Structural Model for Phenylalkylamine Binding to L-type Calcium Channels

Phenylalkylamines (PAAs), a major class of L-type calcium channel (LTCC) blockers, have two aromatic rings connected by a flexible chain with a nitrile substituent. Structural aspects of ligand-channel interactions remain unclear. We have built a KvAP-based model of LTCC and used Monte Carlo energy minimizations to dock devapamil, verapamil, gallopamil, and other PAAs. The PAA-LTCC models have the following common features: (i) the meta-methoxy group in ring A, which is proximal to the nitrile group, accepts an H-bond from a PAA-sensing Tyr_IIIS6; (ii) the meta-methoxy group in ring B accepts an H-bond from a PAA-sensing Tyr_IVS6; (iii) the ammonium group is stabilized at the focus of P-helices; and (iv) the nitrile group binds to a Ca²⁺ ion coordinated by the selectivity filter glutamates in repeats III and IV. The latter feature can explain Ca²⁺ potentiation of PAA action and the presence of an electronegative atom at a similar position of potent PAA analogs. Tyr substitution of a Thr in IIIS5 is known to enhance action of devapamil and verapamil. Our models predict that the para-methoxy group in ring A of devapamil and verapamil accepts an H-bond from this engineered Tyr. The model explains structure-activity relationships of PAAs, effects of LTCC mutations on PAA potency, data on PAA access to LTCC, and Ca²⁺ potentiation of PAA action. Common and class-specific aspects of action of PAAs, dihydropyridines, and benzothiazepines are discussed in view of the repeat interface concept.L-type calcium channels (LTCCs)² are targets for different drugs. Benzo(thi)azepines (BTZs), dihydropyridines (DHPs), and phenylalkylamines (PAAs) constitute the three major classes of the LTCC ligands (for reviews, see Refs. 1 and 2). All of these ligands bind to overlapping binding sites in the pore-forming domain of the α₁ subunit, but each class demonstrates unique characteristics of action. Depending on their chemical structure, DHPs act as agonists or antagonists (3). All known PAAs and BTZs are antagonists, but they have different access pathways to their binding sites: external for BTZs (4, 5) and predominantly internal for PAAs (6). Clinical use of verapamil in treatments of hypertension and arrhythmias (7) had stimulated intensive electrophysiological, mutational, and pharmacological studies involving PAAs.The pore-forming domain of LTCC includes the pore-lining inner helices S6, the outer helices S5, and the P-loops from all four repeats of the α₁ subunit. According to mutational analyses, the PAA-binding site is located in the interface between repeats III and IV. In particular, residues in transmembrane helices IIIS5, IIIS6, and IVS6 and P-loops of repeats III and IV contribute to binding of PAAs (8–14).Structure-activity relationships of PAAs were intensively studied (15–17). A common feature of potent PAAs is the presence of two methoxylated aromatic rings (named A and B). The rings are connected by a flexible alkylamine chain with a nitrile and an isopropyl group at the chiral tetrasubstituted carbon atom, which is proximal to ring A. Ring B is proximal to the amino group (see Fig. 1).Open in a separate windowFIGURE 1.Structural formulae of PAAs.Despite the fact that some specific contacts between functional groups of PAAs and PAA-sensing residues (residues that, when mutated, affect action of PAAs) have been proposed (10, 14), the flexibility of the ligands did not allow the characterization of the binding mode and the general pattern of ligand-channel interactions. In the absence of such knowledge, it is hardly possible to provide a molecular basis for structure-activity relationships. The problem is further complicated by the dependence of PAA action on the functional state of the channel, the ionic environment, the transmembrane voltage, and other factors. For example, it is generally believed that PAAs bind to the open/inactivated channels with higher affinities than to the closed state (for review, see Ref 1). However, the molecular basis for this state dependence is unclear.Lipkind and Fozzard (18) docked devapamil in a KcsA-based homology model of the L-type Ca²⁺ channel. They suggested an angular conformation of the drug, with ring B extended into the III/IV repeat interface and ring A in the central cavity. They also suggested that the protonated amino group of devapamil interacts directly with the selectivity filter glutamates. This model explains the effect of some mutations, particularly those in the P-loops and IVS6. However, other important aspects of PAA action such as the role of the nitrile group, the Ca²⁺ potentiation effect, and the effects of mutations in IIIS6 and IIIS5 remain unexplained.The gap between the amount of experimental data on PAA action and the level of understanding of the atomic level mechanisms necessitates further studies. In the absence of x-ray structures of Ca²⁺ channels, molecular modeling is the only available approach to address the structural aspects of PAA-LTCC interactions. Recently, we proposed molecular models for the action of other classes of L-type channel ligands. In the BTZ-LTCC models (19), the main body of the ligands binds in the repeat interface, whereas the amino group protrudes into the inner pore, where it is stabilized by nucleophilic C-terminal ends of the pore helices. In the DHP-LTCC models (20), the ligands also bind in the interface between repeats III and IV, whereas the moieties that differ between agonist and antagonists extend to the pore. Both models suggest direct interactions between the ligands and a Ca²⁺ ion bound to the selectivity filter glutamates in repeats III and IV.In this work, we elaborate molecular models for PAA·LTCC complexes that agree with a large body of experimental data. We further discuss common and different aspects of action of different ligands on LTCC and propose that certain aspects of the ligand action may be relevant to other P-loop channels.     

