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.     

Regulation of metabolism and contraction by cytoplasmic calcium in the intestinal smooth muscle

Reduced pyridine nucleotides (PNred) and oxidized flavoproteins (FPox) were measured fluorometrically in the intestinal smooth muscle strip of guinea pig taenia caeci simultaneously with contractile tension. Cytoplasmic free Ca2+ levels ([Ca2+]cyt) were also measured by a fura-2-Ca2+ fluorescence technique. PNred, FPox, and [Ca2+]cyt increased during spontaneous contraction or upon the addition of high K+ or carbachol and decreased upon the removal of these stimulants. [Ca2+]cyt increased before the increase in muscle tension. PNred increased almost simultaneously with or immediately after the onset of contraction, while FPox increased before the initiation of contraction. Both PNred and FPox decreased a few seconds after the initiation of relaxation. In the K+-depolarized, Ca2+-depleted muscle, graded elevation of external Ca2+ increased PNred, FPox, and muscle tension. The sensitivity to Ca2+ was in the order of FPox greater than PNred greater than muscle tension. Changes in PNred were inhibited when glycolysis was inhibited by substitution of external glucose with oxaloacetate, pyruvate, or beta-hydroxybutylate, but not when oxidative phosphorylation was inhibited by N2 bubbling or by NaCN. In contrast to this, changes in the FPox were inhibited by N2 bubbling or NaCN, but not by the inhibition of glycolysis. These results suggest that an elevation of intracellular Ca2+ activates carbohydrate metabolism and contractile elements independently, resulting in the reduction of cytoplasmic pyridine nucleotides, oxidation of mitochondrial flavoproteins, and development of tension in the intestinal smooth muscle.     

The structure and function of drug pumps

Resistance to drugs has emerged in biological systems as diverse as cancer cells undergoing chemotherapy and microbial pathogens undergoing treatment with antimicrobials. This medical problem is escalating and there is an urgent need for the development of new classes of drugs. In the case of pathogenic bacteria, we are rapidly approaching a scenario where there will be no effective antibiotics in the armoury of drugs available for treating the infectious diseases that these bacteria cause, returning us to the pre-antibiotic era when infectious diseases were rife because they were untreatable. One of the most frequently employed resistance strategies in both prokaryotes and eukaryotes is the transmembrane-protein-catalysed extrusion of drugs from the cell, with these proteins acting like bilge pumps, reducing the intracellular drug concentration to subtoxic levels. There is currently much scientific interest in understanding how these pumps operate, so that we might design transport inhibitors that would block them, allowing a renaissance for drugs that are no longer effective owing to their efflux.     

Remnant cationic dendrimers block RNA migration in electrophoresis after monophasic lysis

Cationic dendrimers such as poly(amidoamine) (PAMAM) and poly(propyleneimine) (PPI) have attractive characteristics for the delivery of nucleic acid and various biomedical applications. Most studies have focused on cationic dendrimer-based intracellular delivery, and very few studies have focused on the non-specific interaction of remnant cationic dendrimers with total RNA after isolation directly from cells in vitro. We examined RNA isolation using the common method of monophasic lysis from human macrophage-like cells (U937) and mouse fibroblast cells (NIH/3T3) that had been exposed to dendrimers and DNA/dendrimer complexes using gel electrophoresis. We found that PAMAM and PPI dendrimers strongly altered the mobility of RNA in the gels. In addition, the extent of dendrimer-induced alteration in RNA mobility was directly dendrimer-generation-dependent: the alteration was greater with higher-generation dendrimers. We also found that DNA/dendrimer complexes at higher dendrimer to DNA ratios interacted with RNA after isolation while gene expression was maintained. The interactions between RNA and remnant dendrimers after isolation were caused by electrostatic bindings, and we recovered total RNA using high ionic strength solvents (2M NaCl solution) to disrupt the electrostatic forces binding dendrimers to RNA. Because RNA isolation is routinely used for biological applications, such dendrimer-induced alteration in RNA mobility should be accounted for in the further processing of RNA-related applications.     

Cloning and characterization of novel snake venom proteins that block smooth muscle contraction.

