Differential activation of dendritic cells by Toll-like receptor agonists isolated from the Gram-positive vaccine vector Streptococcus gordonii.

The oral commensal bacterium Streptococcus gordonii has been gathering interest as a candidate live mucosal vaccine delivery vector. S. gordonii has been shown to be capable of activating antigen presenting immune cells in a manner which leads to their activation and maturation, yet the mechanism used by S. gordonii to do so is poorly understood. The aim of this work was to investigate the immunostimulatory components of S. gordonii in inducing murine dendritic cell (DC) activation and maturation. Lipoteichoic acid (LTA), lipoprotein (LP), peptidoglycan (PGN), and DNA were isolated from S. gordonii, and used to stimulate murine DC. Cytokine production and DC surface marker upregulation in response to the bacterial components was quantified by enzyme-linked immunosorbent assay and flow cytometry respectively. The results were contrasted against data obtained from DC derived from MyD88, TRIF [TIR(Toll/Interleukin-1 Receptor)-domain-containing adapter-inducing interferon-beta] or toll-like receptor-2 (TLR-2) knockout mice. The four S. gordonii bacterial components were found to differentially induce cytokine production and surface marker upregulation by murine DC. Activation of DC by both whole S. gordonii cells and the four bacterial components was abrogated in the absence of MyD88, but not in the absence of TRIF. LTA, LP and PGN, but not DNA and whole S. gordonii, required TLR-2 to induce a DC response. The results collectively indicate that S. gordonii activates DC predominantly through a MyD88-dependent and TRIF-independent pathway. This activation can be attributed to multiple immunostimulatory components present within S. gordonii bacterial cells.

A phase II trial of olanzapine for the prevention of chemotherapy-induced nausea and vomiting: a Hoosier Oncology Group study.

In a previous phase I study, olanzapine was demonstrated to be a safe and effective agent for the prevention of delayed emesis in chemotherapy-naïve cancer patients receiving cyclophosphamide, doxorubicin, and/or cisplatin. Using the maximum tolerated dose of olanzapine in the phase I trial, a phase II trial was performed for the prevention of chemotherapy-induced nausea and vomiting in chemotherapy-naïve patients. The regimen was 5 mg/day of oral olanzapine on the 2 days prior to chemotherapy, 10 mg on the day of chemotherapy, day 1, (added to intravenous granisetron, 10 mcg/kg and dexamethasone 20 mg), and 10 mg/day on days 2-4 after chemotherapy (added to dexamethasone, 8 mg p.o. BID days 2 and 3, and 4 mg p.o. BID day 4). Thirty patients (median age 58.5 years, range 25-84; 23 women; ECOG PS 0, 1) consented to the protocol, and all were evaluable. Complete response (CR) (no emesis, no rescue) was 100% for the acute period (24 h postchemotherapy), 80% for the delayed period (days 2-5 postchemotherapy), and 80% for the overall period (0-120 h postchemotherapy) in ten patients receiving highly emetogenic chemotherapy (cisplatin > or =70 mg/m(2)). CR was also 100% for the acute period, 85% for the delayed period, and 85% for the overall period in 20 patients receiving moderately emetogenic chemotherapy (doxorubicin > or =50 mg/m(2)). Nausea was very well controlled in the patients receiving highly emetogenic chemotherapy, with no patient having nausea [0 on scale of 0-10, M.D. Anderson Symptom Inventory (MDASI)] in the acute or delayed periods. Nausea was also well controlled in patients receiving moderately emetogenic chemotherapy, with no nausea in 85% of patients in the acute period and 65% in the delayed and overall periods. There were no grade 3 or 4 toxicities and no significant pain, fatigue, disturbed sleep, memory changes, dyspnea, lack of appetite, drowsiness, dry mouth, mood changes, or restlessness experienced by the patients. Complete response and control of nausea in subsequent cycles of chemotherapy (25 patients, cycle 2; 25 patients, cycle 3; 21 patients, cycle 4) were equal to or greater than cycle 1. Olanzapine is safe and highly effective in controlling acute and delayed chemotherapy-induced nausea and vomiting in patients receiving highly and moderately emetogenic chemotherapy.

Structural basis for AMPA receptor activation and ligand selectivity: crystal structures of five agonist complexes with the GluR2 ligand-binding core.