Proteomic basis of the antibody response to monkeypox virus infection examined in cynomolgus macaques and a comparison to human smallpox vaccination

Monkeypox is a zoonotic viral disease that occurs primarily in Central and West Africa. A recent outbreak in the United States heightened public health concerns for susceptible human populations. Vaccinating with vaccinia virus to prevent smallpox is also effective for monkeypox due to a high degree of sequence conservation. Yet, the identity of antigens within the monkeypox virus proteome contributing to immune responses has not been described in detail. We compared antibody responses to monkeypox virus infection and human smallpox vaccination by using a protein microarray covering 92-95% (166-192 proteins) of representative proteomes from monkeypox viral clades of Central and West Africa, including 92% coverage (250 proteins) of the vaccinia virus proteome as a reference orthopox vaccine. All viral gene clones were verified by sequencing and purified recombinant proteins were used to construct the microarray. Serum IgG of cynomolgus macaques that recovered from monkeypox recognized at least 23 separate proteins within the orthopox proteome, while only 14 of these proteins were recognized by IgG from vaccinated humans. There were 12 of 14 antigens detected by sera of human vaccinees that were also recognized by IgG from convalescent macaques. The greatest level of IgG binding for macaques occurred with the structural proteins F13L and A33R, and the membrane scaffold protein D13L. Significant IgM responses directed towards A44R, F13L and A33R of monkeypox virus were detected before onset of clinical symptoms in macaques. Thus, antibodies from vaccination recognized a small number of proteins shared with pathogenic virus strains, while recovery from infection also involved humoral responses to antigens uniquely recognized within the monkeypox virus proteome.     

Modeling noncompetitive antagonism of a nicotinic acetylcholine receptor

Models of closed and open channel pores of a muscle-type nicotinic acetylcholine receptor (nAChR) channel comprising M1 and M2 segments are presented. A model of the closed channel is proposed in which hydrophobic residues of the Equatorial Leucine ring screen the oxygen domain formed by the Serine ring, thereby preventing ion flux without completely occluding the pore. This model demonstrates a high similarity with the structure derived from a recent electron microscopy study. We propose that hydrophobic residues of the Equatorial Leucine ring are retracted when the pore is open. Our models provide a possible resolution of the nAChR gate controversy. We have also obtained explanations for the complex mechanisms underlying inhibition of nAChR by philanthotoxins (PhTXs). PhTX-343, containing a spermine moiety with a charge of +3, binds deep in the pore near the Serine ring where classical open channel blockers of nAChR bind. In contrast, PhTX-(12), which has a single charged amino group is unable to reach deeply located rings because of steric restrictions. Both philanthotoxins may bind to a hydrophobic site located close to the external entrance of the pore in a region that includes residues associated with the regulation of desensitization.     

