ALS and SAD-like nicotinic acetylcholine receptor subunit genes are widely distributed in insects

Segments of nicotinic acetylcholine receptor α sub-unit genes have been isolated from a panel of insect species by polymerase chain reaction, using degenerate oligonucleotide primers designed to recognize conserved regions of the Drosophila melanogaster ALS and SAO genes. The amplified segments encode elements of typical a-subunits anticipated to play roles in ligand binding and ion channel formation. Each is also clearly either ALS or SAD-like. The predicted protein sequences display extremely high levels of conservation (over 85% for each subtype) even though derived from very distantly related insect species.     

Roles for nicotinic acetylcholine receptor subunit large cytoplasmic loop sequences in receptor expression and function

To evaluate possible physiological roles of the large cytoplasmic loops (C2) and neighboring transmembrane domains of nicotinic acetylcholine receptor (nAChR) subunits, we generated novel fusion constructs in which human nAChR alpha4, beta2, or beta4 subunit C2 or C2 and neighboring sequences were replaced by corresponding sequences from the mouse serotonin type 3A (5-HT3A) receptor subunit. Following stable expression in human SH-EP1 cells, we found that extensive sequence substitutions involving third and fourth transmembrane domains and neighboring "proximal" C2 sequences (e.g., beta2 H322-V335 and V449-R460) did not allow functional expression of nAChR containing chimeric subunits. However, expression of functional nAChR was achieved containing wild-type alpha4 subunits and chimeric beta2 (beta2chi) subunits whose "nested" C2 domain sequences K336-S448 were replaced with the corresponding 5-HT3A subunit sequences. Whereas these findings suggested indispensable roles for M3/M4 transmembrane and/or proximal C2 sequences in alpha4beta2-nAChR function, nested C2 sequences in the beta2 subunit are not essential for functional receptor expression. Ligand-binding analyses also revealed only subtle differences in pharmacological profiles of alpha4beta2-nAChR compared with alpha4beta2chi-nAChR. Nevertheless, there was heightened emergence of agonist-mediated self-inhibition of alpha4beta2chi function, greater sensitivity to functional blockade by a number of antagonists, and faster and more complete acute desensitization of alpha4beta2chi-nAChR than for alpha4beta2-nAChR. These studies are consistent with unexpected roles of nested C2 sequences in nAChR function.     

The nicotinic acetylcholine receptor subunit Mdalpha6 from Musca domestica is diversified via post-transcriptional modification

Recent studies showed that deletion of a nicotinic acetylcholine receptor (nAChR) subunit gene, Dalpha6 in Drosophila melanogaster results in a strain that is resistant to spinosad, indicating that Dalpha6 is important for the toxic action of this insecticide. To determine if spinosad resistance in house flies was due to a mutation(s) of Mdalpha6 (the orthologue of Dalpha6 from house flies), cDNAs were cloned and characterized from an insecticide-susceptible and a spinosad-resistant strain of the house fly, Musca domestica. The cDNAs contain a 1470-bp open reading frame encoding 490 amino acid residues, 415-bp 5' untranslated region (UTR) and a polymorphic 3'-UTR of approximately 371 bp. The predicted mature protein possesses 468 amino acid residues, has the typical features of a nAChR alpha subunit and is 97% identical to Dalpha6. Quantitative real-time PCR analysis revealed that Mdalpha6 was expressed in the head and the thorax at 1300- and 26-fold higher levels, respectively, than in the abdomen. There was no difference in the expression level of Mdalpha6 between spinosad-resistant and susceptible strains. Ten isoforms arising from alternative splicing were characterized, with isoform II being most common. A-to-I RNA editing was examined and found at 12 sites: editing at 11 of these sites resulted in an amino acid substitution. Mdalpha6 is linked to autosome 1 (spinosad resistance was previously shown to be linked to autosome 1). Single nucleotide polymorphisms, alternative splicing, mRNA levels and A-to-I RNA editing were compared between head and thorax and between insecticide-susceptible and spinosad-resistant strains. These comparisons indicate that Mdalpha6 is not responsible for spinosad resistance in house flies.     

