Rat brain cholinergic, dopaminergic, noradrenergic and serotonergic neurons express GABAA receptors derived from the alpha3 subunit

In order to study the most abundant GABAA receptor subtypes expressed in cholinergic, dopaminergic, noradrenergic and serotonergic neurons (i.e., in neurons of the so-called "global" projection systems), we employed double-immunocytochemical techniques combining the labeling of GABAA receptor alpha1, alpha2 and alpha3 subunit with markers for these cells. Cholinergic neurons in the striatum, habenula, and pedunculo-pontine nucleus were immunonegative for the alpha1 subunit, and most were also alpha2-immunonegative. However, cholinergic neurons in the striatum, septum and pedunculo-pontine nucleus were alpha3 immunopositive. Dopaminergic neurons in the substantia nigra pars compacta were highly immunopositive for the alpha3, and noradrenergic neurons in the locus coeruleus were immunoreactive for the alpha3 and the alpha2-subunit; although neurons of these areas were negative for alpha1. Similarly, serotonergic neurons in raphe also showed a high level of labeling of alpha3, while there was a lack of immunoreactivity for the alpha1-subunit, and only some individual neurons were positive for the alpha2 subunit. As the presence of different alpha-subunits confers specific physiological and pharmacological properties to GABAA receptors, the abundance of receptors containing the alpha3 subunit (and the scarcity of receptor subtypes including the other alpha-subunits studied) may have important implications for the GABAergic regulation of brain "global" or "diffuse" projection systems.     

Monospecific antibodies as probes for the stoichiometry of recombinant GABA(A) receptors

GABA(A) receptors composed of alpha1beta3 gamma2 and alpha1beta3 subunits were expressed in insect Sf9 cells and solubilized in 1% Triton X100. In sucrose density gradients, [3H]-Ro15-1788 binding activity, in the case of alpha1beta3 gamma2, and [3H]-muscimol binding activity, in the case of alpha1beta3 containing receptors sedimented as a single sharp peak suggesting the formation of receptors containing a defined number of subunits. When alpha1beta3gamma2 -containing receptors were incubated with an alpha-subunit specific antibody (bd24), a single class of antibody receptor complex was formed irrespective of the receptor-antibody ratio. This is consistent with two alpha subunits cross-linked within the receptor by the antibody. Similar results were obtained using a beta-subunit specific antibody (bd17). Several classes of antibody-receptor complex were formed when receptors were pre-incubated with a gamma specific antibody (anti gamma(2) 1-15 Cys). This profile is consistent with the presence of a single gamma subunit in each complex. Experiments with alpha1beta3 subunit containing receptors and antibody bd24 produced a profile similar to that seen with alpha1beta3 gamma2 receptors, consistent with two alpha subunits per receptor complex. In this case, the anti-beta subunit antibody, bd17, produced a unique and complex profile consistent with three beta subunits per receptor. This method permits the rapid determination of subunit stoichiometries of homogeneous receptor populations     

Stoichiometry of recombinant heteromeric glycine receptors revealed by a pore-lining region point mutation

Heteromeric glycine receptors mediate synaptic inhibition in the caudal areas of the adult mammalian central nervous system (CNS). These channels resemble other receptors in the nicotinic superfamily in that they are pentamers, but may differ in that they contain alpha and beta subunits in a 3:2 rather than a 2:3 ratio. Evidence in favor of a 3alpha:2beta stoichiometry of heteromeric glycine receptors comes from biochemical data and from the expression of chimeric subunits. We investigated this question using a potentially more direct approach and mutated the highly conserved hydrophobic residues in the middle (position 9') of the pore-lining domain. This mutation increases agonist potency in all channels in the nicotinic superfamily and its effects are in first approximation proportional to the number of mutant subunit incorporated into the receptor. We expressed in HEK 293 cells wild-type glycine alpha1beta receptors or receptors bearing the 9' mutation on either the alpha or the beta subunit, using an alpha:beta plasmid ratio of 1:40 in the transfection. This resulted in negligible levels of contamination by homomeric alpha1 receptors, as proven by low picrotoxin potency and by the extreme rarity of high conductances in single channel recording. Our data show that the effects of the 9' mutation on the receptor sensitivity to glycine were more marked when the alpha subunit bore the mutation. The magnitude of the leftward shift in the agonist dose-response curve for the two mutant combinations was in agreement with a subunit stoichiometry of 3alpha:2beta.     

