eckhardt M. Bukalo O. Chazal G. Wang L. Goridis C. Schachner M. Gerardy-Schahn R. Cremer H. Dityatev A. J. Neurosci. 2000; 20: 5234-5244Crossref PubMed Google Scholar). The mechanisms underlying an increase in synaptic efficacy during long term potentiation in this region probably involve changes in the number and functional properties of AMPA-Rs (23Barria A. Muller D. Derkach V. Griffith L.C. Soderling T.R. Science. 1997; 276: 2042-2045Crossref PubMed Scopus (869) Google Scholar, 24Benke T.A. Luthi A. Isaac J.T. Collingridge G.L. Nature. 1998; 393: 793-797Crossref PubMed Scopus (422) Google Scholar, 25Shi S.H. Hayashi Y. Petralia R.S. Zaman S.H. Wenthold R.J. Svoboda K. Malinow R. Science. 1999; 284: 1811-1816Crossref PubMed Scopus (1045) Google Scholar, 26Malinow R. Malenka R.C. Annu. Rev. Neurosci. 2002; 25: 103-126Crossref PubMed Scopus (2029) Google Scholar). In this context, we set out to test the effects of bacterially derived PSA, colominic acid, on the activity of purified AMPA-Rs reconstituted in artificial lipid bilayers. This technique was used, since it allowed us to address the possibility of a direct modulation of AMPA receptors by PSA. Our single channel recordings in lipid bilayers demonstrate that colominic acid can dramatically prolong AMPA-R channel open time and increase its bursting activity. Colominic acid also increased AMPA-R currents in immature but not in mature CA1 pyramidal cells. Thus, our data reveal an age-dependent interaction between PSA and AMPA-Rs that may modulate neuronal transmission and plasticity in the developing central nervous system. Purification of AMPA Receptors—Membrane fractions were prepared according to methods described elsewhere (27Bahr B.A. Vodyanoy V. Hall R.A. Suppiramaniam V. Kessler M. Sumikawa K. Lynch G. J. Neurochem. 1992; 59: 1979-1982Crossref PubMed Scopus (14) Google Scholar). Brains from adult Sprague-Dawley rats were homogenized in 0.32 m sucrose, 5 mm HEPES, 0.1 mm EDTA, 20 μg/ml antipain, 1 μg/ml aprotinin, 1 μg/ml pepstatin A, 20 μg/ml N-tosyl-l-phenylalanine chloromethylketone (Sigma); 40 μg/ml calpain inhibitor I (Calbiochem); 1 mg/ml leupeptin (Chemicon, San Diego, CA); and 35 μg/ml phenylmethylsulfonyl fluoride (Chemicon; added fresh) (pH 7.4, 4 °C). The homogenate was centrifuged at 1,090 × g for 10 min, and the supernatant was centrifuged again at 14,600 × g for 20 min. The pellet was lysed in 10 mm Tris in the presence of the protease inhibitors described above, at pH 8.1, 4 °C, for 60 min and successively centrifuged at 11,400 × g with 10-min resuspension cycles. Final pellets were resuspended in buffer A (30 mm HEPES, 5 mm EDTA, 1 mm EGTA, 0.02% NaN3, pH 7.4). Membrane Solubilization—Lysed membranes (1.5 mg of protein/ml) were solubilized in ice-cold buffer A with 4% (w/v) n-octyl-α-d-glucopyranoside, 10% (w/v) glycerol, and 0.12% (w/v) phosphatidylcholine (PC). The suspension was homogenized in an etched glass Potter-Elvehjem tissue grinder for 30 s at high speed and then placed on ice for 1 h. After centrifugation at 50,400 × g for 40 min at 4 °C, the supernatant was stored at –80 °C. Receptor Purification—AMPA-Rs were partially purified by a sequence of chromatographic steps: DEAE anion exchange, wheat germ lectin affinity, and polyethyleneimine anion exchange as described below. Solubilized membranes were diluted with an equal volume of buffer A with 10% glycerol and applied to a DEAE-Sepharose column equilibrated at 4 °C with buffer A containing 1% n-octyl-α-d-glucopyranoside, 10% glycerol, and 0.05% PC (buffer B). After the column was washed with 3 column volumes of buffer B at a flow rate of 2 ml/min, AMPA-Rs were eluted with a 500-ml linear salt gradient from 0 to 2000 mm KSCN in buffer B. The AMPA-R fractions were applied at a 1 ml/min flow rate to a wheat germ lectin affinity column in buffer B containing 0.1 m NaCl. The column was washed, and AMPA-Rs were eluted with 0.7 mN-acetylglucosamine in buffer B. An aliquot of AMPA-Rs was injected into the HPLC polyethyleneimine column in buffer B with a flow rate of 1 ml/min. After wash with buffer B and a linear salt gradient from 0 to 350 mm KSCN, fractions were tested with anti-GluR1 immunoblotting (28Bahr B.A. Hoffman K.B. Kessler M. Hennegriff M. Park G.Y. Yamamoto R.S. Kawasaki B.T. Vanderklish P.W. Hall R.A. Lynch G. Neuroscience. 