Glutamate is the principal excitatory neurotransmitter within the mammalian CNS, playing an important role in many different functions in the brain such as learning and memory. In this study, a combination of molecular biology, X-ray structure determinations, as well as electrophysiology and binding experiments, has been used to increase our knowledge concerning the ionotropic glutamate receptor GluR2 at the molecular level. Five high-resolution X-ray structures of the ligand-binding domain of GluR2 (S1S2J) complexed with the three agonists (S)-2-amino-3-[3-hydroxy-5-(2-methyl-2H-tetrazol-5-yl)isoxazol-4-yl]propionic acid (2-Me-Tet-AMPA), (S)-2-amino-3-(3-carboxy-5-methylisoxazol-4-yl)propionic acid (ACPA), and (S)-2-amino-3-(4-bromo-3-hydroxy-isoxazol-5-yl)propionic acid (Br-HIBO), as well as of a mutant thereof (S1S2J-Y702F) in complex with ACPA and Br-HIBO, have been determined. The structures reveal that AMPA agonists with an isoxazole moiety adopt different binding modes in the receptor, dependent on the substituents of the isoxazole. Br-HIBO displays selectivity among different AMPA receptor subunits, and the design and structure determination of the S1S2J-Y702F mutant in complex with Br-HIBO and ACPA have allowed us to explain the molecular mechanism behind this selectivity and to identify key residues for ligand recognition. The agonists induce the same degree of domain closure as AMPA, except for Br-HIBO, which shows a slightly lower degree of domain closure. An excellent correlation between domain closure and efficacy has been obtained from electrophysiology experiments undertaken on non-desensitising GluR2i(Q)-L483Y receptors expressed in oocytes, providing strong evidence that receptor activation occurs as a result of domain closure. The structural results, combined with the functional studies on the full-length receptor, form a powerful platform for the design of new selective agonists.

Mechanisms for ligand binding to GluR0 ion channels: crystal structures of the glutamate and serine complexes and a closed apo state.

High-resolution structures of the ligand binding core of GluR0, a glutamate receptor ion channel from Synechocystis PCC 6803, have been solved by X-ray diffraction. The GluR0 structures reveal homology with bacterial periplasmic binding proteins and the rat GluR2 AMPA subtype neurotransmitter receptor. The ligand binding site is formed by a cleft between two globular alpha/beta domains. L-Glutamate binds in an extended conformation, similar to that observed for glutamine binding protein (GlnBP). However, the L-glutamate gamma-carboxyl group interacts exclusively with Asn51 in domain 1, different from the interactions of ligand with domain 2 residues observed for GluR2 and GlnBP. To address how neutral amino acids activate GluR0 gating we solved the structure of the binding site complex with L-serine. This revealed solvent molecules acting as surrogate ligand atoms, such that the serine OH group makes solvent-mediated hydrogen bonds with Asn51. The structure of a ligand-free, closed-cleft conformation revealed an extensive hydrogen bond network mediated by solvent molecules. Equilibrium centrifugation analysis revealed dimerization of the GluR0 ligand binding core with a dissociation constant of 0.8 microM. In the crystal, a symmetrical dimer involving residues in domain 1 occurs along a crystallographic 2-fold axis and suggests that tetrameric glutamate receptor ion channels are assembled from dimers of dimers. We propose that ligand-induced conformational changes cause the ion channel to open as a result of an increase in domain 2 separation relative to the dimer interface.

Identification of RFC(Ctf18p, Ctf8p, Dcc1p): an alternative RFC complex required for sister chromatid cohesion in S. cerevisiae.

We have identified and characterized an alternative RFC complex RFC(Ctf18p, Ctf8p, Dcc1p) that is required for sister chromatid cohesion and faithful chromosome transmission. Ctf18p, Ctf8p, and Dcc1p interact physically in a complex with Rfc2p, Rfc3p, Rfc4p, and Rfc5p but not with Rfc1p or Rad24p. Deletion of CTF18, CTF8, or DCC1 singly or in combination (ctf18Deltactf8Deltadcc1Delta) leads to sensitivity to microtubule depolymerizing drugs and a severe sister chromatid cohesion defect. Furthermore, temperature-sensitive mutations in RFC4 result in precocious sister chromatid separation. Our results highlight a novel function of the RFC proteins and support a model in which sister chromatid cohesion is established at the replication fork via a polymerase switching mechanism and a replication-coupled remodeling of chromatin.