Evidence for voltage-gated potassium channel beta-subunits with oxidoreductase motifs in human and rodent pancreatic beta cells

Voltage-gated K(+) channel alpha subunits (K(V) alpha) have been previously identified in pancreatic islet beta-cells where it has been suggested they have a role in membrane repolarization and insulin secretion. Here we report the cloning of the three mammalian K(V) beta subunits, including splice variants of these subunits, from both human and rat pancreatic islets and from the rat insulinoma cell line INS-1. Two of the splice variants, K(V) beta1a and K(V) beta3, previously reported to be neuronal tissue specific, are expressed in islets and INS-1 cells. In addition, a splice variant of K(V) beta2 that lacks two potential protein kinase C phosphorylation sites at the amino terminus is present. Immunoblot analysis suggests a high level of K(V) beta2 subunit protein in rat pancreatic islets and immunoprecipitation with anti-K(V) beta2 antibody pulls down a protein from INS-1 cells that reacts with anti-aldose reductase antibody. The K(V) beta subunits, which are attached to the cytoplasmic face of the alpha subunits and are members of the aldose reductase superfamily of NADPH oxidoreductases, may have an as yet undetermined role in the regulation of insulin secretion by the intracellular redox potential. Finally, we suggest that a systematic nomenclature for K(V) beta subunits first proposed by McCormack et al. be adopted for this family of potassium channel subunits as it corresponds with the nomenclature used for their cognate K(V) alpha subunits.     

Cloning and expression of a jellyfish calcium channel beta subunit reveal functional conservation of the alpha1-beta interaction.

In high voltage-activated calcium channels, the binding between the pore-forming alpha1 subunit and the modulatory beta subunit is mediated by interaction domains in each molecule that are highly conserved among most known subunits. However, the interaction domain within CyCaalpha1, an alpha1 subunit cloned from the jellyfish Cyanea capillata, matches the canonical sequence of the alpha1 interaction domain at only four of nine sites. We have now cloned a cDNA from Cyanea neuromuscular tissue that encodes a Ca2+ channel beta subunit. The subunit, named CyCabeta, shares 47-54% identity with vertebrate beta subunit isoforms, but is most highly conserved within its interaction domain. Coexpression of CyCabeta with CyCaalpha1 in Xenopus oocytes increases the amplitude of the CyCaalpha1 current and shifts its activation to more hyperpolarized potentials. These responses are mimicked by coexpression of the rat beta2a subunit, demonstrating that the alpha1 beta interaction is functionally conserved between cnidarians and mammals. CyCabeta also markedly accelerates the rate of recovery of CyCaalpha1 from inactivation, an action that is modestly duplicated by beta2a and may represent an additional mechanism by which beta subunit isoforms differentially modulate alpha1 subunits. These findings establish that limited conservation within the alpha1 interaction domain is sufficient to allow full modulation by a beta subunit, as well as altered regulation by different beta isoforms.     

The bradykinin B2 receptor couples less efficiently than the angiotensin AT1 receptor to the G protein Gq in transiently transfected COS-7 cells

The purpose of this study was to compare the efficiency of two different Gq protein-coupled receptors (AT1 receptor for angiotensin II and B2 receptor for bradykinin) to activate phospholipase C (PLC). When the receptors were expressed at a similar level of 0.5 pmol/mg of protein, inositol trisphosphate (IP) accumulation elicited by AT1 receptor was four times higher than that elicited by B2 receptor. Genistein and pertussis toxin did not modify AT1 receptor- or B2 receptor-induced IP accumulation. These results indicate that in COS-7 cells, the two receptors activate PLC beta through G proteins of the Gq family. AT1 or B2 receptors were co-expressed with the alpha subunit of either Gq or G11. Both alpha subunits potentiated to the same extent AT1 receptor-induced IP accumulation. alpha 11 was also as efficient as alpha q to potentiate B2 receptor-induced response. Interestingly, however, the potentiating effect of alpha q and alpha 11 was more important (by 5-fold) on AT1 receptor-mediated response than on B2 receptor-mediated response. These results demonstrate that the extent of activation of PLC beta by different Gq-coupled receptors depends on the level of expression of these receptors and on their coupling efficiency. These are important parameters that determine the relative contribution of specific hormones to different biological processes.     

Voltage-dependent Ca2+ channel alpha2delta auxiliary subunit: structure, function and regulation.