Genetic essential tremor in gamma-aminobutyric acidA receptor alpha1 subunit knockout mice

Essential tremor is the most common movement disorder and has an unknown etiology. Here we report that gamma-aminobutyric acidA (GABA(A)) receptor alpha1-/- mice exhibit postural and kinetic tremor and motor incoordination that is characteristic of essential tremor disease. We tested mice with essential-like tremor using current drug therapies that alleviate symptoms in essential tremor patients (primidone, propranolol, and gabapentin) and several candidates hypothesized to reduce tremor, including ethanol; the noncompetitive N-methyl-D-aspartate receptor antagonist MK-801; the adenosine A1 receptor agonist 2-chloro-N6-cyclopentyladenosine (CCPA); the GABA(A) receptor modulators diazepam, allopregnanolone, and Ro15-4513; and the L-type Ca2+ channel antagonist nitrendipine. Primidone, propranolol, and gabapentin reduced the amplitude (power) of the pathologic tremor. Nonsedative doses of ethanol eliminated tremor in mice. Diazepam, allopregnanolone, Ro15-4513, and nitrendipine had no effect or enhanced tremor, whereas MK-801 and CCPA reduced tremor. To understand the etiology of tremor in these mice, we studied the electrophysiological properties of cerebellar Purkinje cells. Cerebellar Purkinje cells in GABA(A) receptor alpha1-/- mice exhibited a profound loss of all responses to synaptic or exogenous GABA, but no differences in abundance, gross morphology, or spontaneous synaptic activity were observed. This genetic animal model elucidates a mechanism of GABAergic dysfunction in the major motor pathway and potential targets for pharmacotherapy of essential tremor.     

Aging: physiological aspects and clinical implications

Aging is not a new phenomenon, yet the physiology of aging is little understood. Several theories have been formulated to explain the aging process, ranging from immunological to free-radical theories of aging. Although the etiology of aging is important to understand, equally important is the differentiation of normal physiological changes from those associated with disease. The clinician's inability to recognize these differences may result in unnecessary testing, misdiagnoses and mismanagement of the elderly client. In addition, many of the changes associated with aging (e.g., decrease in visual acuity, impaired hearing) are potential hazards. One major role for the health care provider is to anticipate these changes, prepare the elderly client for them, and assist the elderly client in adapting to them. This article summarizes the physiological aspects of aging in the absence of disease and discusses some of the clinical implications related to these changes. Each system is addressed separately, as are the clinical implications related to that system.     

Molecular and pharmacological characterization of GABA(A) receptor alpha1 subunit knockout mice

GABA(A) receptors mediate fast inhibitory neurotransmission in the central nervous system (CNS), and approximately half of these receptors contain alpha1 subunits. GABA(A) receptor alpha1 subunits are important for receptor assembly and specific pharmacological responses to benzodiazepines. Plasticity in GABA(A) receptor alpha1 subunit expression is associated with changes in CNS excitability observed during normal brain development, in animal models of epilepsy, and upon withdrawal from alcohol and benzodiazepines. To examine the role of alpha1 subunit-containing GABA(A) receptors in vivo, we characterized receptor subunit expression and pharmacological properties in cerebral cortex of knockout mice with a targeted deletion of the alpha1 subunit. The mice are viable but exhibit an intention tremor. Western blot analysis confirms the complete loss of alpha1 subunit peptide expression. Stable adaptations in the expression of several GABA(A) receptor subunits are observed in the fifth to seventh generations, including decreased expression of beta2/3 and gamma2 subunits and increased expression of alpha2 and alpha3 subunits. There was no change in alpha4, alpha5, or delta subunit peptide levels in cerebral cortex. Knockout mice exhibit loss of over half of GABA(A) receptors measured by [(3)H]muscimol, [(3)H]2-(3-carboxyl)-3-amino-6-(4-methoxyphenyl)-pyridazinium bromide ([(3)H]SR-95531), and t-butylbicyclophosphoro[(35)S]thionate ([(35)S]TBPS) binding. [(3)H]Ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo[1,5-a][1,4]benzodiazepine-3-carboxylate ([(3)H]Ro15-4513) binding is reduced by variable amounts in different regions across brain. GABA(A) receptor alpha1(-/-) mice lose all high-affinity [(3)H]zolpidem binding and about half of [(3)H]flunitrazepam binding in the cerebral cortex. The potency and maximal efficacy of muscimol-stimulated (36)Cl(-) uptake in cerebral cortical synaptoneurosomes are reduced in alpha1(-/-) mice. Furthermore, knockout mice exhibit increased bicuculline-induced seizure susceptibility compared with wild-type mice. These data emphasize the significance of alpha1 subunit expression and its involvement in the regulation of CNS excitability.     