In this study, we isolated a 25-kDa novel snake venom protein, designated ablomin, from the venom of the Japanese Mamushi snake (Agkistrodon blomhoffi). The amino-acid sequence of this protein was determined by peptide sequencing and cDNA cloning. The deduced sequence showed high similarity to helothermine from the Mexican beaded lizard (Heloderma horridum horridum), which blocks voltage-gated calcium and potassium channels, and ryanodine receptors. Ablomin blocked contraction of rat tail arterial smooth muscle elicited by high K+-induced depolarization in the 0.1-1 microm range, but did not block caffeine-stimulated contraction. Furthermore, we isolated three other proteins from snake venoms that are homologous to ablomin and cloned the corresponding cDNAs. Two of these homologous proteins, triflin and latisemin, also inhibited high K+-induced contraction of the artery. These results indicate that several snake venoms contain novel proteins with neurotoxin-like activity.     

Allometric scaling relationship between frequency of intestinal contraction and body size in rodents and rabbits

This study aimed to establish an allometric scaling relationship between the frequency of intestinal contractions and body mass of different mammalian species. The frequency of intestinal contractions of rabbit, guinea pig, rat and mouse were measured using an isolated organ system. The isolated rings were prepared from proximal segments of jejunums and the frequency of contractions was recorded by an isometric force procedure. The coefficients of the obtained allometric equation were ascertained by computation of least squares after logarithmic transformation of both body mass and frequency. Significant differences (p?<?0.001) were shown in the frequency of contractions between different species. The highest frequency that corresponded to the mice was 57.7 min^?1 and the 95% confidence interval (CI) ranged from 45.4 to 70, while rabbits showed the lowest frequency (12.71 min^?1, CI: 8.6–16.8). Logarithms of frequency were statistically proportional to logarithms of body mass (r?=?0.99; p?<?0.001). The data fitted an equation $ \mathrm{F}=18.51{{\mathrm{B}}^{-0.31 }} $ and the 95% confidence interval of the exponent ranged from ?0.30 to ?0.32. The results of this study suggest that it is probably possible to extrapolate the intestinal contraction frequency of other mammalian species by the means of allometry scaling.     

The mechanochemistry of V-ATPase proton pumps

The vacuolar H(+)-ATPases (V-ATPases) are a universal class of proton pumps that are structurally similar to the F-ATPases. Both protein families are characterized by a membrane-bound segment (V(o), F(o)) responsible for the translocation of protons, and a soluble portion, (V(1), F(1)), which supplies the energy for translocation by hydrolyzing ATP. Here we present a mechanochemical model for the functioning of the V(o) ion pump that is consistent with the known structural features and biochemistry. The model reproduces a variety of experimental measurements of performance and provides a unified view of the many mechanisms of intracellular pH regulation.     

A plant Kunitz-type inhibitor mimics bradykinin-induced cytosolic calcium increase and intestinal smooth muscle contraction

Abstract: BbKI is a kallikrein inhibitor with a reactive site sequence similar to that of kinins, the vasoactive peptides inserted in kininogen moieties. This structural similarity probably contributes to the strong interaction with plasma kallikrein, the enzyme that releases, from high-molecular weight kininogen (HMWK), the proinflammatory peptide bradykinin, which acts on B2 receptors (B2R). BbKI was examined on smooth muscle contraction and Ca2+ mobilization, in which the kallikrein-kinin system is involved. Contrary to expectations, BbKI (1.8 μm) increased [Ca2+]cand contraction, as observed with BK (2.0 μm). Not blocked by B1 receptors (B1R), the BbKI agonistic effect was blocked by the B2R antagonist, HOE-140 (6 μm), and the involvement of B2R was confirmed in B2R-knockout mice intestine. The same tissue response was obtained using a synthetic peptide derived from the BbKI reactive site structure, more resistant than BK to angiotensin I-converting enzyme (ACE) hydrolysis. Depending on the concentration, BbKI has a dual effect. At a low concentration, BbKI acts as a potent kallikrein inhibitor; however, due to the similarity to BK, in high concentrations, BbKI greatly increases Ca2+ release from internal storages, as a consequence of its interaction with B2R. Therefore, the antagonistic and agonistic effects of BbKI may be considered in conditions of B2R involvement.     

Neuropeptide Y effects on vasorelaxation and intestinal contraction in the Atlantic cod Gadus morhua

Neuropeptide Y (NPY) has prominent cardiovascular effects in mammals and sharks, but no such effect has previously been demonstrated in any teleost fish. In the Atlantic cod, we found that cod NPY (10(-10)-10(-6) M) relaxed celiac arteries precontracted with epinephrine, and weak contractions were elicited in intestinal ring preparations. A few NPY-immunoreactive nerve fibers were present along small gut arteries. The results suggest that cod NPY produces vasorelaxation both by a direct action on smooth muscle and by release of prostaglandins, but with no involvement of nitric oxide, leukotrienes, or endothelium-derived relaxing factors. An additional indirect effect involving another neurotransmitter may occur. Cod NPY (10(-7) M) and human NPY (10(-7) M) had identical effects on the vessels. Small differences only in the effects of porcine [Leu(31),Pro(34)]NPY, NPY-(13-36), and cod NPY suggest the presence of a Y(1) subfamily receptor, similar to the zebrafish Ya receptor. A physiological role for NPY in teleost vasculature is concluded, but surprisingly the effect, a vasodilation, is opposite to that in mammals and is mediated by prostaglandins.     

