Sports drinks and tooth decay.
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Beverages↗
Biomedical subjects
S J Moss
Publications and source records attributed to S J Moss.
Subcellular localization of gamma-aminobutyric acid type A receptors is determined by receptor beta subunits.
gamma-aminobutyric acid type A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are constructed from four subunit classes with multiple members: alpha (1-6), beta (1-4), gamma (1-4), and delta (1). The contribution of subunit diversity in determining receptor subcellular targeting was examined in polarized Madin-Darby canine kidney (MDCK) cells. Significant detection of cell surface homomeric receptor expression by a combination of both immunological and electrophysiological methodologies was only found for the beta 3 subunit. Expression of alpha/beta binary combinations resulted in a nonpolarized distribution for alpha 1 beta 1 complexes, but specific basolateral targeting of both alpha 1 beta 2 and alpha 1 beta 3 complexes. The polarized distribution of these alpha/beta complexes was unaffected by the presence of the gamma 2S subunit. Interestingly, delivery of receptors containing the beta 3 subunit to the basolateral domain occurs via the apical surface. These results show that beta subunits can selectively target GABAA receptors to distinct cellular locations. Changes in the spatial and temporal expression of beta-subunit isoforms may therefore provide a mechanism for relocating GABAA receptor function between distinct neuronal domains. Given the critical role of these receptors in mediating synaptic inhibition, the contribution of different beta subunits in GABAA receptor function, may have implications in neuronal development and for receptor localization/clustering.
Animals↗
Proton sensitivity of the GABA(A) receptor is associated with the receptor subunit composition.
1. Modulation of GABA(A) receptors by external H(+) was examined in cultured rat sympathetic neurones, and in Xenopus laevis oocytes and human embryonic kidney (HEK) cells expressing recombinant GABA(A) receptors composed of combinations of alpha 1, beta 1, beta 2, gamma 2S and delta subunits. 2. Changing the external pH from 7.4 reduced GABA-activated currents in sympathetic neurones. pH titration of the GABA-induced current was fitted with a pH model which predicted that H(+) interact with two sites (PK(a) values of 6.4 and 7.2). 3. For alpha 1 beta 1 GABA(A) receptors, low external pH (< 7.4) enhanced responses to GABA. pH titration predicted the existence of two sites with PK(a) values of 6.6 and 7.5. The GABA concentration-response curve was shifted to the left by low pH and non-competitively inhibited at high pH (> 7.4). 4. alpha 1 beta 1 gamma 2S receptor constructs were not affected by external pH, whereas exchanging the beta 1 subunit for beta 2 conferred a sensitivity to pH, with predicted PK(a) values of 5.16 and 9.44. 5. Low pH enhanced the responses to GABA on alpha 1 beta 1 delta subunits, whilst high pH caused an inhibition (PK(a) values of 6.6 and 9.9). The GABA concentration-response curves were enhanced (pH 5.4) or reduced (pH 9.4) with no changes in the GABA EC(50). 6. Immunoprecipitation with subunit and epitope-specific antisera to alpha 1, beta 1 and delta subunits demonstrated that these subunits could co-assemble in cell membranes. 7. Expression of alpha 1 beta 1 gamma 2S delta constructs resulted in a 'bell-shaped' pH titration relationship. Increasing or decreasing external pH inhibited the responses to GABA. 8. The pH sensitivity of recombinant GABA(A) receptors expressed in HEK cells was generally in accordance with data accrued from Xenopus oocytes. However, rapid application of GABA to alpha 1 beta 1 constructs at high pH (> 7.4) caused an increased peak and reduced steady-state current, with a correspondingly increased rate of desensitization. 9. Modulation of GABA(A) receptor function was apparently unaffected by the internal pH. Moreover, pH values between 5 and 9.5 did not significantly affect the charge distribution on the zwitterionic GABA molecules. 10. In conclusion, this study demonstrates that external pH can either enhance, have little effect, or reduce GABA-activated responses, and this is apparently dependent on the receptor subunit composition. The potential importance of H(+) sensitivity of GABA(A) receptors is discussed.