1996; 74: 707-721Crossref PubMed Scopus (29) Google Scholar), and the most GluR1-enriched fractions were pooled. Finally, equal volumes of buffer A were added to portions of each AMPA-R pool to dilute the n-octyl-α-d-glucopyranoside to 0.5%, and pure PC (1 mg/ml) and phosphatidylserine (50 μg/ml) were added. AMPA-Rs were immunoprecipitated from the solubilized membranes with anti-GluR1 antibodies, as previously described (28Bahr B.A. Hoffman K.B. Kessler M. Hennegriff M. Park G.Y. Yamamoto R.S. Kawasaki B.T. Vanderklish P.W. Hall R.A. Lynch G. Neuroscience. 1996; 74: 707-721Crossref PubMed Scopus (29) Google Scholar). Anti-GluR1 antibodies, covalently cross-linked to protein A-Sepharose CL-4B (Sigma), were incubated with solubilized membranes for 6–8 h at 4 °C. The immobilized receptors were washed thoroughly in 30 mm Tris, 0.5 m NaCl, 0.2% Triton X-100; extracted from the immunosupport with diethylamine/deoxycholate at pH 11.5; and immediately neutralized with 0.5 m Tris, pH 6. Purity of the preparation was verified by silver staining and Western blotting. The purified AMPA-Rs retained many of the properties of the receptors in their native membrane microenvironment (27Bahr B.A. Vodyanoy V. Hall R.A. Suppiramaniam V. Kessler M. Sumikawa K. Lynch G. J. Neurochem. 1992; 59: 1979-1982Crossref PubMed Scopus (14) Google Scholar, 28Bahr B.A. Hoffman K.B. Kessler M. Hennegriff M. Park G.Y. Yamamoto R.S. Kawasaki B.T. Vanderklish P.W. Hall R.A. Lynch G. Neuroscience. 1996; 74: 707-721Crossref PubMed Scopus (29) Google Scholar, 29Vodyanoy V. Bahr B.A. Suppiramaniam V. Hall R.A. Baudry M. Lynch G. Neurosci. Lett. 1993; 50: 80-84Crossref Scopus (16) Google Scholar, 30Suppiramaniam V. Bahr B.A. Sinnarajah S. Owens K. Rogers G. Yilma S. Vodyanoy V. Synapse. 2001; 40: 154-158Crossref PubMed Scopus (41) Google Scholar). Like native AMPA receptors, reconstituted purified AMPA receptors are activated by glutamate and AMPA, although with higher affinity. They are potentiated by ampakine and blocked by CNQX. The open channel probability obtained for purified receptors is similar to that observed in native retinal cells, and their conductance corresponds to maximal conductances characteristic of native AMPA receptors (31Morkve S.H. Veruki M.L. Hartveit E. J. Physiol. 2002; 542: 147-165Crossref PubMed Scopus (56) Google Scholar, 32Smith T.C. Wang L.Y. Howe J.R. J. Neurosci. 2000; 20: 2073-2085Crossref PubMed Google Scholar). Production and Purification of PSA-NCAM-Fc—Mouse PSA-NCAM-Fc was produced according to Vutskits et al. (33Vutskits L. Djebbara-Hannas Z. Zhang H. Paccaud J.P. Durbec P. Rougon G. Muller D. Kiss J.Z. Eur. J. Neurosci. 2001; 13: 1391-1402Crossref PubMed Google Scholar) using a stably transfected TE671 cell line kindly provided by Dr. G. Rougon (Laboratoire de Genetique et Physiologie du Developpement, Marseille, France). Coomassie staining and Western blotting of purified molecules showed a single broad band with a molecular mass above 200 kDa that is immunopositive with NCAM and PSA antibodies (Fig. 1A). Acute Isolation of Hippocampal Neurons—Hippocampal neurons were freshly isolated as previously reported (34Weber M. Dietrich D. Gräsel I. Reuter G. Seifert G. Steinhäuser C. J. Neurochem. 2001; 77: 1108-1115Crossref PubMed Scopus (27) Google Scholar, 35Seifert G. Zhou M. Dietrich D. Schumacher T.B. Dybek A. Weiser T. Wienrich M. Wilhelm D. Steinhäuser C. Neuropharmacology. 