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.     

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.     

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.     

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     

Phylogenomic analysis and evolution of the potassium channel gene family

Potassium channels govern the permeability of cells to potassium ions, thereby controlling the membrane potential. In metazoa, potassium channels are encoded by a large, diverse gene family. Previous analyses of this gene family have focused on its diversity in mammals. Here we have pursued a more comprehensive study in Caenorhabditis elegans, Drosophila melanogaster, and mammalian genomes. The investigation revealed 164 potassium channel encoding genes in C. elegans, D. melanogaster, and mammals, classified into seven conserved families, which we applied to phylogenetic analysis. The trees are discussed in relation to the assignment of orthologous relationships between genes and vertebrate genome duplication.     

Genomic organization of a voltage-gated Na+ channel in a hydrozoan jellyfish: insights into the evolution of voltage-gated Na+ channel genes

Voltage-gated Na+ channels are responsible for fast propagating action potentials. The structurally simplest animals known to contain rapid, transient, voltage-gated currents carried exclusively by Na+ ions are the Cnidaria. The Cnidaria are thought to be close to the origin of the metazoan radiation and thus are pivotal organisms for studying the evolution of the Na+ channel gene. Here we describe the genomic organization of the Na+ channel alpha subunit, PpSCN1, from the hydrozoan jellyfish, Polyorchis penicillatus. We show that most of the 20 intron sites in this diploblast are conserved in mammalian Na+ channel genes, with some even shared by Ca2+ channels. One of these conserved introns is spliced by a rare U 12-type spliceosome. Such conservation places the origin of the primary exon arrangement of Na+ channels and different intron splicing mechanisms to at least the common ancestors of diploblasts and triploblasts, approximately 600 million-1 billion years ago.     

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.     

A novel computational approach for simultaneous tracking and feature extraction of C. elegans populations in fluid environments.

The nematode Caenorhabditis elegans (C. elegans) is a genetic model widely used to dissect conserved basic biological mechanisms of development and nervous system function. C. elegans locomotion is under complex neuronal regulation and is impacted by genetic and environmental factors; thus, its analysis is expected to shed light on how genetic, environmental, and pathophysiological processes control behavior. To date, computer-based approaches have been used for analysis of C. elegans locomotion; however, none of these is both high resolution and high throughput. We used computer vision methods to develop a novel automated approach for analyzing the C. elegans locomotion. Our method provides information on the position, trajectory, and body shape during locomotion and is designed to efficiently track multiple animals (C. elegans) in cluttered images and under lighting variations. We used this method to describe in detail C. elegans movement in liquid for the first time and to analyze six unc-8, one mec-4, and one odr-1 mutants. We report features of nematode swimming not previously noted and show that our method detects differences in the swimming profile of mutants that appear at first glance similar.     

Structural and functional characterization of Kv6.2 a new gamma-subunit of voltage-gated potassium channel.

We have cloned and functionally expressed Kv6.2, a new member of the Kv6 subfamily of voltage-gated potassium channel subunits. The human Kv6.2 (KCNF2) gene was mapped at 18q22-18q23. Kv6.2 mRNA is preferentially expressed in rat and human myocard. Rat and human Kv6.2 subunits appear to be unable to form functional Kv channels in a heterologous expression system, but, when coexpressed with Kv2.1 alpha subunits, heteromultimeric Kv channels were formed mediating voltage-activated delayed-rectifier type outward currents. Their kinetics and conductance-voltage relationship were different from those mediated by homomultimeric Kv2.1 channels. Yeast two-hybrid reporter assays indicated that Kv6.2 amino-termini are able to interact specifically with the Kv2.1 amino-terminus. It is proposed that this protein protein interaction underlies Kv2.1/Kv6.2 subunit assembly and the expression of functional heteromultimeric Kv2.1/Kv6.2 channels. The most resiliant feature of the Kv2.1/Kv6.2 channels was their submicromolar sensitivity to the antiarrhythmic drug propafenone. The data suggest that delayed-rectifier type channels containing Kv6.2 subunits may contribute to cardiac action potential repolarization.     

Immunocytochemical study of alpha 1 and beta 2/3 subunits of GABAA receptors in freehand isolated vestibular Deiters' neurons

Vestibular Deiters' neurons have been isolated from bovine brain by the Hydén's freehand dissection technique and challenged with monoclonal antibodies directed toward the alpha 1 and beta 2/3 subunits of the GABAA receptors. Subsequent challenge with fluorescent secondary antibodies and confocal microscopy allowed the study of the cellular distribution of such subunits. In Deiters' neurons the beta 2/3 subunit displayed a clear presence all along the cell body profile and the initial parts of the dendrites. The alpha 1 subunit was found highly present all over the cell interior except the nuclear profiles. The strong presence inside the cells possibly masked its presence on the plasma membrane. However, in part of the cells studied a distinct presence on the plasma membrane was evident. This subunit was visualized also all along the long dendrites of these neurons. The approach we describe here, involving freehand isolated mature neurons from adult animals, may allow a better characterization of the tridimensional distribution of different types of neuronal GABAA receptors in the respect of the approach with brain slices.     

