Zinc potentiation of the glycine receptor chloride channel is mediated by allosteric pathways

Molecular mechanisms of zinc potentiation were investigated in recombinant human alpha 1 glycine receptors (GlyRs) by whole-cell patch-clamp recording and [H-3]strychnine binding assays. In the wild-type (WT) GlyR, 1 mu M zinc enhanced the apparent binding affinity of the agonists glycine and taurine and reduced their concentrations required for half-maximal activation. Thus, in the WT GlyR, zinc potentiation apparently occurs by enhancing agonist binding. However, analysis of GlyRs incorporating mutations in the membrane-spanning domain M1-M2 and M2-M3 loops, which are both components of the agonist gating mechanism, indicates that most mutations uncoupled zinc potentiation from glycine-gated currents but preserved zinc potentiation of taurine-gated currents. One such mutation in the M2-M3 loop, L274A, abolished the ability of zinc to potentiate taurine binding but did not inhibit zinc potentiation of taurine-gated currents. In this same mutant where taurine acts as a partial agonist, zinc potentiated taurine-gated currents but did not potentiate taurine antagonism of glycine-gated currents, suggesting that zinc interacts selectively with the agonist transduction pathway. The intracellular M246A mutation, which is unlikely to bind zinc, also disrupted zinc potentiation of glycine currents. Thus, zinc potentiation of the GlyR is mediated via allosteric mechanisms that are independent of its effects on agonist binding.

Monepantel irreversibly binds to and opens Haemonchus contortus MPTL-1 and Caenorhabditis elegans ACR-20 receptors.

Monepantel is a recently developed anthelmintic with a novel mode of action. Parasitic nematodes with reduced sensitivity to monepantel have led to the identification of MPTL-1, a ligand-gated ion-channel subunit of the parasitic nematode Haemonchus contortus, as a potential drug target. Homomeric MPTL-1 channels reconstituted in Xenopus oocytes are gated by µM concentrations of betaine and mM concentrations of choline. Measurement of reversal potentials indicated that the channel has a similar conductance for Na(+) and K(+) ions and does not permeate Ca(2+). Concentrations of monepantel (amino-acetonitrile derivative [AAD]-2225) >0.1 μM, but not its inactive enantiomer AAD-2224, induced channel opening in an irreversible manner. Currents elicited by monepantel alone were larger than the maximal current amplitudes achieved with betaine or choline, making monepantel a superagonist. Currents elicited by betaine or choline were allosterically potentiated by nM concentrations of monepantel and to a much smaller degree by AAD-2224. We have also reconstituted the Caenorhabditis elegans homomeric ACR-20 receptor in Xenopus oocytes. The acr-20 sequence has higher similarity to mptl-1 than acr-23, the primary target for monepantel mode of action in C. elegans. The ACR-20 channel is gated similarly as MPTL-1. Monepantel, but not AAD-2224, was able to induce channel opening in an irreversible manner at similar concentrations as for MPTL-1. Interestingly, the allosteric potentiation measured in the presence of betaine was much smaller than in MPTL-1 receptors. Together, these results establish the mode of action of monepantel in H. contortus and contribute to our understanding of the mode of action of this anthelmintic.

Structural analogues of the natural products magnolol and honokiol as potent allosteric potentiators of GABA(A) receptors.

Biphenylic compounds related to the natural products magnolol and 4'-O-methylhonokiol were synthesized, evaluated and optimized as positive allosteric modulators (PAMs) of GABA(A) receptors. The most efficacious compounds were the magnolol analog 5-ethyl-5'-hexylbiphenyl-2,2'-diol (45) and the honokiol analogs 4'-methoxy-5-propylbiphenyl-2-ol (61), 5-butyl-4'-methoxybiphenyl-2-ol (62) and 5-hexyl-4'-methoxybiphenyl-2-ol (64), which showed a most powerful potentiation of GABA-induced currents (up to 20-fold at a GABA concentration of 3μM). They were found not to interfere with the allosteric sites occupied by known allosteric modulators, such as benzodiazepines and N-arachidonoylglycerol. These new PAMs will be useful as pharmacological tools and may have therapeutic potential for mono-therapy, or in combination, for example, with GABA(A) receptor agonists.