Voltage-dependent Ca2+ channels are heteromultimeric proteins consisting minimally of a alpha1 main subunit and auxiliary alpha2delta, and beta subunits. The alpha1 subunit forms the ion-conducting pore and contains receptor sites for ligands that modify channel activity. The auxiliary subunits appear to be necessary for the expression of the native kinetic properties of the channel. In particular, the alpha2delta complex, with the transmembrane domain-containing delta subunit arising as a result of proteolysis from the C-terminal end of a alpha2delta precursor, has shown to modify the biophysical and pharmacological properties of the alpha1 subunit in different model systems. Structure-function studies have provided insight into the molecular mechanisms through which alpha2delta exerts its actions and revealed that both allosteric modulation and cellular localization of the Ca2+ channel complex are important mechanisms for alpha2delta regulation of ionic current. Recent studies have shown that the alpha2delta subunit can also support pharmacological interactions with therapeutic agents for the treatment of neurological disorders.     

Determining data independence on a digitized membrane in threedimensions

A method for determining whether structures distributed along a cell's membrane represent a random spatial distribution is presented in this paper. Two three-dimensional (3-D) images are acquired from one cell by wide-field digital imaging of cells which have been labeled with two different fluorescent antibodies. Prior to spatial analysis, a constrained regularized least squares restoration of the images is performed. This is followed by registration via fiducial markers (dual-labeled beads). A deformable model is then used to map data near the surface to the surface. Finally, each resulting data set is analyzed to determine whether it is spatially random. To do this, the authors generalize the test for complete spatial randomness of points in a plane, to test voxels distributed along a voxelized membrane in three dimensions. The authors also test whether the distribution of one protein is independent of the distribution of a second protein. The method is applied to compare the distribution of the protein kinase C to that of vinculin. Vinculin is a protein which anchors intracellular filaments to the cell's plasma membrane. It is also used as a (sparse) membrane marker for the deformable model. Protein kinase C facilitates molecular motors inside the cell. These may be associated with actin and myosin filaments     

GABA(A) receptor modulation in rat cerebellum granule cells

The inhibitory GABA(A) receptor is a key element in determining the pattern of nerve cell electrical activity. Thus, modulation of its function is of paramount impact in shaping neuronal functional activity under physiological and pathological conditions. This applies to cerebellar granule neurons as to all the other neurons in the brain. The culture of cerebellar granules from newborn rats is a convenient means by which to approach these cells for electrophysiological studies provided that they maintain, as far as GABA(A) receptors are concerned, the same characteristics as in situ. Thus, the regulation of GABA(A) receptor activity in these neurons has been studied by the patch-clamp technique, both in the whole-cell and outside-out configuration. An obvious first level of control of such receptors' activity is their desensitization under continued agonist application, with biphasic kinetics. The data do not allow one to conclude whether one is dealing with two different populations of receptors or with a single population with two desensitization phases; although the presence of two GABA(A) receptor populations is suggested by a host of observations. The granule cell GABA(A) receptors are modulated by changes in extracellular pH with lower pH resulting in an enhanced receptor activity. They display, under the conditions of whole-cell recording, a run-down phenomenon which is most probably due to a tyrosine phosphatase activity which is in turn under control by a protein serine kinase. Thus, in situ tyrosine phosphorylation is a key element in determining the efficiency of GABA mediated inhibition. Activation of protein kinase A or protein kinase G (PKG) down-regulates GABA(A) receptors' activity. This last event is involved in the depression of those receptors' activity by L-arginine via the production of nitric oxide. In addition, the activity of calmodulin-activated adenylate cyclase I is controlled by GABA(B) receptors. Dendritic GABA(A) receptor activity is partially blocked by previous activation of N-methyl-D-aspartate (NMDA) receptors via calcineurin mediated dephosphorylation/activation of protein tyrosine phosphatase and concomitant production of nitric oxide and PKG activation. The site phosphorylated by PKG is evidently not available for calcineurin-mediated serine dephosphorylation, due to calcineurin-specific membrane localization in respect of the GABA(A) receptor. Overall, a complex network of biochemical signals appear to keep granule cells GABA(A) receptors under a fine balance between up- and down-regulatory mechanisms. The overall data appear also to indicate the presence of two GABA(A) receptor populations: a dendritic one which can be modulated by Ca++ entering via NMDA receptors and a cell body one. The two populations are probably different in terms of desensitization kinetics and benzodiazepine sensitivity.     