Remote effects of occupational and non-occupational exposure to electromagnetic fields of power-line frequency. Epidemiological studies

A retrospective cohort study of mortality in the personnel of power-supply plants in the European regions of Russia was carried out. The exposure of the personnel to electromagnetic fields of power-line frequency (PF) was taken into account. Statistically non-significant raise of mortality from leukemia was found, compared to low mortality rates due to all other causes including cancer of any type. Standardized mortality ratio (SMR) was equal to 2.03 (95% CI = 0.23-7.31). In the retrospective case-control study the haemoblastosis development risk under occupational PF EMF exposure was evaluated. The data of 571 "cases" and 1208 "controls" interview showed that odd ratio (OR) was 1.64 (95% CI = 0.8-3.1). In another retrospective case-control study the risk of the haemoblastosis development in children due to parents PF EMF occupational exposure was evaluated. The data of 208 "cases" and 319 "controls" interview showed that the odd ratio (OR) was 1.69 (95% CI = 0.7-3.3). A retrospective cohort study of mortality in a settlement situated near a high-voltage (500 kV) substation, which took into account PF EMF levels in residential areas, revealed low mortality rates, except leukemia mortality (SMR 1.3; 95% CI = 0.2-7.0). The obtained data do not allow excluding a possibility of PF EMF leukogenic effect.     

Endochitinase activity determination using N-fluorescein-labeled chitin

A fluorimetric method for the determination of endochitinolytic activity using N-fluorescein-labeled chitin (FITC-Chitin) is proposed, and a procedure for FITC-Chitin preparation with a degree of FITC content of 2.2 mol% (one FITC molecule per 45 glucosamine residues) is described. FITC-Chitin is capable to distinguish endochitinase and exochitinase (beta-N-acetylglucosaminidase) activities.     

Electrostatic interactions in catalytic centers of F1-ATPase

The first part of this paper is a brief review of works concerned with the mechanisms of functioning of F0F1-ATP synthases. F0F1-ATP syntheses operate as rotating molecular machines that provide the synthesis of ATP from ADP and inorganic phosphate (Pi) in mitochondria, chloroplasts, and bacteria at the expense of the energy of electrochemical gradient of hydrogen ions generated across energy-transducing mitochondrial, chloroplast or, bacterial membranes. A distinguishing feature of these enzymes is that they operate as rotary molecular motors. In the second part of the work, we calculated the contribution of electrostatic interactions between charged groups of a substrate (MgATP), reaction products (MgADP and Pi), and charged amino acid residues of the F1-ATPase molecule to energy changes associated with the binding of ATP and its chemical transformations in the catalytic centers located at the interface of the alpha- and beta-subunits of the enzyme (oligomer complex alpha 3 beta 3 gamma of bovine mitochondrial ATPase). The catalytic cycle of ATP hydrolysis considered in the work includes conformational changes of alpha- and beta-subunits caused by unidirectional rotations of the central gamma-subunit. The results of our calculations are consistent with the idea that the energetically favorable process of ATP binding to the "open" catalytic center of F1-ATPase initiates the rotation of the gamma-subunit followed by ATP hydrolysis in another ("closed") catalytic center of the enzyme.     

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 GABA_A-receptor mediated currents evoked by application of -aminobutyric acid (GABA) and to antagonize the enhancing effect of the benzodiazepine agonist flurazepam (FZP) on the GABA_A 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 GABA_A-benzodiazepine receptor. This suggests that FLU acts as a partial agonist on GABA_A receptors.     

Recombinant human insulin IX. Investigation of factors,influencing the folding of fusion protein-S-sulfonates,biotechnological precursors of human insulin

The peculiarities of molecular structures and the influence of reaction conditions on the folding efficiency of fusion proteins-biotechnological precursors of human insulin, expressed in Escherichia coli as inclusion bodies have been investigated. The fusion proteins contained proinsulin sequence with various leader peptides connected by an Arg residue to the insulin B-chain. The kind and the size of leader peptide do not have essential influence on folding efficiency. However, the efficiency of protein folding depends on the location of the (His)6 site, which is used for metal-chelating affinity chromatography. In our study the protein folding depends on the reaction medium composition (including additives), the presence of accompanied cell components, pH, temperature, concentrations of protein, and redox agents. A negative influence of nucleic acid and heavy metal ions on folding has been found. S-sulfonated fusion protein has proinsulin-like secondary structure (by CD-spectroscopy data) that is the key point for 95% efficient folding proceeding. Folded fusion proteins are transformed into insulin by enzymatic cleavage.