Animals↗
Assembly and cell surface expression of heteromeric and homomeric gamma-aminobutyric acid type A receptors.
The ability of differing subunit combinations of gamma-aminobutyric acid type A (GABAA) receptors produced from murine alpha 1, beta 2, and gamma 2L subunits to form functional cell surface receptors was analyzed in both A293 cells and Xenopus oocytes using a combination of molecular, electrophysiological, biochemical, and morphological approaches. The results revealed that GABAA receptor assembly occurred within the endoplasmic reticulum and involved the interaction with the chaperone molecules immunoglobulin heavy chain binding protein and calnexin. Despite all three subunits possessing the ability to oligomerize with each other, only alpha 1 beta 2 and alpha 1 beta 2 gamma 2L subunit combinations could produce functional surface expression in a process that was not dependent on N-linked glycosylation. Single subunits and the alpha 1 gamma 2L and beta 2 gamma 2L combinations were retained within the endoplasmic reticulum. These results suggest that receptor assembly occurs by defined pathways, which may serve to limit the diversity of GABAA receptors that exist on the surface of neurons.
Amino Acid Sequence↗
Phosphorylation of the predicted major intracellular domains of the rat and chick neuronal nicotinic acetylcholine receptor alpha 7 subunit by cAMP-dependent protein kinase.
The predicted major intracellular domains of the chick and rat neuronal nicotinic acetylcholine receptor alpha 7 subunits were expressed in E. coli as glutathione-S-transferase fusion proteins. These proteins were then purified to near homogeneity by chromatography on immobilized glutathione. The intracellular domains of the alpha 7 subunit from both species were phosphorylated to high stoichiometry by cAMP-dependent protein kinase, but not by protein kinase C, cGMP-dependent protein kinase, or calcium/calmodulin-dependent protein kinase. Phosphorylation occurred on serine residues only within an identical single tryptic peptide for both proteins. This conserved phosphorylation site was identified as Ser 342 utilizing site-directed mutagenesis. These results demonstrate that the intracellular domain of the alpha 7 subunit is a substrate of PKA, and suggest a role for protein phosphorylation in mediating cellular regulation upon neuronal AChRs containing this subunit.
Amino Acid Sequence↗
A functional comparison of the antagonists bicuculline and picrotoxin at recombinant GABAA receptors.
Allosteric modulation of GABAA receptor function by a number of ligands has been shown to be dependent on the subunit composition of the receptor complex. In this respect, modulation of GABAA receptors by the antagonists bicuculline and picrotoxin was examined in Xenopus laevis oocytes expressing recombinant GABAA receptors composed of combinations of murine alpha 1, beta 1, gamma 2S and gamma 2L subunits. Bicuculline and picrotoxin reduced GABA-activated responses mediated by GABAA receptors composed of alpha 1 beta 1, alpha 1 beta 1 gamma 2S and alpha 1 beta 1 gamma 2L subunits in a dose-dependent manner. GABA equilibrium concentration-response curves for each receptor construct were shifted to the right by increasing concentrations of bicuculline in a competitive manner, whereas picrotoxin induced a slight lateral shift as well as a depression of the maximum response consistent with a mixed/non-competitive inhibitory mechanism. GABA concentration-response curves in the absence and presence of bicuculline were subjected to Schild analysis, which revealed similar pKB values of approximately 5.9 for alpha 1 beta 1, alpha 1 beta 1 gamma 2S and alpha 1 beta 1 gamma 2L receptor constructs. Concentration inhibition curves were used to estimate IC50 for picrotoxin were relatively unaffected by the GABAA receptor isoforms used in this study, and in particular, by the absence of the gamma 2 subunit in the alpha 1 beta 1 GABAA receptor complex. The similarity of the pKBs reported in this study to those previously reported using native neuronal preparations, which are likely to represent heterogeneous GABAA receptor populations, further indicates the lack of dependence on receptor subunit composition for the inhibitory action of bicuculline.
Animals↗
Modulation of amino acid-gated ion channels by protein phosphorylation.
