Allosteric Block of KCa2 Channels by Apamin

Activation of small conductance calcium-activated potassium (K_Ca2) channels can regulate neuronal firing and synaptic plasticity. They are characterized by their high sensitivity to the bee venom toxin apamin, but the mechanism of block is not understood. For example, apamin binds to both K_Ca2.2 and K_Ca2.3 with the same high affinity (K_D ∼ 5 pm for both subtypes) but requires significantly higher concentrations to block functional current (IC₅₀ values of ∼100 pm and ∼5 nm, respectively). This suggests that steps beyond binding are needed for channel block to occur. We have combined patch clamp and binding experiments on cell lines with molecular modeling and mutagenesis to gain more insight into the mechanism of action of the toxin. An outer pore histidine residue common to both subtypes was found to be critical for both binding and block by the toxin but not for block by tetraethylammonium (TEA) ions. These data indicated that apamin blocks K_Ca2 channels by binding to a site distinct from that used by TEA, supported by a finding that the onset of block by apamin was not affected by the presence of TEA. Structural modeling of ligand-channel interaction indicated that TEA binds deep within the channel pore, which contrasted with apamin being modeled to interact with the channel outer pore by utilizing the outer pore histidine residue. This multidisciplinary approach suggested that apamin does not behave as a classical pore blocker but blocks using an allosteric mechanism that is consistent with observed differences between binding affinity and potency of block.     

Synthesis and biological evaluation of N-methyl-laudanosine iodide analogues as potential SK channel blockers

Neuronal action potentials are followed by an afterhyperpolarisation (AHP), which is mediated by small conductance Ca2+-activated K+ channels (SK channels or KCa2 channels). This AHP plays an important role in regulating neuronal activity and agents modulating AHP amplitude could have a potential therapeutic interest. It was previously shown that N-methyl-bicuculline iodide blocks SK channels but its GABA) activity represents a serious drawback. In view of the structural analogy between bicuculline and laudanosine 14, several N-quaternary analogues of the latter were developed. It was shown that N-methyl-laudanosine 15 (NML) and N-ethyl-laudanosine 16 induce a reversible and relatively specific blockade of the apamin sensitive AHP in dopaminergic neurones with mean IC50s of 15, and 47 microM, respectively. Laudanosine 14, N-butyl-17 and N-benzyl-18 derivatives were less potent. In order to find pharmacophore elements, modifications were performed at different positions such as C-1, C-6 and C-7. Intracellular recordings on rat midbrain dopaminergic neurones were made in order to evaluate the putative blockade of SK channels by these molecules. Simplified structures such as tetrahydroisoquinoline derivatives with H or Me at C-1 1-6 presented no significant activity at 300 microM. The presence of a 1-(3,4-dimethoxybenzyl) moiety seems an important feature. Indeed, compound 8 showed a blockade of the AHP of only 33% at 300 microM while compound 13 blocked it by 67%, respectively, at the same concentration. Binding experiments were also performed. Binding affinities for SK channels are in good agreement with electrophysiological data. These results indicate that the presence of a charged nitrogen group is an essential point for the affinity on SK channels. Finally, because of the similar activity of both enantiomers of NML 19 and 20, the interaction site may present a symmetrical configuration.     

Evolution of the WANCY region in amniote mitochondrial DNA

In most vertebrate mitochondrial genomes, the site for initiation of light-strand replication, OL, is found within a cluster of five transfer RNA (tRNA) genes (tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), and tRNA(Tyr)). This region and part of the adjacent cytochrome c oxydase subunit I (COI) gene were sequenced for two crocodilian, two turtle, and one snake species and for Sphenodon punctatus; part of the adjacent nicotinamide adenine dinucleotide dehydrogenase subunit 2 (ND2) gene was also sequenced for the crocodilian and turtle species. All had the typical vertebrate gene order. The turtles and the snake have a lengthy noncoding sequence between the tRNA(Asn) and tRNA(Cys) genes that we assumed to be homologous to the mammalian OL. The crocodilians and Sphenodon lack such a sequence, a condition they share with birds. Most proposed phylogenies for the amniotes require that OL at this position was lost at least twice during their diversification or was evolved independently more than once. Within the five tRNA genes, frequencies of substitutions are much higher in loops than in stems. Many loops vary dramatically in size among the species; in the most extreme case, the D-arm of the Sphenodon tRNA(Cys) is a "D-arm replacement" loop of seven nucleotides. Frequency of transitions in stems is relatively uniform across tRNAs, but frequency of transversions varies greatly. Mismatches in stems are infrequent, and their relative frequency in a specific tRNA is unrelated to the frequency of substitution in the corresponding gene. Several features of mammalian mitochondrial tRNAs are conserved in WANCY tRNAs throughout amniotes. The inferred initiation codon for COI is GTG in crocodilians, turtles, and the snake, a condition they share with fishes, certain amphibians, and birds. TTG appears to be the initiation codon for COI in Sphenodon; if correct, this would be a novel initiation codon for vertebrate mitochondrial DNA. Phylogenetic analyses of the inferred amino acid sequences of ND2 and COI support the sister-group relationship of birds and crocodilians and suggest that mammals are an early derived lineage within the amniotes.      

