Gating pore currents,a new pathological mechanism underlying cardiac arrhythmias associated with dilated cardiomyopathy

The ability of cells to reliably fire action potentials is critically dependent upon the maintenance of a hyperpolarized resting potential, which allows voltage-gated Na⁺ and Ca²⁺ channels to recover from inactivation and open in response to a subsequent stimulus. Hodgkin and Huxley first recognized the functional importance a small, steady outward leak of K⁺ ions to the resting potential, action potential generation and cellular excitability, and we now appreciate the contribution of inward rectifier-type K⁺ channels (Kir or KCNJ channels) to this process. More recently, however, it has become evident that two-pore domain K⁺ (K2P) channels also contribute to the steady outward leak of K⁺ ions, and thus, maintenance of the resting potential. Molecular cloning efforts have demonstrated that K2P channel exist in yeast to humans, and represent a major branch in the K⁺ channel superfamily. Humans express 15 types of K2P channels, which are grouped into six subfamilies, based on similarities in amino acid sequence and functional properties. Although K2P channels are not voltage-gated, due to the absence of a canonical voltage sensor domain, their activity can be regulated by a variety of stimuli, including mechanical force, polyunsaturated fatty acids (PUFAs) (e.g., arachidonic acid), volatile anesthetics, acidity/pH, pharmacologic agents, heat and signaling events, such as phosphorylation and protein-protein interactions. K2P channels thus represent important regulators of cellular excitability by virtue of their impact on the resting potential, and as such, have garnered considerable attention in recent years.     

Functional formation of heterotypic gap junction channels by connexins-40 and -43

Connexin40 (Cx40) and connexin43 (Cx43) are co-expressed in the cardiovascular system, yet their ability to form functional heterotypic Cx43/Cx40 gap junctions remains controversial. We paired Cx43 or Cx40 stably-transfected N2a cells to examine the formation and biophysical properties of heterotypic Cx43/Cx40 gap junction channels. Dual whole cell patch clamp recordings demonstrated that Cx43 and Cx40 form functional heterotypic gap junctions with asymmetric transjunctional voltage (V_j) dependent gating properties. The heterotypic Cx43/Cx40 gap junctions exhibited less V_j gating when the Cx40 cell was positive and pronounced gating when negative. Endogenous N2a cell connexin expression levels were 1,000-fold lower than exogenously expressed Cx40 and Cx43 levels, measured by real-time PCR and Western blotting methods, suggestive of heterotypic gap junction formation by exogenous Cx40 and Cx43. Imposing a [KCl] gradient across the heterotypic gap junction modestly diminished the asymmetry of the macroscopic normalized junctional conductance – voltage (G_j-V_j) curve when [KCl] was reduced by 50% on the Cx43 side and greatly exacerbated the V_j gating asymmetries when lowered on the Cx40 side. Pairing wild-type (wt) Cx43 with the Cx40 E9,13K mutant protein produced a nearly symmetrical heterotypic G_j-V_j curve. These studies conclusively demonstrate the ability of Cx40 and Cx43 to form rectifying heterotypic gap junctions, owing primarily to alternate amino-terminal (NT) domain acidic and basic amino acid differences that may play a significant role in the physiology and/or pathology of the cardiovascular tissues including cardiac conduction properties and myoendothelial intercellular communication.     

Interaction of the BKCa channel gating ring with dendrotoxins

Two classes of small homologous basic proteins, mamba snake dendrotoxins (DTX) and bovine pancreatic trypsin inhibitor (BPTI), block the large conductance Ca²⁺-activated K⁺ channel (BK_Ca, KCa1.1) by production of discrete subconductance events when added to the intracellular side of the membrane. This toxin-channel interaction is unlikely to be pharmacologically relevant to the action of mamba venom, but as a fortuitous ligand-protein interaction, it has certain biophysical implications for the mechanism of BK_Ca channel gating. In this work we examined the subconductance behavior of 9 natural dendrotoxin homologs and 6 charge neutralization mutants of δ-dendrotoxin in the context of current structural information on the intracellular gating ring domain of the BK_Ca channel. Calculation of an electrostatic surface map of the BK_Ca gating ring based on the Poisson-Boltzmann equation reveals a predominantly electronegative surface due to an abundance of solvent-accessible side chains of negatively charged amino acids. Available structure-activity information suggests that cationic DTX/BPTI molecules bind by electrostatic attraction to site(s) on the gating ring located in or near the cytoplasmic side portals where the inactivation ball peptide of the β2 subunit enters to block the channel. Such an interaction may decrease the apparent unitary conductance by altering the dynamic balance of open versus closed states of BK_Ca channel activation gating.     

P50 Sensory Gating in Infants

Attentional deficits are common in a variety of neuropsychiatric disorders including attention deficit-hyperactivity disorder, autism, bipolar mood disorder, and schizophrenia. There has been increasing interest in the neurodevelopmental components of these attentional deficits; neurodevelopmental meaning that while the deficits become clinically prominent in childhood or adulthood, the deficits are the results of problems in brain development that begin in infancy or even prenatally. Despite this interest, there are few methods for assessing attention very early in infancy. This report focuses on one method, infant auditory P50 sensory gating.Attention has several components. One of the earliest components of attention, termed sensory gating, allows the brain to tune out repetitive, noninformative sensory information. Auditory P50 sensory gating refers to one task designed to measure sensory gating using changes in EEG. When identical auditory stimuli are presented 500 ms apart, the evoked response (change in the EEG associated with the processing of the click) to the second stimulus is generally reduced relative to the response to the first stimulus (i.e. the response is "gated"). When response to the second stimulus is not reduced, this is considered a poor sensory gating, is reflective of impaired cerebral inhibition, and is correlated with attentional deficits.Because the auditory P50 sensory gating task is passive, it is of potential utility in the study of young infants and may provide a window into the developmental time course of attentional deficits in a variety of neuropsychiatric disorders. The goal of this presentation is to describe the methodology for assessing infant auditory P50 sensory gating, a methodology adapted from those used in studies of adult populations.     