The major excitatory and inhibitory amino acid receptors in the mammalian central nervous system are considered to be glutamate, gamma-aminobutyric acid type A (GABAA), and glycine receptors. These receptors are widely acknowledged to participated in fast synaptic neurotransmission, which ultimately is responsible for the control of neuronal excitability. In addition to these receptors being regulated by endogenous factors, including the natural neurotransmitters, they also form target substrates for phosphorylation by a number of protein kinases, including serine/threonine and tyrosine kinases. The process of phosphorylation involves the transfer of a phosphate group(s) from adenosine triphosphate to one or more serine, threonine, or tyrosine residues, which are invariably found in an intracellular location within the receptor Phosphorylation is an important means of receptor regulation since it represents a covalent modification of the receptor structure, which can have important implications for ion channel function. This chapter reviews the current molecular and biochemical evidence regarding the sites of phosphorylation for both native neuronal and recombinant glutamate, GABAA and glycine receptors, and also reviews the functional electrophysiological implications of phosphorylation for receptor function.
Amino Acid Sequence↗
Thiocyanate ions inhibit AMPA-activated currents in recombinant non-NMDA receptors expressed in Xenopus laevis oocytes: the role of the GluR2 subunit.
The functional interaction of thiocyanate (SCN-) ions with recombinant non-N-methyl-D-aspartate receptors was examined by studying alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and kainic acid (KA)-activated currents in Xenopus laevis oocytes. Recombinant receptors were expressed after microinjection of oocytes with combinations of cDNAs or cRNAs encoding for GluR1, GluR2, GluR2(R586Q), GluR3 or GluR6 subunits. When the GluR2 subunit was expressed with GluR1, SCN- (2 mM) inhibited the responses to 50 microM AMPA, whereas responses to 100 microM KA were slightly increased and responses to 200 microM L-glutamate were unaffected. Equilibrium concentration-response curves for AMPA were antagonized in a non-competitive manner by SCN- with a reduction in the EC50. The inhibitory effects of SCN- were unaffected by prior reduction of receptor desensitization with either 10 microM Concanavalin-A or 0.5 mM diazoxide. AMPA-activated currents recorded from homomeric GluR1 or Glur3 receptors were not affected by SCN-, and GluR6 homomeric receptors, which are sensitive to KA but not to AMPA, were also unaffected. In contrast, AMPA activation of homomeric GluR2(R586Q) subunit receptors, or combinations of GluR1 or GluR3 + GluR2(R586Q) subunits, were markedly inhibited by SCN-. In addition, the inhibitory effect of AMPA on KA-activated responses on these heteromeric receptors, was enhanced by SCN-. These results indicate that SCN- exert an inhibitory effect on 'AMPA receptors' but only when the recombinant non-NMDA receptor is a GluR2 homomer, or when GluR2 subunits are present as part of a heteromeric combination. Moreover, this inhibitory effect was unaffected by the 'Q/R' site in the presumed second transmembrane domain, since currents mediated by the GluR2(R586Q) subunit were also susceptible to inhibition by SCN-. Thus the inhibition was not related to the rectification properties or calcium permeability of the non-NMDA receptors. It is suggested that the GluR2 subunit may have a specific binding site for anions which could modulate the function of non-NMDA receptors and that SCN- may be a useful probe for the detection of these subunits in native neurons.
Animals↗
Homomeric beta 1 gamma-aminobutyric acid A receptor-ion channels: evaluation of pharmacological and physiological properties.
