Structural model of the Ca V 1.2 pore

Understanding the structure and functional mechanisms of voltage-gated calcium channels remains a major task in membrane biophysics. In the absence of three dimensional structures, homology modeling techniques are the method of choice, to address questions concerning the structure of these channels. We have developed models of the open Ca(V)1.2 pore, based on the crystal structure of the mammalian voltage-gated potassium channel K(V)1.2 and a model of the bacterial sodium channel NaChBac. Our models are developed to be consistent with experimental data and modeling criteria. The models highlight major differences between voltage-gated potassium and calcium channels in the P segments, as well as the inner pore helices. Molecular dynamics simulations support the hypothesis of a clockwise domain arrangement and experimental observations of asymmetric calcium channel behavior. In the accompanying paper these models were used to study structural effects of a channelopathy mutation.     

Models of voltage-dependent conformational changes in NaChBac channels

Models of the transmembrane region of the NaChBac channel were developed in two open/inactivated and several closed conformations. Homology models of NaChBac were developed using crystal structures of Kv1.2 and a Kv1.2/2.1 chimera as templates for open conformations, and MlotiK and KcsA channels as templates for closed conformations. Multiple molecular-dynamic simulations were performed to refine and evaluate these models. A striking difference between the S4 structures of the Kv1.2-like open models and MlotiK-like closed models is the secondary structure. In the open model, the first part of S4 forms an α-helix, and the last part forms a 3₁₀ helix, whereas in the closed model, the first part of S4 forms a 3₁₀ helix, and the last part forms an α-helix. A conformational change that involves this type of transition in secondary structure should be voltage-dependent. However, this transition alone is not sufficient to account for the large gating charge movement reported for NaChBac channels and for experimental results in other voltage-gated channels. To increase the magnitude of the motion of S4, we developed another model of an open/inactivated conformation, in which S4 is displaced farther outward, and a number of closed models in which S4 is displaced farther inward. A helical screw motion for the α-helical part of S4 and a simple axial translation for the 3₁₀ portion were used to develop models of these additional conformations. In our models, four positively charged residues of S4 moved outwardly during activation, across a transition barrier formed by highly conserved hydrophobic residues on S1, S2, and S3. The S4 movement was coupled to an opening of the activation gate formed by S6 through interactions with the segment linking S4 to S5. Consistencies of our models with experimental studies of NaChBac and K_v channels are discussed.     

Excess of Gbetae over Gqalphae in vivo prevents dark, spontaneous activity of Drosophila photoreceptors

Drosophila melanogaster photoreceptor cells are capable of detecting single photons. This utmost sensitivity is critically dependent on the maintenance of an exceedingly low, dark, spontaneous activity of photoreceptor cells. However, the underlying mechanisms of this hallmark of phototransduction are not fully understood. An analysis of the Drosophila visual heterotrimeric (alphabetagamma) Gq protein revealed that wild-type Drosophila flies have about a twofold excess of Gbeta over Galpha subunits of the visual Gq protein. Studies of Gbetae mutants in which the excess of Gbeta was genetically eliminated showed dramatic dark, spontaneous activity of the photoreceptor cells, whereas concurrent genetic reduction of the Galpha subunit, which restored the excess of Gbeta, abolished this effect. These results indicate that an excess of Gbeta over Galpha is a strategy used in vivo for the suppression of spontaneous activity, thereby yielding a high signal to noise ratio, which is characteristic of the photoreceptor light response. This mechanism could be relevant to the regulation of G protein signaling in general.     

Air-sampled Filter Analysis for Endotoxins and DNA Content

Outdoor aerosol research commonly uses particulate matter sampled on filters. This procedure enables various characterizations of the collected particles to be performed in parallel. The purpose of the method presented here is to obtain a highly accurate and reliable analysis of the endotoxin and DNA content of bio-aerosols extracted from filters. The extraction of high molecular weight organic molecules, such as lipopolysaccharides, from sampled filters involves shaking the sample in a pyrogen-free water-based medium. The subsequent analysis is based on an enzymatic reaction that can be detected using a turbidimetric measurement. As a result of the high organic content on the sampled filters, the extraction of DNA from the samples is performed using a commercial DNA extraction kit that was originally designed for soils and modified to improve the DNA yield. The detection and quantification of specific microbial species using quantitative polymerase chain reaction (q-PCR) analysis are described and compared with other available methods.     

