Denis B. Tikhonov

Kinked-Helices Model of the Nicotinic Acetylcholine Receptor Ion Channel and Its Complexes with Blockers: Simulation by the Monte Carlo Minimization Method

A model of the nicotinic acetylcholine receptor ion channel was elaborated based on the data from electron microscopy, affinity labeling, cysteine scanning, mutagenesis studies, and channel blockade. A restrained Monte Carlo minimization method was used for the calculations. Five identical M2 segments (the sequence EKMTLSISVL10LALTVFLLVI20V) were arranged in five-helix bundles with various geometrical profiles of the pore. For each bundle, energy profiles for chlorpromazine, QX-222, pentamethonium, and other blocking drugs pulled through the pore were calculated. An optimal model obtained allows all of the blockers free access to the pore, but retards them at the rings of residues known to contribute to the corresponding binding sites. In this model, M2 helices are necessarily kinked. They come into contact with each other at the cytoplasmic end but diverge at the synaptic end, where N-termini of M1 segments may contribute to the pore. The kinks disengage alpha-helical H-bonds between Ala12 and Ser8. The uncoupled lone electron pairs of Ser8 carbonyl oxygens protrude into the pore, forming a hydrophilic ring that may be important for the permeation of cations. A split network of H-bonds provides a flexibility to the chains Val9-Ala12, the numerous conformations of which form only two or three intrasegment H-bonds. The cross-ectional dimensions of the interface between the flexible chains vary essentially at the level of Leu11. We suggest that conformational transitions in the chains Val9-Ala12 are responsible for the channel gating, whereas rotations of more stable alpha-helical parts of M2 segments may be necessary to transfer the channel in the desensitized state.

Effect of Flumazenil on GABAA Receptors in Isolated Rat Hippocampal Neurons

Using whole cell patch-clamp recordings from pyramidal cells acutely dissociated from rat hippocampal slices, Ro-15 1788 (flumazenil, FLU) was shown to enhance the GABAA-receptor mediated currents evoked by application of γ-aminobutyric acid (GABA) and to antagonize the enhancing effect of the benzodiazepine agonist flurazepam (FZP) on the GABAA response. Both FLU and FZP increased the peak and the steady-state components of the responses and accelerated the current decay. This suggests that both agents act via a common mechanism on GABA transmission. It is concluded that FLU possesses high affinity for the binding site, but low efficacy on the GABAA-benzodiazepine receptor. This suggests that FLU acts as a partial agonist on GABAA receptors.

Intersegment hydrogen bonds as possible structural determinants of the N/Q/R site in glutamate receptors.

Specific electrophysiological and pharmacological properties of ionic channels in NMDA, AMPA, and kainate subtypes of ionotropic glutamate receptors (GluRs) are determined by the Asn (N), Gln (Q), and Arg (R) residues located at homologous positions of the pore-lining M2 segments (the N/Q/R site). Presumably, the N/Q/R site is located at the apex of the reentrant membrane loop and forms the narrowest constriction of the pore. Although the shorter Asn residues are expected to protrude in the pore to a lesser extent than the longer Gln residues, the effective dimension of the NMDA channel (corresponding to the size of the largest permeant organic cation) is, surprisingly, smaller than that of the AMPA channel. To explain this paradox, we propose that the N/Q/R residues form macrocyclic structures (rings) stabilized by H-bonds between a NH(2) group in the side chain of a given M2 segment and a C==O group of the main chain in the adjacent M2 segment. Using Monte Carlo minimization, we have explored conformational properties of the rings. In the Asn, but not in the Gln ring, the side-chain oxygens protruding into the pore may facilitate ion permeation and accept H-bonds from the blocking drugs. In this way, the model explains different electrophysiological and pharmacological properties of NMDA and non-NMDA GluR channels. The ring of H-bonded polar residues at the pore narrowing resembles the ring of four Thr(75) residues observed in the crystallographic structure of the KcsA K(+) channel.

Folding similarity of the outer pore region in prokaryotic and eukaryotic sodium channels revealed by docking of conotoxins GIIIA, PIIIA, and KIIIA in a NavAb-based model of Nav1.4

Analyses of toxin binding to a homology model of Nav1.4 indicate similar folding of the outer pore region in eukaryotic and prokaryotic sodium channels.

Ligand action on sodium, potassium, and calcium channels: role of permeant ions.

Ion channels are targets for many naturally occurring toxins and small-molecule drugs. Despite great progress in the X-ray crystallography of ion channels, we still do not have a complete understanding of the atomistic mechanisms of channel modulation by ligands. In particular, the importance of the simultaneous interaction of permeant ions with the ligand and the channel protein has not been the focus of much attention. Considering these interactions often allows one to rationalize the highly diverse experimental data within the framework of relatively simple structural models. This has been illustrated in earlier studies on the action of local anesthetics, sodium channel activators, as well as blockers of potassium and calcium channels. Here, we discuss the available data with a view to understanding the use-, voltage-, and current carrying cation-dependence of the ligand action, paradoxes in structure--activity relationships, and effects of mutations in these ion channels.