The ubiquitous distribution of gamma-aminobutyric acid A (GABAA) receptor beta subunits throughout the central nervous system is in accord with a vital role in receptor structure and function. Homomeric beta subunits have been reported to be either GABA-gated or capable of forming anion-selective channels that lacked GABA-gating properties. With electrophysiological recording techniques, we examined the properties of the murine Beta 1 subunit, addressed whether the homomeric receptor is expressed independently from the host cell's genome, and investigated whether these channels can open spontaneously. Murine beta 1 subunits, expressed in Xenopus oocytes or A293 cells, were unaffected by GABA or bicuculline; however, the resting membrane conductances were reduced by picrotoxin, zinc, or penicillin-G. In comparison, the expression of bovine beta1 subunits formed GABA-gated C1- channels. For murine beta 1 subunits, both pentobarbitone and propofol increased the membrane conductance, although the benzodiazepine ligands flurazepam, flumazenil, and methyl-6,7-dimethoxy-4 ethyl-beta-carboline-3-carboxylate were inactive. Oocytes injected with murine beta 1 cRNA in the presence of actinomycin D (to block host cell DNA transcription) expressed beta1 channels that were indistinguishable from those derived from previous cDNA injections in cells capable of normal transcription. Single-channel recording from murin beta 1 cDNA-injected oocytes revealed spontaneously opening channels with a main state conductance of 18 pS. Picrotoxin inhibited the channel openings by reducing the probability of opening. We concluded that murine beta 1 subunits can form functional ion channels that are not gated by GABA but can be closed by some noncompetitive GABA antagonists. Interestingly, previous observations of spontaneously opening ion channels with properties similar to those found for the murine beta 1 receptor suggest that a limited expression of homomeric beta subunit-ion channels may exist in vivo.
Animals↗
Modulation of GABAA receptors by tyrosine phosphorylation.
gamma-Aminobutyric acid type-A (GABAA) receptors are the major sites of fast synaptic inhibition in the brain. They are presumed to be pentameric heteroligomers assembled from four classes of subunits with multiple members: alpha (1-6), beta (1-3), gamma (1-3) and delta (1). Here, GABAA receptors consisting of alpha 1, beta 1 and gamma 2L subunits, coexpressed in mammalian cells with the tyrosine kinase vSRC (the transforming gene product of the Rous sarcoma virus), were phosphorylated on tyrosine residues within the gamma 2L and beta 1 subunits. Tyrosine phosphorylation enhanced the whole-cell current induced by GABA. Site-specific mutagenesis of two tyrosine residues within the predicted intracellular domain of the gamma 2L subunit abolished tyrosine phosphorylation of this subunit and eliminated receptor modulation. A similar modulation of GABAA receptor function was observed in primary neuronal cultures. As GABAA receptors are critical in mediating fast synaptic inhibition, such a regulation by tyrosine kinases may therefore have profound effects on the control of neuronal excitation.
Amino Acid Sequence↗
Clinical implications of recent advances in salivary research.
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Fluorides↗
Early loss of pit and fissure sealant: a clinical and SEM study.
The rate of early loss of preventive resin restoration and single application of pit and fissure sealant was determined by a clinical survey. The rate of partial and complete sealant loss on the observed surfaces was 14.4% (43/299) at the 3-month recall, with a further loss of 7.0% (12/171) between the 3- and 6-month recalls. Typically, a complete loss of sealant was observed on maxillary molars and on the buccal surface of mandibular molars, while partial loss caused on the occlusal surface of mandibular molars. Scanning electron microscopic observation of in vivo tooth replicas revealed partial gap formations and small fractures of sealant indicating failure or degradation of adhesion. The presence of unetched areas after routine cleaning and acid etching, especially in and around pits and fissures could be a major cause of early sealant loss.
Bicuspid↗
Platelet-derived growth factor receptor is a novel modulator of type A gamma-aminobutyric acid-gated ion channels.