Structural similarities between glutamate receptor channels and K(+) channels examined by scanning mutagenesis.

The pores of glutamate receptors and K(+) channels share sequence homology, suggesting a conserved secondary structure. Scanning mutagenesis with substitution of alanine and tryptophan in GluR6 channels was performed based on the structure of KcsA. Our assay used disruption of voltage-dependent polyamine block to test for changes in the packing of pore-forming regions. Alanine scanning from D567 to R603 revealed reduced rectification resulting from channel block in two regions. A periodic pattern from F575 to M589 aligned with the pore helix in KcsA, whereas a cluster of sensitive positions around Q590, a site regulated by RNA editing, mapped to the selectivity filter in KcsA. Tryptophan scanning from D567 to R603 revealed similar patterns, but with a complete disruption of spermine block for 7 out of the 37 positions and a pM dissociation constant for Q590W. Molecular modeling with KcsA coordinates showed that GluR6 pore helix mutants disrupting polyamine block pack against M1 and M2, and are not exposed in the ion channel pore. In the selectivity filter, tryptophan creates an aromatic cage consistent with the pM dissociation constant for Q590W. A scan with glutamate substitution was used to map the cytoplasmic entrance to the pore based on charge neutralization experiments, which established that E594 was uniquely required for high affinity polyamine block. In E594Q mutants, introduction of glutamate at positions S593-L600 restored polyamine block at positions corresponding to surface-exposed residues in KcsA. Our results reinforce proposals that the pore region of glutamate receptors contains a helix and pore loop analogous to that found in K(+) channels. At the cytoplasmic entrance of the channel, a negatively charged amino acid, located in an extended loop with solvent-exposed side chains, is required for high affinity polyamine block and probably attracts cations via a through space electrostatic mechanism.

Barriers to care among racial/ethnic groups under managed care.

We describe barriers to care reported by racial/ethnic groups and explore the extent to which barriers vary between persons enrolled in managed care and those in non-managed care plans, using data from the 1996 Medical Expenditure Panel Survey (MEPS). Most respondents expressed satisfaction with their care; however, a substantial percentage reported experiencing barriers. Minorities, particularly Hispanics and Asian Americans, were more likely than non-Hispanic whites were to report barriers. Managed care enrollees across racial/ethnic groups faced different types of barriers than non-managed care enrollees did. Although managed care enrollees were more likely to report having a usual source of care and greater continuity of care, they also reported more difficulties obtaining care and less satisfaction with their care.

The effects of Medicaid expansions and reimbursement increases on dentists' participation.

North Carolina Medicaid increased nominal Medicaid reimbursement to dentists 23% from 1988 to 1991 and doubled enrollment through eligibility expansions from 1985 to 1991. Using Medicaid claims data and panel data techniques, this analysis investigates the effect of these policy changes on the probability that a dentist participated in Medicaid, and on the number of Medicaid children seen per provider per quarter. The results suggest that eligibility expansions and reimbursement rate increases were only marginally effective in increasing access to dental services for the Medicaid population.

Money illusion among health care providers: should we adjust for inflation in analyses of provider behavior?

This analysis questions the appropriateness of inflation adjustment in analyses of provider behavior by comparing results from estimations using adjusted financial variables with those from estimations using unadjusted financial variables. Using Medicaid claims from 1984-1991, we explored the effects of Medicaid reimbursement increases on dentists' participation. Using results from inflation adjusted analyses, we would conclude that a 23% nominal increase in Medicaid reimbursement rates yields no increase in the number of Medicaid children seen by dentists. In contrast, estimations based on unadjusted reimbursement rates suggest that this same 23% nominal increase in reimbursement leads to an expected 16-person (15.4%) increase in the number of Medicaid patients seen per provider per year. These analyses demonstrate that results are sensitive to adjustment for inflation. While adjusting for inflation is a generally accepted practice in health services research, doing so without evidence that providers respond to adjusted reimbursement may be unjustified. More research is needed to determine the appropriateness of inflation adjustment in analyses of provider behavior, and the circumstances under which it should or should not be done.

