Modulation of the voltage sensor of L-type Ca2+ channels by intracellular Ca2+

Both intracellular calcium and transmembrane voltage cause inactivation, or spontaneous closure, of L-type (CaV1.2) calcium channels. Here we show that long-lasting elevations of intracellular calcium to the concentrations that are expected to be near an open channel (>/=100 microM) completely and reversibly blocked calcium current through L-type channels. Although charge movements associated with the opening (ON) motion of the channel's voltage sensor were not altered by high calcium, the closing (OFF) transition was impeded. In two-pulse experiments, the blockade of calcium current and the reduction of gating charge movements available for the second pulse developed in parallel during calcium load. The effect depended steeply on voltage and occurred only after a third of the total gating charge had moved. Based on that, we conclude that the calcium binding site is located either in the channel's central cavity behind the voltage-dependent gate, or it is formed de novo during depolarization through voltage-dependent rearrangements just preceding the opening of the gate. The reduction of the OFF charge was due to the negative shift in the voltage dependence of charge movement, as previously observed for voltage-dependent inactivation. Elevation of intracellular calcium concentration from approximately 0.1 to 100-300 microM sped up the conversion of the gating charge into the negatively distributed mode 10-100-fold. Since the "IQ-AA" mutant with disabled calcium/calmodulin regulation of inactivation was affected by intracellular calcium similarly to the wild-type, calcium/calmodulin binding to the "IQ" motif apparently is not involved in the observed changes of voltage-dependent gating. Although calcium influx through the wild-type open channels does not cause a detectable negative shift in the voltage dependence of their charge movement, the shift was readily observable in the Delta1733 carboxyl terminus deletion mutant, which produces fewer nonconducting channels. We propose that the opening movement of the voltage sensor exposes a novel calcium binding site that mediates inactivation.     

Complete suppression of Htt fibrilization and disaggregation of Htt fibrils by a trimeric chaperone complex

Huntington's disease (HD) is a neurodegenerative disorder caused by an expanded CAG trinucleotide repeat in the huntingtin gene (HTT). Molecular chaperones have been implicated in suppressing or delaying the aggregation of mutant Htt. Using in vitro and in vivo assays, we have identified a trimeric chaperone complex (Hsc70, Hsp110, and J‐protein) that completely suppresses fibrilization of HttExon1Q₄₈. The composition of this chaperone complex is variable as recruitment of different chaperone family members forms distinct functional complexes. The trimeric chaperone complex is also able to resolubilize Htt fibrils. We confirmed the biological significance of these findings in HD patient‐derived neural cells and on an organismal level in Caenorhabditis elegans. Among the proteins in this chaperone complex, the J‐protein is the concentration‐limiting factor. The single overexpression of DNAJB1 in HEK293T cells is sufficient to profoundly reduce HttExon1Q₉₇ aggregation and represents a target of future therapeutic avenues for HD.     

Statistical analysis of systematic errors in high-throughput screening

High-throughput screening (HTS) is an efficient technology for drug discovery. It allows for screening of more than 100,000 compounds a day per screen and requires effective procedures for quality control. The authors have developed a method for evaluating a background surface of an HTS assay; it can be used to correct raw HTS data. This correction is necessary to take into account systematic errors that may affect the procedure of hit selection. The described method allows one to analyze experimental HTS data and determine trends and local fluctuations of the corresponding background surfaces. For an assay with a large number of plates, the deviations of the background surface from a plane are caused by systematic errors. Their influence can be minimized by the subtraction of the systematic background from the raw data. Two experimental HTS assays from the ChemBank database are examined in this article. The systematic error present in these data was estimated and removed from them. It enabled the authors to correct the hit selection procedure for both assays.     

Direct synthesis and crystal structures of new heteropolynuclear complexes containing aminoalcohol ligands: From heterobi- (Co/Zn) to heterotrimetallic (Cu/Co/Zn) compounds

Three novel heterometallic complexes [Co₂Zn₂(Dea)₂(HDea)₂(NCS)₄] · 4dmso (1), [CuCoZn₂(Dea)₃Cl₃(HOMe)] · MeOH (2) and [CuCoZn(Me₂Ea)₄(NCS)₂(OAc)] (3) have been prepared from zerovalent zinc, cobalt thiocyanate and a dimethylsulfoxide solution of diethanolamine (1), zerovalent copper, cobalt chloride, zinc chloride and a methanol solution of diethanolamine (2), zerovalent copper, cobalt thiocyanate, zinc acetate and an acetonitrile solution of 2-(dimethylamino)ethanol in air (3) [H₂Dea is diethanolamine and HMe₂Ea is 2-(dimethylamino)ethanol]. The structures of all complexes were determined by single crystal X-ray analysis. Compound 1 consists of a centrosymmetric molecule based on a Co₂Zn₂O₆ core. In contrast to 1, the coordination cores of the structures of the heterotrimetallic complexes, CuCoZn₂O₆ (for 2) and CuCoZnO₄ (for 3) have no inversion centre. Both 2 and 3 possess a similar CuCoZnO₄ fragment that can be viewed as an incomplete distorted cube.     