Adenosine triphosphate acts as both a competitive antagonist and a positive allosteric modulator at recombinant N-methyl-D-aspartate receptors

ATP and glutamate are fast excitatory neurotransmitters in the central nervous system acting primarily on ionotropic P2X and glutamate [N-methyl-D-aspartate (NMDA) and non-NMDA] receptors, respectively. Both neurotransmitters regulate synaptic plasticity and long-term potentiation in hippocampal neurons. NMDA receptors are responsible primarily for the modulatory action of glutamate, but the mechanism underlying the modulatory effect of ATP remains uncertain. In the present study, the effect of ATP on recombinant NR1a + 2A, NR1a + 2B, and NR1a + 2C NMDA receptors expressed in Xenopus laevis oocytes was investigated. ATP inhibited NR1a + 2A and NR1a + 2B receptor currents evoked by low concentrations of glutamate but potentiated currents evoked by saturating glutamate concentrations. In contrast, ATP potentiated NR1a + 2C receptor currents evoked by nonsaturating glutamate concentrations. ATP shifted the glutamate concentration-response curve to the right, indicating a competitive interaction at the agonist binding site. ATP inhibition and potentiation of glutamate-evoked currents was voltage-independent, indicating that ATP acts outside the membrane electric field. Other nucleotides, including ADP, GTP, CTP, and UTP, inhibited glutamate-evoked currents with different potencies, revealing that the inhibition is dependent on both the phosphate chain and nucleotide ring structure. At high concentrations, glutamate outcompetes ATP at the agonist binding site, revealing a potentiation of the current. This effect must be caused by ATP binding at a separate site, where it acts as a positive allosteric modulator of channel gating. A simple model of the NMDA receptor, with ATP acting both as a competitive antagonist at the glutamate binding site and as a positive allosteric modulator at a separate site, reproduced the main features of the data.

Allosteric Control of Gating Mechanisms Revisited: The Large Conductance Ca(2+)-Activated K(+) Channel

Large-conductance Ca(2+)-activated K(+) channels (BK) play a fundamental role in modulating membrane potential in many cell types. The gating of BK channels and its modulation by Ca(2+) and voltage has been the subject of intensive research over almost three decades, yielding several of the most complicated kinetic mechanisms ever proposed. A large number of open and closed states disposed, respectively, in two planes, named tiers, characterize these mechanisms. Transitions between states in the same plane are cooperative and modulated by Ca(2+). Transitions across planes are highly concerted and voltage-dependent. Here we reexamine the validity of the two-tiered hypothesis by restricting attention to the modulation by Ca(2+). Large single channel data sets at five Ca(2+) concentrations were simultaneously analyzed from a Bayesian perspective by using hidden Markov models and Markov-chain Monte Carlo stochastic integration techniques. Our results support a dramatic reduction in model complexity, favoring a simple mechanism derived from the Monod-Wyman-Changeux allosteric model for homotetramers, able to explain the Ca(2+) modulation of the gating process. This model differs from the standard Monod-Wyman-Changeux scheme in that one distinguishes when two Ca(2+) ions are bound to adjacent or diagonal subunits of the tetramer.

Acute effects and postactivation potentiation in the special judo fitness test

Miarka, B, Del Vecchio, FB, and Franchini, E. Acute effects and postactivation potentiation in the special judo fitness test. J Strength Cond Res 25(2): 427-431, 2011-The purpose of this study was to compare the acute short-term effects of (1) plyometric exercise, (2) combined strength and plyometric exercise (contrast), and (3) maximum strength performance in the Special Judo Fitness Test (SJFT). Eight male judo athletes (mean +/- SD, age, 19 +/- 1 years; body mass, 60.4 +/- 5 kg; height, 168.3 +/- 5.4 cm) took part in this study. Four different sessions were completed; each session had 1 type of intervention: (a) SJFT control, (b) plyometric exercises + SJFT, (c) maximum strength + SJFT, and (d) contrast + SJFT. The following variables were quantified: throws performed during series A, B, and C; total number of throws; heart rate immediately and 1 minute after the test; and test index. Significant differences were found in the number of throws during series A: the plyometric exercise (6.4 +/- 0.5 throws) was superior (p < 0.05) to the control condition (5.6 +/- 0.5 throws). Heart rate 1 minute after the SJFT was higher (p < 0.01) during the plyometric exercise (192 +/- 8 bpm) than during the contrast exercise (184 +/- 9 bpm). The contrast exercise (13.58 +/- 0.72) resulted in better index values than the control (14.67 +/- 1.30) and plyometric exercises (14.51 +/- 0.54). Thus, this study suggests that contrast and plyometric exercises performed before the SJFT can result in improvements in the test index and anaerobic power of judo athletes, respectively.