GABAA receptor alpha 1 and beta 2 subunit null mutant mice: behavioral responses to ethanol

Mice lacking either the alpha1 or beta 2 subunit of the GABAA receptor were tested for ethanol, saccharin, or quinine consumption, ethanol-conditioned place preference, ethanol-conditioned taste aversion, ethanol-simulated motor activity, and handling-induced seizures following chronic consumption of an ethanol liquid diet. The alpha1 null mutants showed decreased ethanol and saccharin consumption, increased aversion to ethanol, and a marked stimulation of motor activity after injection of ethanol. The beta 2 null mutants showed decreased consumption of saccharin and quinine, but not ethanol. Surprisingly, neither mutant showed marked changes in handling induced seizures before or after withdrawal of ethanol. The unique effects of deletion of these two GABAA receptor subunits on ethanol responses are discussed in terms of the distinct changes in different populations of GABAA receptors.     

Adrenergic receptor knockout mice: distinct functions of 9 receptor subtypes

The biological effects of epinephrine and norepinephrine are mediated via 9 different adrenergic receptor subtypes, which all belong to the superfamily of G protein-coupled receptors. Although pharmacological ligands for adrenergic receptors have an important place in medical therapy, the full therapeutic potential of the 9 adrenergic receptor subtypes has not been explored yet. To dissect the physiological relevance of adrenergic receptor subtype diversity, gene-targeted mouse models carrying deletions in these receptor genes ("knockout mice") have been generated. This review gives an overview of the phenotypes observed in mice deficient in adrenergic receptors and discusses the therapeutic relevance of subtype-specific drug therapy.     

Functional activity of the M2 and M4 receptor subtypes in the spinal cord studied with muscarinic acetylcholine receptor knockout mice

Stimulation of spinal muscarinic acetylcholine receptors (mAChRs) produces potent analgesia. Both M(2) and M(4) mAChRs are coupled to similar G proteins (G(i/o) family) and play a critical role in the analgesic action of mAChR agonists. To determine the relative contribution of M(2) and M(4) subtypes to activation of G(i/o) proteins in the spinal cord, we examined the receptor-mediated guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) binding in M(2) and M(4) subtype knockout (KO) mice. Basal [(35)S]GTPgammaS binding in the spinal cord was similar in the wild-type controls, M(2) and M(4) single-KO, and M(2)/M(4) double-KO mice. The spinal [(35)S]GTPgammaS binding stimulated by either muscarine or oxotremorine-M was not significantly different among three groups of wild-type mouse strains. In M(2) single-KO and M(2)/M(4) double-KO mice, the agonist-stimulated [(35)S]GTPgammaS binding was completely abolished in the spinal cord. Furthermore, the agonist-stimulated [(35)S]GTPgammaS binding in the spinal cord of M(4) single-KO mice was significantly reduced ( approximately 15%), compared with that in wild-type controls. On the other hand, the spinal [(35)S]GTPgammaS binding stimulated by a mu-opioid agonist was not significantly different between wild-type and M(2) and M(4) KO mice. This study provides complementary new evidence that M(2) is the most predominant mAChR subtype coupled to the G(i/o) proteins in the spinal cord. Furthermore, these data suggest that a small but functionally significant population of M(4) receptors exists in the mouse spinal cord. The functional activity of these M(4) receptors seems to require the presence of M(2) receptors.     

Dopamine receptor functions: lessons from knockout mice [corrected].