2000; 39: 931-942Crossref PubMed Scopus (19) Google Scholar). Briefly, FVB mice or Wistar rats (1–4 days old or 2 months old) were anesthetized and decapitated, and frontal brain slices of 300-μm thickness were cut with a vibratome (FTB, Plano, Marburg, Germany) in a solution containing 90 mm NaCl, 3 mm KCl, 2 mm MgSO4, 2 mm CaCl2, 1 mm sodium pyruvate, 10 mm glucose, 10 mm HEPES, 90 mm sucrose (4 °C, pH 7.4). Subsequently, slices were allowed to recover for at least 30 min in artificial cerebrospinal fluid (35 °C, pH adjusted to 7.4 by gassing with carbogen). Artificial cerebrospinal fluid contained 132 mm NaCl, 3 mm KCl, 1.25 mm NaH2PO4, 2 mm MgCl2, 2 mm CaCl2, 20 mm NaHCO3, 10 mm glucose. For enzymatic treatment, slices were transferred into a Pronase on PG1 (1–2 mg/ml)-supplemented HEPES-buffered solution containing 150 mm NaCl, 5 mm KCl, 2 mm MgSO4, 2 mm CaCl2, 10 mm glucose, 10 mm HEPES (O2 aeration). Incubation time varied between 5 and 6 min at 35 °C (juvenile mice) and 20 min at 35 °C plus 10 min at room temperature (21–24 °C, adult). After wash, the stratum pyramidale of the CA1 region of the hippocampus was dissected, and cells were isolated as described (35Seifert G. Zhou M. Dietrich D. Schumacher T.B. Dybek A. Weiser T. Wienrich M. Wilhelm D. Steinhäuser C. Neuropharmacology. 2000; 39: 931-942Crossref PubMed Scopus (19) Google Scholar). Electrophysiological Analysis of Isolated Cells—Membrane currents were analyzed with the patch clamp technique in the whole-cell mode (room temperature, holding potential VH = –70 mV). Currents were filtered at 3 or 10 kHz and sampled at 10 or 100 kHz (EPC7; List, Darmstadt, Germany). Recording pipettes were fabricated from borosilicate capillaries (Hilgenberg, Germany) and had resistances of 2–4 MΩ. Pipette solution consisted of 120 mm CsCl, 10 mm tetraethylammonium chloride, 10 mm HEPES, and 3 mm Na2-ATP, pH 7.3. Membrane capacitance and series resistance were compensated (40–50%) to improve voltage clamp control. The bath solution contained 150 mm NaCl, 5 mm KCl, 2 mm MgSO4, 2 mm CaCl2, 10 mm glucose, 10 mm HEPES, supplemented with 0.5 μm tetrodotoxin (TTX), 4 mm 4-AP, 0.5 mm (S)-α-methyl-carboxyphenylglycine, and 50 μm APV. Chemicals were of analytical grade. TTX was purchased from Alomone (Jerusalem, Israel), Na2-ATP was from Fluka (Taufkirchen, Germany), AMPA (hydrobromide salt) was from RBI (Natick, MA), GYKI 53655 was from Tocris (Bristol, UK), and colominic acid with a molecular mass of 23 kDa was from Fluka. Chondroitin sulfate A and all other reagents were from Sigma. The coefficient of variation was calculated for glutamate responses at –70 mV as the following ratio: CV = (variance(glutamate response) – variance(base line))½/(mean(glutamate response) – mean(base line)). Two hundred data points were used for calculation of mean and variance values. Reconstitution of AMPA Receptors in Lipid Bilayers—To reconstitute affinity-purified AMPA-Rs and analyze single channel currents, the "tip-dip" method was used (36Wilmsen U. Methfessel F. Hanke W. Boheim G. Spach G. Physical Chemistry of Transmembrane Ion Motions. Elsevier, Amsterdam1983: 479-485Google Scholar, 37Suarez-Isla B.A. Wan K. Lindstrom J. Montal M. Biochemistry. 1983; 22: 2319-2323Crossref PubMed Scopus (114) Google Scholar, 38Coronado R. Lattore R. Biophys. J. 1983; 43: 231-236Abstract Full Text PDF PubMed Scopus (222) Google Scholar). The formation of artificial lipid bilayers was initiated by forming small bilayers on the tips of polished glass patch pipettes (World Precision Instruments Inc., Sarasota, FL). The BB-CH-PC electronic micropipette puller (Mechanex S.A., Switzerland) was used to pull patch pipettes with 100-MΩ resistance. The patch bilayer was formed in asymmetric saline conditions with the pseudoextracellular fluid (ECF) on one side, and the pseudointracellular fluid (ICF) on the other side. The ECF contained 125 mm NaCl, 5 mm KCl, 1.25 mm NaH2PO4, and 5 mm Tris-HCl (pH 7.4), whereas the ICF contained 110 mm KCl, 4 mm NaCl, 2 mm NaHCO3, 0.1 mm CaCl2, 1 mm MgCl2, 2 mm MOPS (pH 7.4) (Fisher). Lipid bilayers in the "outside-out" configuration were initiated by lowering both the patch pipette, which was filled with ICF, and the reference electrode into the bathing solution using the attached micromanipulator. The bathing solution consisted of 300 μl of the ECF. Formation of lipid bilayers was initiated by adding 5 μl of the synthetic phospholipid 