Using Medicaid claims to construct dental service market areas.

OBJECTIVE: To use Medicaid claims data to construct patient origin-based market areas for dental services and compare constructed market areas with those based on the practice county. DATA SOURCES: North Carolina Medicaid claims, eligibility, and provider files, the Cooperative Health Information Systems' dentist licensure files, and the Log Into North Carolina data. STUDY DESIGN: A visit-level file was created from the Medicaid claims data and aggregated by provider practice county and patient county of residence. Using the aggregated file and an algorithm based on the Elzinga-Hogarty approach, patient travel patterns were used to construct mutually exclusive patient origin market areas. DATA ANALYSIS: Market area characteristics were compared across definitions using Pearson correlation coefficients. In addition, estimations of provider participation were performed using market area characteristics as control variables. The beta coefficients associated with market area characteristics were compared across market area definitions. PRINCIPAL FINDINGS: Medicaid claims data, when combined with provider licensure files, can be used to construct market areas based on patient origin data. However, measures of market area characteristics are correlated highly between the two types of market areas studied. Furthermore, beta coefficients on market area variables in models of provider participation are similar in sign, significance, and magnitude across market definitions. CONCLUSIONS: Compared with market areas constructed using patient origin data, county-based market areas adequately proxy for dental markets. Using the county as the market area also avoids the time and computational costs associated with using a patient origin-based approach and facilitates the use of widely available data.

Amino acid substitutions in the pore of rat glutamate receptors at sites influencing block by polyamines.

1. The effect on polyamine block of mutations at the Q/R site and the conserved negative charge +4 site in AMPA and kainate receptors was studied using the rat glutamate receptor GluR6 expressed in Xenopus oocytes and human embryonic kidney (HEK) cells. 2. Introduction of negative charge at the Q/R site increased the equilibrium dissociation constant at 0 mV (Kd(0)) for spermine from 1.3 to 4.0 microM (Q590E); the smaller side chains Q590D and Q590N had Kd(0) values of 47 and 20 microM. Reductions in spermine affinity were also obtained for the small hydrophobic residues Q590V and Q590A, with Kd(0) values of 3.6 and 8.8 microM. Positively charged side chains produced outward rectifying responses similar to those recorded for GluR6(Q) with polyamine-free conditions, suggesting a complete absence of voltage-dependent block by spermine. 3. Substitution of tryptophan at the Q/R site produced high-affinity block with a Kd(0) of 190 pM. In Xenopus oocytes no outward current was observed at potentials up to +200 mV. A much smaller increase in affinity was observed for Q590F and Q590Y, which had Kd(0) values of 0.28 and 0.83 microM respectively. 4. The Q590H mutant gave weakly birectifying responses strikingly different from those for other mutants. When ionization of the His group was increased by raising the external hydrogen ion concentration, responses became outward rectifying. The ratios of the conductance at 100 mV over that at -100 mV for Q590H were 0.52 at pH 8.3 and 2.5 at pH 5.3. 5. Neutralization of charge or aromatic residues at the +4 site produced a large reduction of spermine affinity, with Kd(0) values for E594N, E594Q and E594W of 109, 1020 and 2150 microM, respectively. In the absence of polyamines, E594K and E594R produced strongly inward rectifying responses while E594Q, E594A and E594W were birectifying. 6. A model for permeant block allowed quantitative comparisons between mutants. Despite large changes in well depth and barrier heights, there was little change in the voltage dependence of block for both Q/R and +4 site mutants. We propose a model with a distributed binding site for polyamines in which the +4 site is located near the entrance to the channel.

Heteromeric kainate receptors formed by the coassembly of GluR5, GluR6, and GluR7.