Influence of strength training background on postactivation potentiation response

Batista, MAB, Roschel, H, Barroso, R, Ugrinowitsch, C, and Tricoli, V. Influence of strength training background on postactivation potentiation response. J Strength Cond Res 25(9): 2496-2502, 2011-The aim of this study was to evaluate the influence of the subjects` level of maximal dynamic strength and training background on postactivation potentiation (PAP). A group of 23 subjects, composed of power track-and-field athletes (PT = 8), bodybuilders (BB = 7), and physically active subjects (PA = 8), participated in the study. Maximal dynamic strength (1 repetition maximum test) was assessed in the leg press exercise for subjects` characterization. Their countermovement vertical jump (CMJ) performance was assessed before and after 2 different conditioning activity (CA) protocols (1 or 3 maximum voluntary isometric contractions [MVICs] of 5-second duration in the leg press exercise) or after control (no CA), performed on separate days. No significant differences among groups were found for CMJ height or take-off velocity after any of the CA protocols (p <= 0.05). However, individual analysis showed that some subjects increased performance in response to the CA, despite their previous training history. We concluded that subjects` level of maximal dynamic strength and training background have no influence on PAP manifestation. Our data suggest that coaches should individually identify the athletes that are PAP responders before introducing MVICs as part of their warm-up routines.

Potentiation of dapsone induced methemoglobinemia by N-acetylcysteinc in rats

Dapsone (DDS) (4,4` diaminodiphenylsulfone), the drug of choice for the treatment of leprosy, frequently induces hemolytic anemia and methemoglobinemia. N-hydroxylation, one of the major pathways of biotransformation, has been constantly related to the methemeglobinemia after the use of the drug. In order to prevent the dapsone-induced hemotoxicity, N-acetylcysteine, a drug precursor of glutathione, was administered in combination with DDS to male Wistar rats, weighting 220-240 g. The animals were then anaesthetized and blood was collected from the aorta for determination of plasma DDS concentration by HPLC, determination of methemoglobinemia and glutathione by spectrophotometry, and for biochemical and hematological parameters. Our results showed that N-acetylcysteine enhanced dapsone-induced methemoglobinemia due to increased dapasone plasmatic concentration and consequent increased N-hydroxylamine formation. We concluded that drug interactions with dapsone require individually studies in order to avoid undesirable effects of dapsone.

Long-term potentiation of synaptic transmission in the avian hippocampus

The avian hippocampus plays a pivotal role in memory required for spatial navigation and food storing. Here we have examined synaptic transmission and plasticity within the hippocampal formation of the domestic chicken using an in vitro slice preparation. With the use of sharp microelectrodes we have shown that excitatory synaptic inputs in this structure are glutamatergic and activate both NMDA-and AMPA-type receptors on the postsynaptic membrane. In response to tetanic stimulation, the EPSP displayed a robust long-term potentiation (LTP) lasting >1 hr. This LTP was unaffected by blockade of NMDA receptors or chelation of postsynaptic calcium. Application of forskolin increased the EPSP and reduced paired-pulse facilitation: (PPF), indicating an increase in release probability. In contrast, LTP was not associated with a change in the PPF ratio. Induction of LTP did not occlude the effects of forskolin. Thus, in contrast to NMDA receptor-independent LTP in the mammalian brain, LTP in the chicken hippocampus is not attributable to a change in the probability of transmitter release and does not require activation of adenylyl cyclase, These findings indicate that a novel form of synaptic plasticity might underlie learning in the avian hippocampus.