1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids Inc., Alabaster, AL) to the ECF. The synthetic phospholipid was prepared by dissolving it in anhydrous hexane (Aldrich) at a concentration of 1 mg/ml (39Vodyanoy V. Brand J.G. Teeter J.H. Receptor Events and Transduction Mechanisms in Taste and Olfaction. Marcel Dekker, New York1989: 319-345Google Scholar, 40Vodyanoy V. Hong F.T. Molecular Electronics: Biosensors and Biocomputers. Plenum Publishing Corp., New York1989: 317-328Crossref Google Scholar). A suspension of affinity-purified AMPA-Rs was fused into the lipid bilayer on the tip of the patch pipette by gentle stirring of the extracellular fluid using a small magnetic stirring bar placed at the bottom of the microbeaker. Single Channel Recordings—Single channel activity evoked by 290 nm AMPA alone and in the presence of 0.5, 1, 2, 3, and 10 μg/ml colominic acid was recorded on a videotape (Sony Corp., New York) using a VR-10B Digital Data recorder (Instrutech Corp., Port Washington, NY). For single channel analysis, data were digitally lowpass filtered at 2 kHz and compressed to final sampling rates of 20 kHz and read in pClamp software (Axon Instruments, Union City, CA). Data processing of the patch clamp recordings was essentially as described previously (41Sinnarajah S. Suppiramaniam V. Kumar K.P. Hall R.A. Bahr B.A. Vodyanoy V. Synapse. 1999; 31: 203-209Crossref PubMed Scopus (18) Google Scholar). All of the digitized traces were carefully inspected for artifacts and base-line drift before any quantitative analysis was performed. Transitions to and from the major conductance level were analyzed. The various peaks in the amplitude histogram were fitted with Gaussian curves to determine the maxima. AMPA-R currents were plotted as a function of membrane voltage. The channel conductance was determined according to the equation g = I/(V – V0), where I is the current, V is the voltage, and V0 is the reversal potential estimated from the I/V graph. To calculate the equivalent valence of the AMPA-R channel gate (z) in the presence and absence of colominic acid, we used the method described by Sinnarajah and co-workers (41Sinnarajah S. Suppiramaniam V. Kumar K.P. Hall R.A. Bahr B.A. Vodyanoy V. Synapse. 1999; 31: 203-209Crossref PubMed Scopus (18) Google Scholar), approximating the voltage dependence of channel open time as log (τopen) = –(zF/2.303RT)ΔV + log(τo). Open and closed time distributions (or the probability density functions, pdf) were assumed to be a sum of several exponential functions (i.e. pdf(t) = A1exp(–λ1t) + A2exp(–λ2t) + A3exp(–λ3t), where λ1, λ2, and λ3 are rate constants. The time constants for closed and open states t1 (equal to 1/λ1), t2 (equal to 1/λ2), and t3 (equal to 1/λ3) within bursts were derived from fits of the respective histograms by a sum of three or two exponential functions using the Marquardt least squares method (PSTAT module of Axon pClamp 6.0 software). For burst analyses, only experiments in which patches exhibiting single channel current levels were chosen. The full-size openings and the burst delimiter were determined from the plot of burst delimiter versus closings per burst as previously described (42Sigurdson W.J. Morris C.E. Brezden B.L. Gardner D.R. J. Exp. Biol. 1987; 127: 191-209Crossref Google Scholar). Burst duration time constants were derived from exponential fits obtained using the maximum likelihood method. The estimated rate constants λ1, λ2, and λ3 depend on fundamental transition rate constants k between connected states in the ion channel gating scheme. In order to derive individual rate constants for steps underlying activation of AMPA-Rs by colominic acid, we used the QuB program (available on the World Wide Web at www.qub.buffalo.edu). The digitized data were first idealized using the segmental k-means algorithm. Segmental k-means uses hidden Markov models to both find the most likely sequence of events in the data set and estimate model parameters. The maximum likelihood interval analysis program was used to compute the likelihood of the experimental