In the CNS kainate subtype glutamate receptors (GluRs) are likely to be heteromeric assemblies containing multiple gene products. However, although recombinant kainate receptors from the GluR5-GluR7 gene family have been studied extensively in their homomeric forms, there have been no tests to determine whether these subunits can coassemble with each other. We used the GluR5 selective agonists (RS)-2-amino-3-(3-hydroxy-5-tertbutylisoxazol-4-yl)propanoic acid (ATPA) and (S)-5-iodowillardiine (I-will) to test for the coassembly of GluR5 with GluR6 and GluR7 by measuring changes in rectification that occur for heteromeric receptors containing both edited and unedited Q/R site subunits. Birectifying ATPA and I-will responses resulting from polyamine block for homomeric GluR5(Q) became outwardly rectifying when GluR6(R) was coexpressed with GluR5(Q), although GluR6 was not activated by ATPA or I-will, indicating the formation of heteromeric receptors. Similar approaches showed the coassembly of GluR7 with GluR6 and GluR5. Heteromeric kainate receptors containing both GluR5 and GluR6 subunits exhibited novel functional properties, including reduced desensitization and faster recovery from desensitization than those recorded for homomeric GluR5. Coexpression of GluR6 with GluR5 also enhanced the magnitude of responses to GluR5 selective agonists. In contrast, the coassembly of GluR7 with GluR6 markedly decreased the amplitude of agonist responses. Our results indicate that, similar to AMPA receptors, the kainate receptor subunits GluR5-GluR7 exhibit promiscuous coassembly. The formation of heteromeric kainate receptors may help to explain why the functional properties of native kainate receptors differ from those that have been reported for recombinant kainate receptors.

The role of state policies and programs in buffering the effects of poverty on children's immunization receipt.

OBJECTIVES: This study assessed the influence of public policies on the immunization status of 2-year old children in the United States. METHODS: Up-to-dateness for the primary immunization series was assessed in a national sample of 8100 children from the 1988 National Maternal and Infant Health Survey and its 1991 Longitudinal Follow-Up. RESULTS: Documented immunization rates of this sample were 33% for poor children and 44% for others. More widespread Medicated coverage was associated with greater likelihood of up-to-dateness among poor children. Up-to-dateness was more likely for poor children with public rather than private sources of routine pediatric care, but all children living in states where most immunizations were delivered in the public sector were less likely to be up to date. Poor children in state with partial vaccine replacement programs were less likely to be up to date than those in free-market purchase states. CONCLUSIONS: While state policies can enhance immunization delivery for poor children, heavy reliance on public sector immunization does not ensure timely receipt of vaccines. Public- and private-sector collaboration is necessary to protect children from vaccine-preventable diseases.

Functional characterization of a potassium-selective prokaryotic glutamate receptor.

Ion channels are molecular pores that facilitate the passage of ions across cell membranes and participate in a range of biological processes, from excitatory signal transmission in the mammalian nervous system to the modulation of swimming behaviour in the protozoan Paramecium. Two particularly important families of ion channels are ionotropic glutamate receptors (GluRs) and potassium channels. GluRs are permeable to Na+, K+ and Ca2+, are gated by glutamate, and have previously been found only in eukaryotes. In contrast, potassium channels are selective for K+, are gated by a range of stimuli, and are found in both prokaryotes and eukaryotes. Here we report the discovery and functional characterization of GluR0 from Synechocystis PCC 6803, which is the first GluR found in a prokaryote. GluR0 binds glutamate, forms potassium-selective channels and is related in amino-acid sequence to both eukaryotic GluRs and potassium channels. On the basis of amino-acid sequence and functional relationships between GluR0 and eukaryotic GluRs, we propose that a prokaryotic GluR was the precursor to eukaryotic GluRs. GluR0 provides evidence for the missing link between potassium channels and GluRs, and we suggest that their ion channels have a similar architecture, that GluRs are tetramers and that the gating mechanisms of GluRs and potassium channels have some essential features in common.

The role of hydrophobic interactions in binding of polyamines to non NMDA receptor ion channels.

Block of kainate subtype glutamate receptor channels by internal polyamines was analysed using outside out patches from HEK 293 cells transiently transfected with GluR6(Q). Tetramines with different numbers and spacing of methylene groups between NH2 groups produced biphasic rectification well fit by the Woodhull model for a weakly permeable ion channel blocker. Such analysis revealed an increase in binding energy of 611 cal M(-1) for each methylene group added over the range 6-12 (CH2), suggesting that a major component of block by polyamines involves hydrophobic binding. Isomers with the same number of CH2 groups but different spacing between NH2 groups showed similar affinity. Due to differences in pKa values for protonation of NH2 groups, the average charge on the tetramines studied would be expected to vary from 3.98 to 2.22 at physiological pH; despite this, the voltage dependence of block was similar for all tetramines tested, with a mean value for ztheta of 1.82, similar to values for polyamines with five or six NH2 groups. In contrast, for 1,3-propane diamine (DA3 ztheta 0.83), and the N-propyl- (ztheta 1.42) and N,N'-diethyl- (ztheta 1.37) analogues of DA3, there was an increase in the voltage dependence of block on addition of hydrophobic groups.