Auxin,biosynthesis of ethylene and sex expression in cucumber (Cucumis sativus)

Ethylene production, level of 1-aminocyclopropane-1-carboxylic acid (ACC) and activity of the ethylene forming enzyme (EFE) were higher in apices of gynoecious cucumber (Cucumis sativus cv. Alma) as compared to monoecious cucumber (C. sativus cv. Elem). Application of indole-3-acetic acid (IAA) enhanced ethylene and ACC production in both cultivars. The stimulatory effect of IAA was more pronounced in gynoecious apices. Induction of ethylene production and accumulation of ACC resulting from treatment with IAA were effectively blocked by aminoethoxyvinylglycine (AVG). Content of endogenous IAA, measured by an enzyme immunoassay, was lower in gynoecious cucumber as compared to monoecious one. Treatment of gynoecious plants with the antiauxins -(p-chlorophenoxy)isobutyric acid (PCIB) and -naphthaleneacetic acid (-NAA) did not inhibit female sex expression.It appears that although exogenous IAA enhances ACC and ethylene production, endogenous IAA might not have a major role in the control of sex expression in cucumber of the Beit-Alfa type.Prof. Rudich passed away in May 1986.     

Effect of substance P and eledoisin on K efflux, amylase release and cyclic nucleotide levels in slices of rat parotid gland

The undecapeptides, substance P and eledosin, caused a rapid, concentration-dependent increase in K⁺ efflux and amylase release from parotid tissue slices. The effects were not blocked by β-adrenergic, α-adrenergic, or cholinergic anatagonists. Incubation buffer calcium was required for stimulation of K⁺ efflux and amylase release. The action of the undecapeptides was independent of any effects on parotid cyclic AMP or cyclic GMP levels. Since the actions of the undecapeptides were Ca²⁺ dependent and no effects on cyclic nucleotide levels were discerned it was concluded that Ca²⁺ plays a primary role in agonist regulation of K⁺ efflux from the parotid.     

The level of phytohormones in monoecious and gynoecious cucumbers as affected by photoperiod and ethephon

The endogenous levels of auxin, gibberellin, and inhibitors were followed in monoecious and gynoecious cucumber (Cucumis sativus L.) plants, and in plants treated with the ethylene-releasing compound Ethephon (2-chloroethyl phosphonic acid). Higher auxin inhibitor and lower gibberellin levels were associated with female tendency. The endogenous level of gibberellin and auxin decreased in Ethephon-treated plants. Application of Ethephon induced a rise in abscisic acid. Root application of abscisic acid promoted female tendency of gynoecious cucumbers grown under conditions which increase maleness. High CO₂ levels, which are known to antagonize ethylene, increased maleness of gynoecious cucumbers. The possibility of interrelationship between gibberellin, auxin, ethylene, and abscisic acid on sex expression are discussed.     

Electrophysiological responses to light in the compound eyes of some stored-product insects

That stored-product insects demonstrate behavioral response to light is well-known (Stermer, 1959; Sohi, 1966; Yinon & Shulov, 1966). The question asked is whether these insects, which exist always in conditions of low illumination or darkness, possess a normal visual mechanism. In order to clarify this, electroretinograms have been recorded from the compound eyes of the adults of the yellow mealworm beetle Tenebrio molitor L., the hide beetle Dermestes maculatus De G., and the khapra beetle Trogoderma granarium Everts. Nineteen specimens were the subject of experiments in T. molitor and four in each of the other species. Conventional optical and electronic equipment were used to stimulate the eyes and to record the response (Yinon & Aucrbach, 1969). A computer of average transients was used and permitted accurate diagnosis of the response pattern. Fifty responses for each stimulus type (i.e., light intensity) were averaged. A dark adaptation of 5–10 minutes was given before each experiment.     

Models and mechanisms of repeat expansion disorders: a worm’s eye view

The inappropriate genetic expansion of various repetitive DNA sequences underlies over 20 distinct inherited diseases. The genetic context of these repeats in exons, introns and untranslated regions has played a major role in thinking about the mechanisms by which various repeat expansions might cause disease. Repeat expansions in exons are thought to give rise to expanded toxic protein repeats (i.e. polyQ). Repeat expansions in introns and UTRs (i.e. FXTAS) are thought to produce aberrant repeat-bearing RNAs that interact with and sequester a wide variety of essential proteins, resulting in cellular toxicity. However, a new phenomenon termed ‘repeat-associated nonAUG dependent (RAN) translation’ paints a new and unifying picture of how distinct repeat expansion-bearing RNAs might act as substrates for this noncanonical form of translation, leading to the production of a wide range of repeat sequence-specific-encoded toxic proteins. Here, we review how the model system Caenorhabditis elegans has been utilized to model many repeat disorders and discuss how RAN translation could be a previously unappreciated contributor to the toxicity associated with these different models.