series of open and closed times for a given set of trial rate constants and to search for the rate constants maximizing the likelihood (43Qin F. Auerbach A. Sachs F. Biophys. J. 1996; 70: 264-280Abstract Full Text PDF PubMed Scopus (368) Google Scholar). Modulation of AMPA Receptors Reconstituted in a Lipid Bilayer—To investigate the physiological properties of isolated AMPA-Rs, these were purified from brain homogenates by a sequence of chromatographic steps, culminating with immunoprecipitation using anti-GluR1 antibodies. Silver staining and Western blot analysis of purified proteins revealed a single band with a molecular mass of 105 kDa corresponding to the cognate molecular mass of the GluR1 subunit (Fig. 1B, arrow). A similar staining was found with anti-GluR2/3 antibody. Thus, the immunoprecipitated material probably represents the native heteromeric composition of AMPA-R subunits in the adult brain with an endogenous glycosylation pattern that makes it preferable to the use of recombinant AMPA receptors. Isolated receptors reconstituted in a lipid bilayer expressed single channel currents upon application of 290 nm AMPA (Fig. 2). This activity could be blocked by the antagonist of the AMPA/kainate glutamate receptors, CNQX (1 μm). When 290 nm AMPA was applied together with colominic acid (at 0.5, 1, 2, 3, and 10 μg/ml), a considerable increase in channel activity was observed (Figs. 2 and 3). The effect of colominic acid was reversible; the open probability increased from 0.23 ± 0.04 to 0.83 ± 0.03 after co-application of 2 μg/ml colominic acid and 290 nm AMPA and returned to 0.23 ± 0.05 after a 2-min washout of colominic acid (n = 6). Colominic acid induces no significant changes in the single channel conductance, but there was a profound increase in probability of channels to stay in the open state. Open probability, burst duration, and interburst intervals saturated at concentrations of colominic acid between 3 and 10 μg/ml (Fig. 4). To verify the specificity of colominic acid effects, we tested monomers of sialic acid (5 μg/ml) and another negatively charged polymeric carbohydrate, chondroitin sulfate A (5 μg/ml). Co-application of these compounds with 290 nm AMPA did not potentiate channel activity (open probability of 0.27 ± 0.06 (n = 5) for AMPA with sialic acid and 0.24 ± 0.05 (n = 4) for AMPA with chondroitin sulfate A versus 0.23 ± 0.04 (n = 9) for AMPA alone), implying that only polymers of sialic acid, and not monomers or any negatively charged sugar polymers, modulate AMPA-R activity.Fig. 3Concentration dependence of colominic acid effects on single AMPA-R channels reconstituted in lipid bilayers.A–F, traces (left) and amplitude histograms (right) representing the single channel fluctuations upon application of 290 nm AMPA without colominic acid (A) and in the presence of 0.5 (B), 1 (C), 2 (D), 3 (E), and 10 μg/ml (F) of colominic acid (CA). Channel openings are indicated by upward current deflections. The amplitude histograms show bimodal distributions with peaks corresponding to the stationary current levels (i.e. open and closed states). The maximum unitary current is 3.8 pA (VH = +71). The channel conductance and relative occurrence (in percent) of open states are as follows: 53 pS, 23% (A); 54 pS, 36% (B); 56 pS, 58% (C); 56 pS, 83% (D); 56 pS, 85% (E); 56 pS, 85% (F). The mean burst durations are 960 ms (A), 1523 ms (B), 2300 ms (C), 2850 ms (D), 2985 ms (E), and 3010 ms (F). The interburst intervals are 900 ms (A), 710 ms (B), 420 ms (C), 180 ms (D), 52 ms (E), and 48 ms (F).View Large Image Figure ViewerDownload (PPT)Fig. 4Concentration dependence of colominic acid effects on open probability, burst duration, and interburst interval of AMPA-Rs reconstituted in lipid bilayers.A, open probabilities (□) of AMPA-R channels after co-application of 290 nm AMPA with 0, 0.5, 1, 2, 3, and 10 μg/ml of colominic acid (CA) are 0.23 ± 0.08, 0.35 ± 0.07, 0.58 ± 0.08, 0.83 ± 0.08, 0.85 ± 0.08, and 0.85 ± 0.09, respectively. B, burst durations (▪) after co-application of 290 nm AMPA wi
Referência(s)