Activity-dependent modulation of glutamate receptors by polyamines.

The mechanisms by which polyamines block AMPA and kainate receptors are not well understood, but it has been generally assumed that they act as open-channel blockers. Consistent with this, voltage-jump relaxation analysis of GluR6 equilibrium responses to domoate could be well fit, assuming that spermine, spermidine, and philanthotoxin are weakly permeable open-channel blockers. Analysis of rate constants for binding and dissociation of polyamines indicated that the voltage dependence of block arose primarily from changes in koff rather than kon. Experiments with changes in Na concentration further indicate that the voltage dependence of polyamine block was governed by ion flux via open channels. However, responses to 1 msec applications of L-Glu revealed slow voltage-dependent rise-times, suggesting that polyamines additionally bind to closed states. A kinetic model, which included closed-channel block, reproduced these observations but required that polyamines accelerate channel closure either through an allosteric mechanism or by emptying the pore of permeant ions. Simulations with this model reveal that polyamine block confers novel activity-dependent regulation on calcium-permeable AMPA and kainate receptor responses.

An analysis of philanthotoxin block for recombinant rat GluR6(Q) glutamate receptor channels.

1. The action of philanthotoxin 343 (PhTX) on rat homomeric GluR6(Q) recombinant glutamate receptor channels was analysed using concentration-jump techniques and outside-out patches from HEK 293 cells. Both onset and recovery from block by external PhTX were dependent on the presence of agonist, indicating that channels must open for PhTX to bind and that channel closure can trap PhTX. 2. Block by external PhTX developed with double-exponential kinetics. The rate of onset of the fast component of block showed an exponential increase per 27 mV hyperpolarization over the range -40 to -100 mV. The rate of onset of the slow component of block showed a non-linear concentration dependence indicating a rate-limiting step in the blocking mechanism. 3. The extent of block by 1 microM external PhTX was maximal at -40 mV and did not increase with further hyperpolarization; the rate of recovery from block by external PhTX increased 6-fold on hyperpolarization from -40 to -100 mV suggesting that PhTX permeates at negative membrane potentials. 4. Apparent Kd values for block by external PhTX estimated from dose-inhibition experiments decreased 300-fold on hyperpolarization from +40 mV (Kd, 19.6 microM) to -40 mV (Kd, 69 nM); there was little further increase in affinity with hyperpolarization to -80 mV (Kd, 56 nM), consistent with permeation of PhTX at negative membrane potentials. 5. Block by internal PhTX showed complex kinetics and voltage dependence. Analysis with voltage ramps from -120 to +120 mV indicated a Kd at 0 mV of 20 microM, decreasing e-fold per 16 mV depolarization. However, at +90 mV the extent of block by 1 and 10 microM internal PhTX (73 % and 95 %, respectively) reached a maximum and did not increase with further depolarization. 6. Voltage-jump analysis of block by 100 microM internal PhTX revealed partial trapping. With 100 ms jumps from -100 to -40 mV, onset and recovery from block were complete within 5 ms. With jumps of longer duration the extent of block increased, with a time constant of 8.1 s, reaching 84 % at 30 s. On repolarization to -100 mV, recovery from block showed fast and slow components. 7. The amplitude of the slow component of block by internal PhTX showed a biphasic voltage dependence, first increasing then decreasing with progressive depolarization. Maximum block was obtained at 0 mV. 8. Our results suggest that PhTX acts as an open channel blocker; however, provided that the toxin remains bound to the channel, an allosteric mechanism destabilizes the open state, inducing channel closing and trapping PhTX. Strong depolarization for internal PhTX, or strong hyperpolarization for external PhTX, forces the toxin to permeate before it triggers entry into closed blocked states.

A novel allosteric potentiator of AMPA receptors: 4--2-(phenylsulfonylamino)ethylthio--2,6-difluoro-phenoxyaceta mide.