Overexpression of glutamate decarboxylase in transgenic tobacco plants confers resistance to the northern root-knot nematode

Previous research suggests that the endogenous synthesis of gamma-aminobutyrate (GABA), a naturally occurring inhibitory neurotransmitter, serves as a plant defense mechanism against invertebrate pests. Here, we tested the hypothesis that elevated GABA levels in engineered tobacco confer resistance to the northern root nematode (Meloidogyne hapla). This nematode species was chosen because of its sedentary nature and economic importance in Canada. We derived nine phenotypically normal, homozygous lines of transgenic tobacco (Nicotiana tabacum L.), which contain one or two copies of a full-length, chimeric tobacco glutamate decarboxylase (GAD) cDNA or a mutant version that lacks the autoinhibitory calmodulin-binding domain, under the control of a chimeric octopine synthase/mannopine synthase promoter. Regardless of experimental protocol, uninfected transgenic lines consistently contained higher GABA concentrations than wild-type controls. Growth chamber trials revealed that 9–12 weeks after inoculation of tobacco transplants with the northern root-knot nematode, mature plants of five lines possessed significantly fewer egg masses on the root surface when the data were expressed on both root and root fresh weight bases. Therefore, it can be concluded that constitutive transgenic expression of GAD conferred resistance against the root-knot nematode in phenotypically normal tobacco plants, probably via a GABA-based mechanism.     

Thermal modulation of epicardial Ca2+ dynamics uncovers molecular mechanisms of Ca2+ alternans

Ca²⁺ alternans (Ca-Alts) are alternating beat-to-beat changes in the amplitude of Ca²⁺ transients that frequently occur during tachycardia, ischemia, or hypothermia that can lead to sudden cardiac death. Ca-Alts appear to result from a variation in the amount of Ca²⁺ released from the sarcoplasmic reticulum (SR) between two consecutive heartbeats. This variable Ca²⁺ release has been attributed to the alternation of the action potential duration, delay in the recovery from inactivation of RYR Ca²⁺ release channel (RYR2), or an incomplete Ca²⁺ refilling of the SR. In all three cases, the RYR2 mobilizes less Ca²⁺ from the SR in an alternating manner, thereby generating an alternating profile of the Ca²⁺ transients. We used a new experimental approach, fluorescence local field optical mapping (FLOM), to record at the epicardial layer of an intact heart with subcellular resolution. In conjunction with a local cold finger, a series of images were recorded within an area where the local cooling induced a temperature gradient. Ca-Alts were larger in colder regions and occurred without changes in action potential duration. Analysis of the change in the enthalpy and Q₁₀ of several kinetic processes defining intracellular Ca²⁺ dynamics indicated that the effects of temperature change on the relaxation of intracellular Ca²⁺ transients involved both passive and active mechanisms. The steep temperature dependency of Ca-Alts during tachycardia suggests Ca-Alts are generated by insufficient SERCA-mediated Ca²⁺ uptake into the SR. We found that Ca-Alts are heavily dependent on intra-SR Ca²⁺ and can be promoted through partial pharmacologic inhibition of SERCA2a. Finally, the FLOM experimental approach has the potential to help us understand how arrhythmogenesis correlates with the spatial distribution of metabolically impaired myocytes along the myocardium.     

A QM/QTAIM detailed look at the Watson–Crick↔wobble tautomeric transformations of the 2-aminopurine·pyrimidine mispairs

This work is devoted to the careful QM/QTAIM analysis of the evolution of the basic physico-chemical parameters along the intrinsic reaction coordinate (IRC) of the biologically important 2AP·T(WC)?2AP·T*(w) and 2AP·C*(WC)?2AP·C(w) Watson–Crick(WC)?wobble(w) tautomeric transformations obtained at each point of the IRC using original authors’ methodology. Established profiles reflect the high similarity between the courses of these processes. Basing on the scrupulous analysis of the profiles of their geometric and electron-topological parameters, it was established that the dipole-active WC?w tautomerizations of the Watson–Crick-like 2AP·T(WC)/2AP·C*(WC) mispairs, stabilized by the two classical N3H?N1, N2H?O2 and one weak C6H?O4/N4 H-bonds, into the wobble 2AP·T*(w)/2AP·C(w) base pairs, respectively, joined by the two classical N2H?N3 and O4/N4H?N1 H-bonds, proceed via the concerted stepwise mechanism through the sequential intrapair proton transfer and subsequent large-scale shifting of the bases relative each other, through the planar, highly stable, zwitterionic transition states stabilized by the participation of the four H-bonds – N1⁺H?O4^–/N4^–, N1⁺H?N3^–, N2⁺H?N3^–, and N2⁺H?O2^–. Moreover, it was found out that the 2AP·T(WC)?2AP·T*(w)/2AP·C*(WC)?2AP·C(w) tautomerization reactions occur non-dissociatively and are accompanied by the consequent replacement of the 10 unique patterns of the specific intermolecular interactions along the IRC. Obtained data are of paramount importance in view of their possible application for the control and management of the proton transfer, e.g. by external electric or laser fields.