Role of myosin in terminal web contraction in isolated intestinal epithelial brush borders

We have investigated the role of myosin in contraction of the terminal web in brush borders isolated from intestinal epithelium. At 37 degrees C under conditions that stimulate terminal web contraction (1 microM Ca++ and ATP), most (60-70%) of the myosin is released from the brush border. Approximately 80% of the myosin is also released by ATP at 0 degree C, in the absence of contraction. Preextraction of this 80% of the myosin from brush borders with ATP has no effect on either the time course or extent of subsequently stimulated contraction. However, contraction is inhibited by removal of all of the myosin with 0.6 M KCl and ATP. Contraction is also inhibited by an antibody to brush border myosin, which inhibits both the ATPase activity of brush border myosin and its ability to form stable bipolar polymers. These results indicate that although functional myosin is absolutely required for terminal web contraction only approximately 20% of the brush border myosin is actually necessary. This raises the possibility that there are at least two different subsets of myosin in the terminal web.     

Effect of polymer structure on micelles formed between siRNA and cationic block copolymer comprising thiols and amidines

Small interfering RNA (siRNA) has great therapeutic potential for the suppression of proteins associated with disease, but delivery methods are needed for improved efficacy. Here, we investigated the properties of micellar siRNA delivery vehicles prepared with poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLL) comprising lysine amines modified to contain amidine and thiol functionality. Lysine modification was achieved using 2-iminothiolane (2-IT) [yielding PEG-b-PLL(N2IM-IM)] or dimethyl 3,3'-dithiobispropionimidate (DTBP) [yielding PEG-b-PLL(MPA)], with modifications aimed to impart disulfide cross-linking ability without compromising cationic charge. These two lysine modification reagents resulted in vastly different chemistry contained in the reacted block copolymer, which affected micelle formation behavior and stability along with in vitro and in vivo performance. Amidines formed with 2-IT were unstable and rearranged into a noncharged ring structure lacking free thiol functionality, whereas amidines generated with DTBP were stable. Micelles formed with siRNA and PEG-b-PLL(N2IM-IM) at higher molar ratios of polymer/siRNA, while PEG-b-PLL(MPA) produced micelles only near stoichiometric molar ratios. In vitro gene silencing was highest for PEG-b-PLL(MPA)/siRNA micelles, which were also more sensitive to disruption under disulfide-reducing conditions. Blood circulation was most improved for PEG-b-PLL(N2IM-IM)/siRNA micelles, with a circulation half-life 3× longer than naked siRNA. Both micelle formulations are promising for siRNA delivery applications in vitro and in vivo.     

The efficiency of muscle contraction

When a muscle contracts and shortens against a load, it performs work. The performance of work is fuelled by the expenditure of metabolic energy, more properly quantified as enthalpy (i.e., heat plus work). The ratio of work performed to enthalpy produced provides one measure of efficiency. However, if the primary interest is in the efficiency of the actomyosin cross-bridges, then the metabolic overheads associated with basal metabolism and excitation-contraction coupling, together with those of subsequent metabolic recovery process, must be subtracted from the total heat and work observed. By comparing the cross-bridge work component of the remainder to the Gibbs free energy of hydrolysis of ATP, a measure of thermodynamic efficiency is achieved. We describe and quantify this partitioning process, providing estimates of the efficiencies of selected steps, while discussing the errors that can arise in the process of quantification. The dependence of efficiency on animal species, fibre-type, temperature, and contractile velocity is considered. The effect of contractile velocity on energetics is further examined using a two-state, Huxley-style, mathematical model of cross-bridge cycling that incorporates filament compliance. Simulations suggest only a modest effect of filament compliance on peak efficiency, but progressively larger gains (vis-à-vis the rigid filament case) as contractile velocity approaches Vmax. This effect is attributed primarily to a reduction in the component of energy loss arising from detachment of cross-bridge heads at non-zero strain.