Bacillus stearothermophilus lctB gene gives rise to functional K+ channels in Escherichia coli and in Xenopus oocytes

We have cloned a small K+ channel subunit (LctB) of the gram-positive bacterium Bacillus stearothermophilus (B. stearo.). The B. stearo. LctB protein is only 134 amino acids long. The sequence contains a typical K+ channel P-domain with a K+ channel GYGD signature sequence and two hydrophobic, possibly membrane-spanning segments M1 and M2. Unexpectedly, LctB K+ channels exhibited properties which differed markedly from the ones reported for KcsA channels of the gram-positive bacterium Streptomyces lividans. LctB channels, when expressed in E. coli, were targeted to the outer membrane and not like KcsA channels to the inner membrane. After reconstitution in black lipid membrane, LctB channels mediated K+ currents at neutral pH. They were apparently not gated by pH like KcsA channels. Also, LctB cRNA produced functional LctB channels in the Xenopus oocyte expression system in marked contrast to KcsA. The results demonstrated that heterologous expression produced functional LctB channels both in E. coli and in Xenopus oocytes. It is proposed that bacterial LctB subunits can be properly handled by the Xenopus oocyte leading to the occurrence of functional LctB K+ channels in the oocyte plasma membrane.     

Characterization of human Kv4.2 mediating a rapidly-inactivating transient voltage-sensitive K+ current.

A human cDNA for the voltage-sensitive potassium channel subunit Kv4.2 has been cloned and functionally characterized. The human Kv4.2 (KCND2) gene was mapped at 7q31-32. Kv4.2 mRNA is prominently expressed in human brain. Relatively high concentrations of Kv4.2 mRNA occurred in mRNA preparations of amygdala, caudate nucleus, cerebellum, hippocampus, substantia nigra, and thalamus. Kv4.2 mRNA was not detected in human heart, kidney, liver, lung, pancreas, and skeletal muscle. The derived Kv4.2 open reading frame consists of 630 amino acids. In comparison to rat Kv4.2, the human Kv4.2 sequence is highly conserved showing amino acid sequence differences at five positions only. The Kv4.2 subunits were expressed heterologously in human embryonic kidney (293) cells and mediated a rapidly inactivating, A-type outward K+ current. The gating kinetics of the Kv4.2-mediated currents were very similar to those of rat Kv4.2-mediated currents. Both the Kv4.2 and Kv4.3 subunits have been implicated in mediating the transient outward K+ current Ito in rodent cardiac myocytes. In contrast we did not detect Kv4.2. but solely Kv4.3 mRNA in human heart RNA preparations. This may suggest that Kv4.2 subunits do not contribute to the rapid transient outward K+ current of atrial and ventricular myocytes in humans.     

Titration of KATP channel expression in mammalian cells utilizing recombinant baculovirus transduction

A variety of transfection approaches have been used to deliver plasmids encoding ion channel genes into cells. We have used the baculovirus transduction system, BacMam, to demonstrate transient expression of multi-subunit KATP channels in CHO-K1 and HEK-293 EBNA cells using sulfonylurea receptor 1 (SUR), SUR2A, SUR2B, and KIR 6.2 genes. [3H]-glyburide binding, patch clamp, and DiBAC4(3) measurements of membrane potential changes were used to monitor channel expression. BacMam delivery of each SUR isoform with KIR6.2 was demonstrated based on its pharmacological profiles. Expression levels of SUR1 and KIR6.2 were titrated by varying the viral concentration or time of virus addition, with functional activity measured in as little as 4-6 hours posttransduction. Further increases in BacMam virus induced sufficient KATP expression to dominate membrane potential without pharmacological opening of the channel. Independently altering treatment with virus containing either the SUR1 or KIR6.2 gene revealed interactions among subunits during formation of functional channels in the plasma membrane. This study demonstrates the utility and versatility of BacMam as a valuable gene delivery tool for the study of ion channel function.     

Design of 8-like Non-planar Cavity

For eliminating the negative effects of lock-in region and optical elements in cavity, the non-planar structure is strongly needed in laser gyro. In this paper, the theoretical formula of rotatory effect in non-planar cavity is presented deducing from basic light propagating law. Based on the 8-like plane cavity, a novel non- planar structure is designed and numerically calculated. The results show that for obtaining the required rotatory angle, it just needs to adjust the structural parameters of the original 8-like plane cavity. The four-frequency-differential laser gyro which adopts the non-planar 8-like structure has the same advantages as those with the planar 8-like structure, such as the gain region locates at one leg, the position of Faraday rotator has better spatial symmetry and the coating films of the output mirror is easily designed.     