We report that a novel sulfonylamino compound, 4-[2-(phenylsulfonylamino)ethylthio]-2,6-difluoro-phenoxyacetam ide (PEPA), selectively potentiates glutamate receptors of the AMPA subtype. PEPA (1-200 microM) dose dependently potentiated glutamate-evoked currents in Xenopus oocytes expressing AMPA (GluRA-GluRD), but not kainate (GluR6 and GluR6+KA2) or NMDA (zeta1 + epsilon1-epsilon4), receptor subunits. PEPA was effective at micromolar concentrations and, in contrast to the action of cyclothiazide, preferentially modulated AMPA receptor flop isoforms. At 200 microM, PEPA potentiated glutamate responses by 50-fold in oocytes expressing GluRCflop (EC50 approximately 50 microM) versus only threefold for GluRCflip; a similar preference for flop isoforms was observed for other AMPA receptor subunits. Dose-response analysis for GluRCflop revealed that 100 microM PEPA produced a sevenfold increase in AMPA receptor affinity for glutamate. PEPA produced considerably weaker potentiation of kainate-evoked than glutamate-evoked currents, suggesting modulation of the process of receptor desensitization. In human embryonic kidney 293 cells transfected with AMPA receptor subunits, PEPA either abolished or markedly slowed the rate of onset of desensitization and potentiated steady-state equilibrium currents evoked by glutamate with subunit (GluRC >/= GluRD > GluRA) and splice-variant (flop > flip) selectivity similar to that observed in oocytes. Our results show that PEPA is a novel, flop-preferring allosteric modulator of AMPA receptor desensitization at least 100 times more potent than aniracetam.

Permeation and block of rat GluR6 glutamate receptor channels by internal and external polyamines.

1. Polyamine block of rat GluR6(Q) glutamate receptor channels was studied in outside-out patches from transiently transfected HEK 293 cells. With symmetrical 150 mM Na+ and 30 microM internal spermine there was biphasic voltage dependence with 95% block at +40 mV but only 20% block at +140 mV. Dose-inhibition analysis for external spermine also revealed biphasic block; the Kd at +40 mV (54 microM) was lower than at +80 (167 microM) and -80 mV (78 microM). 2. For internal polyamines relief from block was most pronounced for spermine, weaker for N-(4-hydroxyphenylpropanoyl)-spermine (PPS), and virtually absent for philanthotoxin 343 (PhTX 343), suggesting that permeation of polyamines varies with cross-sectional width (spermine, 0.44 nm; PPS, 0.70 nm; PhTX 343, 0.75 nm). 3. With putrescine, spermidine, or spermine as sole external cations, inward currents at -120 mV confirmed permeation of polyamines. For bi-ionic conditions with 90 mM polyamine and 150 mM Na+i, reversal potentials were -12.4 mV for putrescine (permeability ratio relative to Na+, PPut/PNa = 0.42) and -32.7 mV for spermidine (PSpd/PNa = 0.07). Currents carried by spermine were too small to analyse accurately in the majority of patches. 4. Increasing [Na+]i from 44 to 330 mM had no effect on the potential for 50% block (V1/2) by 30 microM internal spermine; however, relief from block at positive membrane potentials increased with [Na+]i. In contrast, raising [Na+]o from 44 to 330 mM resulted in a depolarizing shift in V1/2, indicating a strong interaction between internal polyamines and external permeant ions. 5. The Woodhull infinite barrier model of ion channel block adequately described the action of spermine at membrane potentials insufficient to produce relief from block. For 30 microM internal spermine such analysis gave Kd(O) = 2.5 microM, z theta = 1.97; block by 30 microM external spermine was weaker and less voltage dependent (Kd(O) = 37.8 microM and z delta = 0.55); delta and theta are electrical distances measured from the outside and inside, respectively. 6. Fits of the Woodhull equation for a permeable blocker adequately described both onset and relief from block by spermine over a wide range of membrane potentials. However, the rate constants and z delta values estimated for block by internal spermine predicted much stronger external block than was measured experimentally, and vice versa. 7. An Eyring rate theory model with two energy wells and three barriers explained qualitatively many characteristic features of the action of polyamines on GluRs, including biphasic I-V relationships, weaker block by external than internal spermine and low permeability.