In the past few years, a number of laboratories have used gene targeting via homologous recombination to generate mice deficient for key molecules involved in dopaminergic (DAergic) transmission. This tremendous effort has resulted in the successful generation and characterization of mice deficient for the neurotransmitter DA, the main terminator of DAergic neurotransmission (the DA transporter), and all five subtypes of DA receptors. This review summarizes the results from studies of the various DA receptor knockout mice and of mice deficient in proteins that mediate DA receptor signaling. It focuses on a comparison of the locomotor phenotypes and responses to drugs of abuse (psychostimulants), and reviews the results of anatomic studies examining the morphological and neurochemical differentiation of the striatum in these mutants. Moreover, an overview of recently published results highlighting the physiological relevance of the interaction between different DA receptors and between DA receptors and other neurotransmitter receptors in the modulation of behavioral and molecular responses to DAergic stimulation is presented. Finally, in view of the recently discovered heteroligomeric assemblies of neurotransmitter receptors that involve DA receptor subtypes, the potential value of knockout mice as a tool for testing the in vivo significance of these heteroligomeric receptors is discussed.     

Muscarinic agonist-mediated increases in serum corticosterone levels are abolished in m(2) muscarinic acetylcholine receptor knockout mice

Muscarinic acetylcholine receptors (M(1)-M(5)) regulate many key functions in the central and peripheral nervous system. Due to the lack of receptor subtype-selective ligands, however, the physiological roles of individual muscarinic receptor subtypes remain to be determined. In this study, we examined the effects of the muscarinic M(2)/M(4) receptor-preferring agonist [5R-(exo)]-6-[4-butylthio-1,2,5-thiadiazol-3-yl]-1-azabicyclo-[3.2.1]-octane (BuTAC) on serum corticosterone levels in M(2) and M(4) receptor single knockout (KO) and M(2,4) receptor double KO mice. Responses were compared with those obtained with the corresponding wild-type (WT) mice. BuTAC (0.03-0.3 mg/kg s.c.) dose dependently and significantly increased serum corticosterone concentrations in WT mice to 5-fold or greater levels compared with vehicle controls. In muscarinic M(2) and M(2,4) KO mice, however, BuTAC had no significant effect on corticosterone concentrations at doses of 0.1, 0.3, and 1 mg/kg s.c. In both WT and muscarinic M(4) KO mice increases in serum corticosterone concentrations induced by BuTAC (0.1 and 0.3 mg/kg) were not significantly different and were blocked by scopolamine. In summary, the muscarinic M(2,4)-preferring agonist BuTAC had no effect on corticosterone levels in mice lacking functional muscarinic M(2) receptors. These data suggest that the muscarinic M(2) receptor subtype mediates muscarinic agonist-induced activation of the hypothalamic-pituitary-adrenocortical axis in mice.     

Glycoproteins: their structure, biosynthesis and possible clinical implications

Glycoproteins carrying asparagine-linked N-glycosyl oligosaccharides have many diverse biological functions. The role of the carbohydrate in these functions is often obscure. However, there is evidence that carbohydrate is involved in stabilization of glycoproteins during passage from the rough endoplasmic reticulum to the cell surface, and in recognition phenomena such as receptor-mediated endocytosis, routing of lysosomal hydrolases to the lysosomes, and the spread of cancer cells to secondary sites. The cell surface carbohydrate of some transformed cell lines tends to be more highly branched than that of the non-transformed controls. The control of branching during synthesis of N-glycosyl oligosaccharides resides in the N-acetylglucosaminyltransferases (GlcNAc-transferases) which initiate these branches. There must be at least seven such GlcNAc-transferases to account for the diversity of structures that have been observed. Our laboratory has developed assays for four of these enzymes. Substrate specificity studies on these enzymes have shed light on some of the control mechanisms involved in the synthesis of highly branched structures. Alterations in these control mechanisms may be important in the pathogenesis of cancer and other disease.     