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.     

Construction risk assessment by linguistics: authors' reply tocomments by J.M. Gleason

In replying to J.M. Gleason's comments (see ibid., vol.38, no.2 p.177-8, 1991) the authors admit that an initial error in calculation of the example that affected subsequent calculations was made in the original paper (see ibid., vol.36, no.2, May 1989). The corrected equations are presented. Taking into account the corrections, it is concluded that among the three predefined fuzzy sets, the set `high' has the closest Euclidean distance to the fuzzy set [S]. Therefore the total risk of this project is `high'     

Cellular signaling mechanisms for muscarinic acetylcholine receptors

Signaling pathways for muscarinic acetylcholine receptors (mAChRs) include several enzymes and ion channels. Recent studies have revealed the importance of various isoforms of both alpha and betagamma subunits of G proteins in initiation of signaling as well as the role of the small monomeric G protein, Rho, in the activation of phospholipase D. Modulation of adenylyl cyclase activity by mAChRs appears more diverse as the interaction of various receptor subtypes with the many isoforms of the enzyme are studied. Both alpha and beta subunits of G(i/o) may be involved. Some mAChR responses arise through release of nitric oxide from nitrergic nerves, including salivary gland secretion and hippocampal slow wave activity. mAChRs utilize a variety of intracellular pathways to activate various mitogen-activated protein kinases. The kinases are involved in cholinergic regulation of kidney epithelial function, catabolism of amyloid precursor protein, hippocampal long-term potentiation, activation of phospholipase A(2), and gene induction. mAChR activation can also stimulate or inhibit cellular growth and apoptosis, dependent on prior levels of cellular activity. Modulation of ion channels by mAChR agonists appears increasingly complex, based on recent studies. K(+) channels may be activated by M(2) and M(4) mAChR stimulation, although in the rat superior cervical ganglion topographical constraints appear to limit the effect to the M(2) mAChR. Another ganglionic K(+) current, the M current, is inhibited by M(1) mAChR activation, but in murine hippocampus inhibition involves another receptor subtype. R-type Ca(2+) channels are both facilitated and inhibited by M(1) and M(2) mAChRs; facilitation being more pronounced with activation of M(1) mAChRs and inhibition with M(2) mAChRs.     

Stratified diagonal layered space-time architectures: signal processing and information theoretic aspects

We consider a multielement antenna system that uses M transmit and N receive antennas [an (M,N) wireless link] impaired by additive white Gaussian noise in a quasistatic flat-fading channel environment. The transmitter, which is subject to a power constraint, does not know the random outcome of the matrix channel but does know the channel statistics. The link operates under a probability of outage constraint. We present a novel architecture using stratified space-time diagonals to express a message for efficient communications. The special message arrangement, which is termed stratified-diagonal-BLAST (SD-BLAST), enables receiver signal processing that substantially mutes self interference caused by multipath without incurring waste of space-time. We investigate the proposed communication structure in important downlink categories, showing that, in theory, the message architecture is optimally efficient for all (M, 1) systems and extremely efficient when M/spl Gt/N. We quantify the capacity performance of SD-BLAST using empirically generated complementary cumulative distribution functions (CCDFs) for (16, 5), (8, 3), and (4, 2) systems to exhibit near optimal performance most especially for the (16, 5) system.     

Structural change of bovine retinal cGMP phosphodiesterase by release of its gamma subunit: direct imaging by improved low angle rotary shadowing

Cyclic GMP phosphodiesterase (PDE), a key enzyme for phototransduction, contains two catalytic subunits, Palpha and Pbeta, and two identical regulatory subunits, Pgammas. Neither the structure of the subunits of PDE nor their changes in structure during PDE regulation have been known. Here, improved low angle rotary shadowing was applied to depict the three-dimensional structure of bovine PDE (Palphabetagammagamma) and its changes by Pgamma release. Palphabetagammagamma and Palphabetagamma were isolated from photoreceptor membranes after treatment with a hydrolysis-resistant GTP analogue, and Palphabeta was prepared from Palphabetagammagamma tryptic digestion. Images of Palphabetagammagamma consisted of two crooked strands. These two strands faced each other to make a ring shape, but this ring structure was bent at the centre line between the two strands. In Palphabetagamma, one of these strands changed its shape toward reducing the central space of the ring structure. This ring appeared to be more bent at the centre line. In Palphabeta, both strands changed their shape such that the ring structure appeared to be a twisted quasi ring shape. These observations suggest that in Palphabetagammagamma each Pgamma is complexed with a catalytic subunit, and that the shapes of Palpha and Pbeta are drastically changed by the Pgamma release. These shape changes are no doubt crucial for various PDE regulations, such as activation of cGMP hydrolysis by Palphabeta, interaction of Palphabeta with GARP2 and a GARP2-like protein and cGMP binding to non-catalytic sites on Palphabeta.     

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.