Platelet-derived growth factor (PDGF) and PDGF receptors (PDGFRs) are ubiquitously expressed in the mammalian central nervous system, where they exert trophic actions on both neuronal and glial cells. However, the acute actions of PDGF on synaptic transmission are unknown. We report a novel regulatory action of PDGF/PDGFR. Activation of PDGFRs inhibited the function of native type A gamma-aminobutyric acid (GABAA) receptors (GABAA-RS) in rat hippocampal CA1 pyramidal neurons and mouse brain membrane vesicles. The mechanism of this inhibition was studied with a panel of mutant PDGFRS-beta coexpressed with cloned human GABAA-Rs in Xenopus oocytes. These experiments revealed that phospholipase C-gamma is the protein that relays the inhibitory signal from PDGFRS to GABAA-Rs. Experiments with microinjected EGTA and inositol-1, 3, 4-triphosphate demonstrated that inhibition of GABAA-Rs depended on a phospholipase C-gamma-mediated increase in intracellular Ca(2+)-levels. The PDGFR-induced inhibitory effect was independent of the subunit composition of GABAA-RS. Moreover, GABAA-RS composed of alpha 1 beta 1 S409A subunits, which do not contain any known protein kinase C phosphorylation sites, were inhibited by PDGF to the same extent as wild-type GABAA-RS. Inhibitors of protein kinase C, CA2+/calmodulin-dependent protein kinase II, calcineurin, and tyrosine phosphatases did not affect the modulatory actions of PDGFR. In conclusion, our results suggest that PDGFRs exert potent modulatory actions on GABAA-R-dependent inhibitory synaptic transmission. These regulatory actions of PDGF could play important roles in the function of the mammalian central nervous system during physiological and pathophysiological conditions.
Animals↗
Differential phosphorylation of intracellular domains of gamma-aminobutyric acid type A receptor subunits by calcium/calmodulin type 2-dependent protein kinase and cGMP-dependent protein kinase.
gamma-Aminobutyric acid type A receptor subunits (GABAA) can be divided into five classes, alpha, beta, gamma, delta, and rho, based on sequence homology. We have used purified fusion proteins of the major intracellular domain of GABAA receptor subunits produced in Escherichia coli to examine the phosphorylation of these subunits by cGMP-dependent protein kinase (PKG) and multifunctional calcium/calmodulin-dependent protein kinase (CAM KII). Both PKG and CAM KII phosphorylated a purified beta 1 subunit fusion. Both of these kinases phosphorylated serine 409 within the beta 1 subunit; in addition, CAM KII also phosphorylated serine 384 as determined by site-specific mutagenesis. Fusion proteins of the major intracellular domains of the gamma 2S and gamma 2L subunits were produced. These proteins differ by 8 amino acids (LLRMFSFK). Both the gamma 2L and gamma 2S fusion proteins were excellent substrates of CAM KII. However, the gamma 2L fusion protein was phosphorylated to higher stoichiometry due to the phosphorylation of serine 343 within this 8-amino acid insertion. Both the gamma 2L and gamma 2S subunits were phosphorylated on common residues by CAM KII identified as serine 348 and threonine 350. These results identify specific sites of phosphorylation for CAM KII and PKG within GABAA receptor subunits, suggesting a role for these two kinases in modulating GABAA receptor function in vivo.
Amino Acid Sequence↗
Regulation of GABAA receptor function by protein kinase C phosphorylation.
GABAA receptors possess consensus sequences for phosphorylation by PKC that are located on the presumed intracellular domains of beta and gamma 2 subunits. PKC phosphorylation sites were analyzed using purified receptor subunits and were located on up to 3 serine residues in beta 1 and gamma 2 subunits. The role of phosphorylation in receptor function was studied using recombinant receptors expressed in kidney cells and Xenopus oocytes and was compared with native neuronal GABAA receptors. For recombinant and native GABAA receptors, PKC phosphorylation caused a reduction in the amplitudes of GABA-activated currents without affecting the time constants for current decay. Selective site-directed mutagenesis of the serine residues reduced the effects of phorbol esters and revealed that serine 343 in the gamma 2 subunit exerted the largest effect on the GABA-activated response. These results indicate that PKC phosphorylation can differentially modulate GABAA receptor function.
Alanine↗
Cyclic AMP and synaptic activity-dependent phosphorylation of AMPA-preferring glutamate receptors.