Comparison of the effect of morphine on locus coeruleus noradrenergic and ventral tegmental area dopaminergic neurons in vitro

Extracellular single-cell recordings were performed on rat brain slices to compare the effects of morphine on noradrenergic neurons of the locus coeruleus (LC) and on dopaminergic neurons of the ventral tegmental area (VTA). Morphine inhibited the firing of LC neurons at very low concentrations. The mean IC50 was 13.4 +/- 1nM (mean +/- SEM) (n = 7). Moreover, the inhibitory effect of morphine was identical in slices obtained from rats anesthetized with chloral hydrate or from non-anesthetized rats. On the contrary, morphine did not have any influence on the firing of most VTA neurons (N = 20) up to 100 microM, and did not modify the sensitivity of their autoreceptors (N = 8). It is concluded that morphine potently inhibits the firing of LC neurons in vitro both in slices of anesthetized and not anesthetized animals and has no direct excitatory effect on VTA dopaminergic neurons of the rat.     

Bis-tetrahydroisoquinoline derivatives: AG525E1, a new step in the search for non-quaternary non-peptidic small conductance Ca(2+)-activated K(+) channel blockers

So far, small conductance Ca(2+)-activated K(+) channel (SK) blockers mostly consist of quaternary ammonium derivatives or peptides. Due to their physicochemical properties, these blockers are not suitable to study the physiological roles of SK channels in the central nervous system in vivo. Herein, we report the discovery of a chiral bis-tertiary amine with SK blocking properties from chemical modulation of laudanosine. AG525E1 has an affinity for SK channels (K(i)=293nM) approximately 100-fold higher than the tertiary compound laudanosine (K(i) approximately 30muM) and similar to the charged compound dequalinium (K(i)=221nM). AG525E1 equipotently blocks SK1, SK2 and SK3 currents in transfected cell lines. Because of its basic and lipophilic properties, it can reach central SK targets.     

Effect of intermittent and continuous exposure to electromagnetic fields on cultured hippocampal cells

This study was designed to assess the effect of 50 Hz electromagnetic fields (EMFs) on hippocampal cell cultures in the presence or absence of either sodium nitroprusside (SNP, a NO donor) or Fe2+ induced oxidative stress. One week old cultured rat hippocampal cells were exposed to either intermittent EMFs (IEMFs, 50 Hz, 0-5 mT, 1 min ON/OFF cycles, repeated 10 times every 2 h, 6 times/day during 48 h) or continuous EMFs (CEMFs, 50 Hz, 0-5 mT for 48 h). In a second set of experiments, the effect on such EMFs applied in combination with oxidative stress induced by 0.5 microM Fe2+ or SNP was estimated. At the end of both sets of experiments, cell mortality was assessed by lactate dehydrogenase measurements (LDH). Neither type of exposure to EMFs was observed to modify the basal rate of cell mortality. The exposure to CEMFs in presence of either NO or Fe2+ did not induce any significant increase in cell death. However, when cells were exposed to EMFs in the presence of NO, we observed a significant increase in cell death of 11 and 23% (P<0.001) at 2.5 and 5 mT, respectively. This effect had some specificity because IEMFs did not modify the effect of Fe2+ on cell mortality. Although the effects of IEMFs reported in this study were only observed at very high intensities, our model may prove valuable in trying to identify one cellular target of EMFs.     