Functional domains of delta- and mu-opioid receptors responsible for adenylyl cyclase inhibition

Opioid receptors (delta, mu) belong to the large superfamily of G protein coupled receptors that inhibit adenylyl cyclase. We have studied the effects of seven synthetic peptides representing selected cytoplasmic regions of the murine delta-opioid receptor on forskolin-mediated adenylyl cyclase activity in membranes of D2 and Neuro(2A) cells stably expressing the delta- and mu-opioid receptors respectively. The entire third intracellular loop (i3), its amino-terminal portion (i3.1) and the carboxyl-terminal region of the second cytoplasmic loop (i2.2) enhanced dose-dependently the agonist-mediated inhibition of cAMP accumulation. The peptide-mediated effects are blocked by pertussis toxin treatment and are not observed in parental cells that lack these receptors. The inhibitory effects of the peptides on adenylyl cyclase were markedly attenuated when membranes from D2 and Neuro(2A) cells were preincubated with antisera against Gi(2) alpha and G beta subunits of G proteins. Our results provide evidence on domains of the delta- and mu-opioid receptors responsible for adenylyl cyclase inhibition.     

An Exposure System for Variable Electromagnetic-Field Orientation Electrophysiological Studies

A TEM system for exposing isolated nerve cells at 2 GHz is described. The system allows for monitoring of transmembrane potentials by means of microelectrodes and variation of the angle between the electric-field vector and the cell. An S-parameter characterization of the system is included along with temperature profile measurements for the energy distribution within the exposure chamber. Additional data on the transient electrical characteristics of microelectrodes upon exposure to microwave pulses in this system are included along with a few examples of the response of Aplysis pacemaker neurons to microwave fields.     

植物细胞膜受体胞吞及其功能的研究进展

细胞膜受体蛋白在细胞应对外部环境变化以及感知相邻细胞信号过程中发挥重要作用.当细胞膜受体蛋白受到配体刺激之后,依赖不同的胞吞机制,形成了两种主要的胞吞途径:网格蛋白介导的胞吞和不依赖网格蛋白介导的胞吞.通过不同胞吞途径进入细胞的细胞膜受体蛋白,会在不同内涵体间转运,从而引发不同的内涵体信号,然后一部分膜受体蛋白重新循环到细胞膜,另一部分则转运到溶酶体或液泡进行降解,最终影响植物的生长发育和免疫等过程.由于膜受体介导的胞吞作用和内涵体信号受到越来越多的关注,因此本文对细胞膜受体介导的胞吞机制及其在植物生长发育和抗病中的作用进行了综述,以期为今后系统开展其功能研究提供理论依据.     

Quantitative evaluation of the surface membrane receptors of leukemic cells to blood serum transcobalamin-II

Serum protein transcobalamin II (TC-II) is responsible for transport of cobalamins into mammalian cells. A method of quantitative estimation of plasma membrane receptors of hemopoietic cells to TC-II cobalamin complex is suggested. Analysis of mouse leukemia L1210 cells includes the saturation of radiolabelled ligand-receptor complex with papain. The number of receptors and 57CoCNCbl content in one cell is determined by differentiated radioactivity count of solubilized protein complexes and of cytoplasm.     

Stable expression of human homomeric and heteromeric AMPA receptor subunits in HEK293 cells

Human homomeric and heteromeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)-type glutamate receptors (GluRs) were stably expressed in HEK293 cells with cDNAs encoding the flip splice variant of GluR1, GluR2, GluR3, GluR4 subunit, and the GluR1/GluR2, GluR3/GluR2, and GluR4/GluR2 combination. The lethal combination of GluR2 and GluR4 subunits was found in high expression levels of both receptors. The AMPA-evoked current voltage relationships demonstrated the functional channel properties, such as a double rectification in GluR1, GluR3, and GluR4 receptors, and a linear relation in receptors assembled from GluR2 alone and coexpression of GluR2 with the other subunits. All the transfectants exhibited higher selectivity for AMPA than glutamate in dose-dependent current responses. [3H]AMPA binding revealed that the homomeric and heteromeric receptors displayed a single binding site in Scatchard analysis, with dissociation constant (Kd) values in the range of 14.5-49.3 nM. The Bmax values were in the range of 0.57-7.66 pmol/mg protein. The ligand displacement potency for [3H]AMPA binding was CNQX > glutamate > NS257 in all of the transfectants. These results suggest that stable transformants expressing human homomeric and heteromeric AMPA receptors will be useful tools to define selectivity and potential site of action for AMPA receptor modulators.     