A new tool for monoclonal antibody analysis

Monoclonal antibody (mAb) products are extraordinarily heterogeneous due to the presence of a variety of enzymatic and chemical modifications, such as deamidation, isomerization, oxidation, glycosylation, glycation, and terminal cyclization. The modifications in different domains of the antibody molecule can result in different biological consequences. Therefore, characterization and routine monitoring of domain-specific modifications are essential to ensure the quality of the therapeutic antibody products. For this purpose, a rapid and informative methodology was developed to examine the heterogeneity of individual domains in mAb products. A recently discovered endopeptidase, IdeS, cleaves heavy chains below the hinge region, producing F(ab')₂ and Fc fragments. Following reduction of disulfide bonds, three antibody domains (LC, Fd, and Fc/2) can be released for further characterization. Subsequent analyses by liquid chromatography/mass spectrometry, capillary isoelectric focusing, and glycan mapping enable domain-specific profiling of oxidation, charge heterogeneity, and glycoform distribution. When coupled with reversed phase chromatography, the unique chromatographic profile of each molecule offers a simple strategy for an identity test, which is an important formal test for biopharmaceutical quality control purposes. This methodology is demonstrated for a number of IgGs of different subclasses (IgG1, IgG2, IgG4), as well as an Fc fusion protein. The presented technique provides a convenient platform approach for scientific and formal therapeutic mAb product characterization. It can also be applied in regulated drug substance batch release and stability testing of antibody and Fc fusion protein products, in particular for identity and routine monitoring of domain-specific modifications.     

The evaluation of survival and proliferation of lymphocytes in autologous mixed leukocyte reaction with dendritic cells. The comparison of incorporation of (3)H-thymidine and differential gating method

Dendritic cells (DCs) play the key role in T-lymphocyte proliferation and induction of antitumour response. The mixed leukocyte reaction (MLR) of T-lymphocytes and DCs is essential instrument for immunological mechanisms studies. Conventionally used method for determination of T-lymphocytes proliferation, ³H-thymidine incorporation, provides only general information. The method of flow cytometry and differential gating seems to be more suitable for quantitative and qualitative analysis of T-lymphocyte proliferation. It is based on time limited acquisition of events and on its distribution according to forward and side scatter values. We decided to compare these two methods and determine mutual correlation and compatibility. Eleven patients were studied and in all cases DCs promoted the survival and proliferation of both CD4 and CD8 lymphocytes. Both methods retained consistency with regard to survival and proliferation of CD4/CD8 lymphocytes. However, the correlation of these methods was not convincing. Therefore, both these methods might be used for evaluation of MLR, but each of them gives specific and complementary information.     

Molecular dynamics study of the primary charge separation reactions in Photosystem I: Effect of the replacement of the axial ligands to the electron acceptor A0

Molecular dynamics (MD) calculations, a semi-continuum (SC) approach, and quantum chemistry (QC) calculations were employed together to investigate the molecular mechanics of ultrafast charge separation reactions in Photosystem I (PS I) of Thermosynechococcus elongatus. A molecular model of PS I was developed with the aim to relate the atomic structure with electron transfer events in the two branches of cofactors. A structural flexibility map of PS I was constructed based on MD simulations, which demonstrated its rigid hydrophobic core and more flexible peripheral regions. The MD model permitted the study of atomic movements (dielectric polarization) in response to primary and secondary charge separations, while QC calculations were used to estimate the direct chemical effect of the A_0A/A_0B ligands (Met or Asn in the 688/668 position) on the redox potential of chlorophylls A_0A/A_0B and phylloquinones A_1A/A_1B. A combination of MD and SC approaches was used to estimate reorganization energies λ of the primary (λ₁) and secondary (λ₂) charge separation reactions, which were found to be independent of the active branch of electron transfer; in PS I from the wild type, λ₁ was estimated to be 390 ± 20 mV, while λ₂ was estimated to be higher at 445 ± 15 mV. MD and QC approaches were used to describe the effect of substituting Met688_PsaA/Met668_PsaB by Asn688_PsaA/Asn668_PsaB on the energetics of electron transfer. Unlike Met, which has limited degrees of freedom in the site, Asn was found to switch between two relatively stable conformations depending on cofactor charge. The introduction of Asn and its conformation flexibility significantly affected the reorganization energy of charge separation and the redox potentials of chlorophylls A_0A/A_0B and phylloquinones A_1A/A_1B, which may explain the experimentally observed slowdown of secondary electron transfer in the M688N_PsaA variant. This article is part of a Special Issue entitled: Photosynthesis research for sustainability: Keys to produce clean energy.     

Buzz pollination in eight bumblebee-pollinated Pedicularis species: does it involve vibration-induced triboelectric charging of pollen grains?

MethodsEight sympatric buzz-pollinated species of Pedicularis that share bumblebee pollinator species were studied, giving a rare opportunity to compare sonication behaviour of a shared pollinator on different plant species.ConclusionsSonication behaviour of B. friseanus differs among Pedicularis species, not only because worker bees assort themselves among plant species by body size, but also because bees of a given size adjust the buzz frequency to achieve a vibration velocity corresponding to the floral traits of each plant species. These findings, and the floral traits that characterize these and other buzz-pollinated species, are compatible with the hypothesis of vibration-induced triboelectric charging of pollen grains.