Several studies have suggested that the function of glutamate receptor channels can be regulated by protein phosphorylation. Furthermore, a basal level of phosphorylation may be necessary to maintain receptor function. Little is known, however, about the phosphorylation state of glutamate receptor channels in neurons and how it is regulated by synaptic activity. In this study, we have investigated the phosphorylation of the AMPA-preferring glutamate receptor subunit GluR1 in cortical neurons in primary culture. These neurons elaborate extensive processes, form functional synapses, and exhibit spontaneous 4-8 sec bursts of synaptic activity every 15-20 sec. In cultures in which this synaptic activity was suppressed by tetrodotoxin and MK-801, the GluR1 protein was phosphorylated on serine residues within a single tryptic phosphopeptide, as determined by phosphoamino acid analysis and phosphopeptide mapping. This same peptide was basally phosphorylated in recombinant GluR1 receptors transiently expressed in human embryonal kidney 293 cells. Treatment of these synaptically inactive cortical neurons with the adenylyl cyclase activator forskolin resulted in a robust increase in phosphorylation on serine residues on a phosphopeptide distinct from the basally phosphorylated peptide. Again, this same phosphopeptide was observed in recombinant GluR1 receptors isolated from 293 cells coexpressing the catalytic subunit of cAMP-dependent protein kinase. Spontaneous synaptic activity in cultures of cortical neurons resulted in a consistent, rapid (within 10-30 sec) increase in phosphorylation on serine and threonine residues. Interestingly, these phosphopeptides were also phosphorylated when neurons from inactive cultures were stimulated with phorbol esters, which activate protein kinase C. These results indicate that AMPA receptors containing the GluR1 subunit may be regulated by extracellular signals working through the cAMP second messenger system as well as by synaptic activity, possibly acting through protein kinase C. Such regulation by protein phosphorylation may be involved in short-term changes in synaptic efficacy thought to involve the functional modulation of AMPA receptors.
1-Methyl-3-isobutylxanthine↗
Phosphorylation of recombinant non-NMDA glutamate receptors on serine and tyrosine residues.
Glutamate receptors are the major excitatory neurotransmitter receptors in the central nervous system. A variety of data has recently suggested that protein phosphorylation of glutamate receptors regulates their function. To examine at a molecular level the role of protein phosphorylation in the modification of glutamate receptors, we have transiently expressed the non-NMDA glutamate receptor subunit GluR1 (flop) in human embryonic kidney 293 cells. Using a polyclonal antipeptide antiserum directed specifically against GluR1, we have immunoprecipitated a 106 kDa phosphoprotein corresponding to the GluR1 subunit. Phosphoamino acid analysis and thermolytic peptide mapping demonstrate that this basal phosphorylation occurs exclusively on serine residues in two phosphopeptides. Application of activators of endogenous cAMP-dependent protein kinase or protein kinase C revealed no consistent changes in the phosphorylation of GluR1. However, co-expression of the GluR1 subunit with the well characterized protein tyrosine kinase v-src results in phosphorylation of GluR1 on tyrosine residues, in a single thermolytic phosphopeptide. These results suggest that GluR1 may be a substrate for protein serine/threonine kinases as well as protein tyrosine kinases in the central nervous system.
Cell Line↗
Functional modulation of GABAA receptors by cAMP-dependent protein phosphorylation.
gamma-Aminobutyric acidA (GABAA) receptors are ligand-gated ion channels that mediate inhibitory synaptic transmission in the central nervous system. The role of protein phosphorylation in the modulation of GABAA receptor function was examined with cells transiently transfected with GABAA receptor subunits. GABAA receptors consisting of the alpha 1 and beta 1 or the alpha 1, beta 1, and gamma 2 subunits were directly phosphorylated on the beta 1 subunit by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA). The phosphorylation decreased the amplitude of the GABA response of both receptor types and the extent of rapid desensitization of the GABAA receptor that consisted of the alpha 1 and beta 1 subunits. Site-specific mutagenesis of the serine residue phosphorylated by PKA completely eliminated the PKA phosphorylation and modulation of the GABAA receptor. In primary embryonic rat neuronal cell cultures, a similar regulation of GABAA receptors by PKA was observed. These results demonstrate that the GABAA receptor is directly modulated by protein phosphorylation and suggest that neurotransmitters or neuropeptides that regulate intracellular cAMP levels may modulate the responses of neurons to GABA and consequently have profound effects on synaptic excitability.
Animals↗