State-dependent inter-repeat contacts of exceptionally conserved asparagines in the inner helices of sodium and calcium channels

Voltage-gated sodium and calcium channels play key roles in the physiology of excitable cells. The alpha-1 subunit of these channels folds from a polypeptide chain of four homologous repeats. In each repeat, the cytoplasmic halves of the pore-lining helices contain exceptionally conserved asparagines. Such conservation implies important roles, which are unknown. Mutations of the asparagines affect activation and inactivation gating as well as the action of pore-targeting ligands, including local anesthetics and steroidal agonists batrachotoxin and veratridine. In the absence of the open-channel structures, underlying mechanisms are unclear. Here, we modeled the pore module of Cav1.2 and Nav1.4 channels and their mutants in the open and closed states using the X-ray structures of potassium and sodium channels as templates. The energy of each model was Monte Carlo-minimized. The asparagines do not face the pore in the modeled states. In the open-channel models, the asparagine residue in a given repeat forms an inter-repeat H-bond with a polar residue, which is typically nine positions downstream from the conserved asparagine in the preceding repeat. The H-bonds, which are strengthened by surrounding hydrophobic residues, would stabilize the open channel and shape the open-pore geometry. According to our calculation, the latter is much more sensitive to mutations of the asparagines than the closed-pore geometry. Rearrangement of inter-repeat contacts may explain effects of these mutations on the voltage dependence of activation and inactivation and action of pore-targeting ligands.

Homology modeling of Kv1.5 channel block by cationic and electroneutral ligands.

The inner pore of potassium channels is targeted by many ligands of intriguingly different chemical structures. Previous studies revealed common and diverse characteristics of action of ligands including cooperativity of ligand binding, voltage- and use-dependencies, and patterns of ligand-sensing residues. Not all these data are rationalized in published models of ligand-channel complexes. Here we have used energy calculations with experimentally defined constraints to dock flecainide, ICAGEN-4, benzocaine, vernakalant, and AVE0118 into the inner pore of Kv1.5 channel. We arrived at ligand-binding models that suggest possible explanations for different values of the Hill coefficient, different voltage dependencies of ligands action, and effects of mutations of residues in subunit interfaces. Two concepts were crucial to build the models. First, the inner-pore block of a potassium channel requires a cationic blocking particle. A ligand, which lacks a positively charged group, blocks the channel in a complex with a permeant ion. Second, hydrophobic moieties of a flexible ligand have a tendency to bind in hydrophobic subunit interfaces.

Analysis of inter-residue contacts reveals folding stabilizers in P-loops of potassium, sodium, and TRPV channels.

The family of P-loop channels includes potassium, sodium, calcium, cyclic nucleotide-gated and TRPV channels, as well as ionotropic glutamate receptors. Despite vastly different physiological and pharmacological properties, the channels have structurally conserved folding of the pore domain. Furthermore, crystallographic data demonstrate surprisingly similar mutual disposition of transmembrane and membrane-diving helices. To understand determinants of this conservation, here we have compared available high-resolution structures of sodium, potassium, and TRPV1 channels. We found that some residues, which are in matching positions of the sequence alignment, occur in different positions in the 3D alignment. Surprisingly, we found 3D mismatches in well-packed P-helices. Analysis of energetics of individual residues in Monte Carlo minimized structures revealed cyclic patterns of energetically favorable inter- and intra-subunit contacts of P-helices with S6 helices. The inter-subunit contacts are rather conserved in all the channels, whereas the intra-subunit contacts are specific for particular types of the channels. Our results suggest that these residue–residue contacts contribute to the folding stabilization. Analysis of such contacts is important for structural and phylogenetic studies of homologous proteins.

Mechanisms of blockade of glutamate receptor ionic channels: Paradox of 9-aminoacridine

Abstract9-Aminoacridine and tacrine differ from other channel blockers of NMDA receptors in that their binding prevents the closing of blocked channels and subsequent dissociation of the agonist. Structural determinants of aminoacridine derivatives underlying the blocking mechanism are still unknown. The aim of this study was to elucidate the effects of a dicationic 9-aminoacridine derivative and some other tricyclic compounds on NMDA receptors of rat hippocampal pyramidal neurons. All the compounds under study are voltage-dependent blockers of NMDA channels; their IC50 values recorded at −80 mV vary from 1 to 50 µM. The dicationic derivatives demonstrate the same voltage dependence of the block as the monocationic derivatives. The monoand dicationic tricyclic compounds under study are weak blockers of AMPA receptor channels and differ from adamantane, phenylcyclohexyl and other dicationic derivatives that exhibit greater voltage dependence of the NMDA channel block and are able to induce effective suppression of AMPA channels. We conclude that the mechanisms of action of the tricyclic and dicationic 9-aminoacridine derivatives are different from that of 9-aminoacridine, since these compounds do not prevent closing of the blocked channels. This suggests that the binding site for 9-aminoacridine has specific properties and high selectivity with respect to ligand structure.

Properties of spontaneous and miniature excitatory postsynaptic currents in neurons of the rat prefrontal cortex

Quantum analysis of postsynaptic currents is important for fundamental and applied studies of synaptic transmission and plasticity. In the present work, we investigated the possibility of using the characteristics of spontaneous excitatory postsynaptic currents (EPSCs) for estimation of quantum parameters of excitatory synaptic transmission in different types of neurons from rat prefrontal cortex slices. By blocking spontaneous spiking activity in slices by tetrodotoxin, we showed that spontaneous and miniature EPSCs in the prefrontal cortex neurons did not differ in their properties. Therefore, both spontaneous and miniature responses can be used for estimation of quantum parameters of excitatory synaptic transmission in this preparation. We also revealed that excitatory spontaneous responses of pyramidal cells were two times lower by amplitude, had a twice lower coefficient of variation and exhibited much slower kinetics than responses of the fast-spiking and regular-spiking interneurons. Possible mechanisms of these differences are discussed.