Ultracytochemical demonstration of Ca2(+)-ATPase activity in the rat saphenous artery and its innervated nerve terminal

The localization of Ca2(+)-ATPase activity was ultracytochemically investigated in the rat saphenous artery and nerve terminals innervating the saphenous artery using a lead citrate method devised by Ando et al. (1981). Intense reaction products in the saphenous arterial endothelial cells were observed inside the caveolae and vesicles along the luminal and abluminal sides. In addition, Ca2(+)-ATPase activity was observed on the external side of the luminal, abluminal and lateral plasma membrane, and the outer membrane of mitochondria. In the smooth muscle cells, intense Ca2(+)-ATPase activity on the inside of caveolae and vesicles was observed, comparing in intensity with that on the plasma membrane of smooth muscle cells. In the nerve terminals innervating the saphenous artery, Ca2(+)-ATPase activity was demonstrated on the plasma membrane of the nerve terminal-Schwann cell interface, the axolemma of unmyelinated axons and the plasma membrane of Schwann cells. It is suggested from the above ultracytochemical results that Ca2(+)-ATPase activity plays an important role in the contraction and relaxation of the saphenous artery, and in the neurotransmitter release.     

Film‐Based Implants for Supporting Neuron–Electrode Integrated Interfaces for The Brain

Neural engineering provides promise for cell therapy by integrating the host brain with brain–machine‐interface technologies in order to externally modulate functions. Long‐term interfaces with the host brain remain a critical challenge due to insufficient graft cell survivability and loss of brain electrode sensitivity over time. Here, integrated neuron–electrode interfaces are developed on thin flexible and transparent silk films as brain implants. Mechanical properties and surface topography of silk films are optimized to promote cell survival and alignment of primary rat cortical cells. Compartmentalized neural cultures and co‐patterned electrode arrays are incorporated on the silk films with built‐in wire connections. Electrical stimulation via electrodes embedded in the films activated surrounding neurons to produce evoked calcium responses. In mice brains, silk film implants show conformal contact capable of modulating host brain cells with minimal inflammatory response and stable indwelling for weeks. The approach of combining cell therapy and brain electrodes could provide sustained functional interfaces with ex vivo control with spatial precision.     

Regulatory mechanism of osteoclast activation

Osteoclasts are multinucleated, terminally differentiated cells which play an essential role in bone resorption. Osteoclasts exhibit high expression of the alpha(v)beta3 integrin, which binds to a variety of extracellular matrix proteins, including vitronectin, osteopontin and bone sialoprotein. RGD (Aug-Gly-Asp)-containing peptides, RGD-mimetics and blocking antibodies to alpha(v)beta3 integrin were shown to inhibit bone resorption in vitro and in vivo, suggesting that this integrin plays an important role in regulating osteoclast function. A number of signalling molecules were found to be involved in the alpha(v)beta3 integrin-dependent signalling pathway, including c-Src, Pyk2 and p130Cas. Both Pyk2 and p130Cas localize to the sealing zone of actively resorbing osteoclasts, suggesting their role in linking the adhesion of osteoclasts to the bone matrix, to cytoskeletal organization, and to the polarization and activation of these cells for bone resorption. In this article, we review the regulatory mechanism of osteoclast activation.     

Stochastic resonance can enhance information transmission in neural networks

Stochastic resonance (SR) is a noise-induced phenomenon whereby signal detection can be improved by the addition of background noise in nonlinear systems. SR can also improve the transmission of information within single neurons. Since information processing in the brain is carried out by neural networks and noise is present throughout the brain, the hypothesis that noise and coupling play an important role in the control of information processing within a population of neurons to control was tested. Using computer simulations, we investigate the effect of noise on the transmission of information in an array of neurons, known as array-enhanced SR (AESR) in an interconnected population of hippocampal neurons. A subthreshold synaptic current (signal) modeled by a filtered homogeneous Poisson process was applied to a distal position in each of the apical dendrites, while background synaptic signals (uncorrelated noise) were presented to the midpoint in the basal dendrite. The transmembrane potentials were recorded in each cell of an array of CA1 neuron models, in order to determine spike firing times and to estimate the total and noise entropies from the spike firing times. The results show that the mutual information is maximized for a specific amplitude of uncorrelated noise, implying the presence of AESR. The results also show that the maximum mutual information increases with increased numbers of neurons and the strength of connections. Moreover, the relative levels of excitation and inhibition modulate the mutual information transfer. It is concluded that uncorrelated noise can enhance information transmission of subthreshold synaptic input currents in a population of hippocampal CA1 neuron models. Therefore, endogenous neural noise could play an important role in neural tissue by modulating the transfer of information across the network.