Calcium-permeable AMPA receptors mediate long-term potentiation in interneurons in the amygdala

Fear conditioning is a paradigm that has been used as a model for emotional learning in animals'. The cellular correlate of fear conditioning is thought to be associative N-methyl-D-aspartate (NMDA) receptor-dependent synaptic plasticity within the amygdala(1-3). Here we show that glutamatergic synaptic transmission to inhibitory interneurons in the basolateral amygdala is mediated solely by alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. In contrast to AMPA receptors at inputs to pyramidal neurons, these receptors have an inwardly rectifying current-voltage relationship, indicative of a high permeability to calcium(4 5), Tetanic stimulation of inputs to interneurons caused an immediate and sustained increase in the efficacy of these synapses. This potentiation required a rise in postsynaptic calcium, but was independent of NMDA receptor activation. The potentiation of excitatory inputs to interneurons was reflected as an increase in the amplitude of the GABAA-mediated inhibitory synaptic current in pyramidal neurons. These results demonstrate that excitatory synapses onto interneurons within a fear conditioning circuit show NMDA-receptor independent long-term potentiation. This plasticity might underlie the increased synchronization of activity between neurons in the basolateral amygdala after fear conditioning(6).

Sequential increase in Egr-1 and AP-1 DNA binding activity in the dentate gyrus following the induction of long-term potentiation

Establishment of long-term potentiation (LTP) at perforant path synapses is highly correlated with increased expression of Egr and AP-1 transcription factors in rat dentate gyrus granule cells. We have investigated whether increased transcription factor levels are reflected in increased transcription factor activity by assessing Egr and AP-I DNA binding activity using gel shift assays. LTP produced an increase in binding to the Egr element, which was NMDA receptor-dependent and correlated closely with our previously reported increase in Egr-1 (zif/268) protein levels. Supershift analysis confirmed involvement of Egr-1, but not Egr-2 in the DNA binding activity. AP-1 DNA binding was also rapidly elevated in parallel with protein levels, however, the peak increase in activity was delayed until 4 h, a time point when we have previously shown that only jun-D protein was elevated. These data indicate that binding of Egr-1 and AP-1 to their response elements is increased in two phases. This may result in activation of distinct banks of target genes which contribute to the establishment of persistent LTP. (C) 2000 Elsevier Science B.V. All rights reserved.

The crustacean gill (Na(+),K(+))-ATPase: Allosteric modulation of high- and low-affinity ATP-binding sites by sodium and potassium

The blue crab, Callinectes danae, tolerates exposure to a wide salinity range employing mechanisms of compensatory ion uptake when in dilute media. Although the gill (Na(+), K(+))-ATPase is vital to hyperosmoregulatory ability, the interactions occurring at the sites of ATP binding on the molecule itself are unknown. Here, we investigate the modulation by Na(+) and K(+) of homotropic interactions between the ATP-binding sites, and of phosphoenzyme formation of the (Na(+),K(+))-ATPase from the posterior gills of this euryhaline crab. The contribution of the high- and low-affinity ATP-binding sites to maximum velocity was similar for both Na(+) and K(+). However, in contrast to Na(+), a threshold K(+) concentration triggers the appearance of the high-affinity binding sites, displacing the saturation curve to lower ATP concentrations. Further, a low-affinity site for phosphorylation is present on the enzyme. These findings reveal notable differences in the catalytic mechanism of the crustacean (Na(+),K(+))-ATPase compared to the vertebrate enzyme. (C) 2008 Elsevier Inc. All rights reserved.

Startle-potentiation during nonaversive human conditioning

Fear-potentiated startle is a well-established measure of emotional learning in nonhuman animals. In humans, startle potentiation in anticipation of an aversive unconditional stimulus (US) has been interpreted as reflecting the same emotional process. This interpretation was supported by previous failures to fmd startle potentiation in anticipation of nonaversive USs, reactiontime tasks. The present research questions these results. Experiment 1 found startle-potentiation in anticipation of an aversive US, which resulted in increased dislike of the conditional stimulus (CS), and in anticipation of a nonaversive US, which did not affect CS valence. Experiment 2 replicated the latter finding, indicating that provision of performance feedback enhanced the salience of the reaction time task USs and thus anticipatory startle potentiation. The present results pose problems for the interpretation of fmdings of potentiated startle in human-aversive conditioning as reflecting emotion. Rather, startle potentiation during aversive and non-aversive conditioning may reflect the attentional processes known to occur during human-associative learning.