Appraisal of the consequences of the DDT-induced bottleneck on the level and geographic distribution of neutral genetic variation in Canadian peregrine falcons, Falco peregrinus

Peregrine falcon populations underwent devastating declines in the mid-20th century due to the bioaccumulation of organochlorine contaminants, becoming essentially extirpated east of the Great Plains and significantly reduced elsewhere in North America. Extensive re-introduction programs and restrictions on pesticide use in Canada and the United States have returned many populations to predecline sizes. A proper population genetic appraisal of the consequences of this decline requires an appropriate context defined by (i) meaningful demographic entities; and (ii) suitable reference populations. Here we explore the validity of currently recognized subspecies designations using data from the mitochondrial control region and 11 polymorphic microsatellite loci taken from 184 contemporary individuals from across the breeding range, and compare patterns of population genetic structure with historical patterns inferred from 95 museum specimens. Of the three North American subspecies, the west coast marine subspecies Falco peregrinus pealei is well differentiated genetically in both time periods using nuclear loci. In contrast, the partitioning of continental Falco peregrinus anatum and arctic Falco peregrinus tundrius subspecies is not substantiated, as individuals from these subspecies are historically indistinguishable genetically. Bayesian clustering analyses demonstrate that contemporary genetic differentiation between these two subspecies is mainly due to changes within F. p. anatum (specifically the southern F. p. anatum populations). Despite expectations and a variety of tests, no genetic bottleneck signature is found in the identified populations; in fact, many contemporary indices of diversity are higher than historical values. These results are rationalized by the promptness of the recovery and the possible introduction of new genetic material.     

A novel phase portrait for neuronal excitability

FIFTY YEARS AGO, FITZHUGH INTRODUCED A PHASE PORTRAIT THAT BECAME FAMOUS FOR A TWOFOLD REASON: it captured in a physiological way the qualitative behavior of Hodgkin-Huxley model and it revealed the power of simple dynamical models to unfold complex firing patterns. To date, in spite of the enormous progresses in qualitative and quantitative neural modeling, this phase portrait has remained a core picture of neuronal excitability. Yet, a major difference between the neurophysiology of 1961 and of 2011 is the recognition of the prominent role of calcium channels in firing mechanisms. We show that including this extra current in Hodgkin-Huxley dynamics leads to a revision of FitzHugh-Nagumo phase portrait that affects in a fundamental way the reduced modeling of neural excitability. The revisited model considerably enlarges the modeling power of the original one. In particular, it captures essential electrophysiological signatures that otherwise require non-physiological alteration or considerable complexification of the classical model. As a basic illustration, the new model is shown to highlight a core dynamical mechanism by which calcium channels control the two distinct firing modes of thalamocortical neurons.     

Synthesis and radioligand binding studies of bis-(8-isopropyl-isoquinolinium) derivatives as ligands for apamin-sensitive sites on cloned SK2 and SK3 channels

A structure-activity relationship study of N-methyl-laudanosine, a SK channel blocker, has indicated that the 6,7-dimethoxy group could be successfully replaced by a hydrophobic moiety such as an isopropyl substituent in position 8 of the isoquinoline ring. In the present study, bis-(8-isopropyl-isoquinolinium) derivatives (2a-e) were synthesized and tested for their affinity for cloned SK2 and SK3 channels in comparison with their 6,7-dimethoxy analogues (4a-f). Several ligands were investigated, both in flexible (propyl, butyl and pentyl) and rigid (m- or p-xylyl) series, the m-xylyl derivative (2d) having the best profile in terms of affinity and selectivity for SK3/SK2 channels. Molecular studies showed that the optimal conformation of compound 2d fits well with our SK pharmacophore model.     

How modeling can reconcile apparently discrepant experimental results: the case of pacemaking in dopaminergic neurons

Midbrain dopaminergic neurons are endowed with endogenous slow pacemaking properties. In recent years, many different groups have studied the basis for this phenomenon, often with conflicting conclusions. In particular, the role of a slowly-inactivating L-type calcium channel in the depolarizing phase between spikes is controversial, and the analysis of slow oscillatory potential (SOP) recordings during the blockade of sodium channels has led to conflicting conclusions. Based on a minimal model of a dopaminergic neuron, our analysis suggests that the same experimental protocol may lead to drastically different observations in almost identical neurons. For example, complete L-type calcium channel blockade eliminates spontaneous firing or has almost no effect in two neurons differing by less than 1% in their maximal sodium conductance. The same prediction can be reproduced in a state of the art detailed model of a dopaminergic neuron. Some of these predictions are confirmed experimentally using single-cell recordings in brain slices. Our minimal model exhibits SOPs when sodium channels are blocked, these SOPs being uncorrelated with the spiking activity, as has been shown experimentally. We also show that block of a specific conductance (in this case, the SK conductance) can have a different effect on these two oscillatory behaviors (pacemaking and SOPs), despite the fact that they have the same initiating mechanism. These results highlight the fact that computational approaches, besides their well known confirmatory and predictive interests in neurophysiology, may also be useful to resolve apparent discrepancies between experimental results.