The N-methyl-D-aspartate receptor subunit NR2B: localization,functional properties,regulation, and clinical implications

The N-methyl-D-aspartate (NMDA) receptor is an example of a heteromeric ligand-gated ion channel that interacts with multiple intracellular proteins by way of different subunits. NMDA receptors are composed of seven known subunits (NR1, NR2A-D, NR3A-B). The present review focuses on the NR2B subunit of the receptor. Over the last several years, an increasing number of reports have demonstrated the importance of the NR2B subunit in a variety of synaptic signaling events and protein-protein interactions. The NR2B subunit has been implicated in modulating functions such as learning, memory processing, pain perception, and feeding behaviors, as well as being involved in a number of human disorders. The following review provides a summary of recent findings regarding the structural features, localization, functional properties, and regulation of the NR2B subunit. The review concludes with a section discussing the role of NR2B in human diseases.     

The nonoxime bispyridinium compound SAD-128 alters the kinetic properties of the nicotinic acetylcholine receptor ion channel: a possible mechanism for antidotal effects

The effects of SAD-128 [1,1'-oxybis(methylene) bis 4-(1,1-dimethylethyl) pyridinium dichloride], a nonoxime bispyridinium compound, were investigated on the nicotinic acetylcholine receptor-ion channels of frog muscle fibers using end-plate current (EPC) and single channel current measurement techniques. SAD-128 decreased the EPC peak amplitude in a concentration-dependent manner and caused nonlinearity in the current-voltage plots. The time constant of EPC decay was prolonged by SAD-128 (10-200 microM) at potentials between +50 and -90 mV without loss of the single exponential decay. However, at -100 mV and below, biphasic decays of the EPCs were observed in the presence of the drug. The time constant of the fast phase of the EPC decay decreased, whereas that of the slow phase increased, with either hyperpolarization or increasing concentration of the drug. SAD-128 weakly inhibited acetylcholinesterase in frog sartorius muscle. At the single-channel current level, SAD-128 reduced the mean channel open time and produced a blocked state evidenced as an additional phase in the closed time distribution. The agent induced a biphasic burst time distribution whose fast component became faster and slow component slower with increasing concentration and hyperpolarization. The present study provides more details regarding the kinetics of the nicotinic acetylcholine receptor ion channel-blocking mechanisms and a correlation between single-channel currents and macroscopic events. The ability of SAD-128 to block the nicotinic acetylcholine receptor may underlie its efficacy in counteracting lethal effect of organophosphorus compounds.     

Identification and pharmacological profile of a new class of selective nicotinic acetylcholine receptor potentiators

Here we report the discovery, by high-throughput screening, of three novel (2-amino-5-keto)thiazole compounds that act as selective potentiators of nicotinic acetylcholine receptors. Compound selectivity was assessed at seven human nicotinic acetylcholine receptors (alpha1beta1gammadelta, alpha2beta4, alpha3beta2, alpha3beta4, alpha4beta2, alpha4beta4, and alpha7) expressed in mammalian cells or Xenopus oocytes. At alpha2beta4, alpha4beta2, alpha4beta4, and alpha7, but not alpha1beta1gammadelta, alpha3beta2, or alpha3beta4, submaximal responses to nicotinic agonists were potentiated in a concentration-dependent manner by all compounds. At similar concentrations, no potentiation of 5-hydroxytryptamine, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid, GABA(A), and N-methyl-d-aspartate receptors or voltage-gated Na(+) and Ca(2+) channels was observed. Furthermore, these compounds did not inhibit acetylcholine esterase. Further profiling revealed that these compounds enhanced the potency and maximal efficacy of a range of nicotinic agonists at alpha4beta2 nicotinic acetylcholine receptors, a profile typical of allosteric potentiators. At concentrations required for potentiation, the compounds did not displace [(3)H]epibatidine from the agonist-binding site, and potentiation was observed at all agonist concentrations, suggesting a noncompetitive mechanism of action. Blockade of common second messenger systems did not affect potentiation. At concentrations higher then required for potentiation the compounds also displayed intrinsic agonist activity, which was blocked by competitive and noncompetitive nicotinic acetylcholine receptor (nAChR) antagonists. These novel selective nicotinic receptor potentiators should help in clarifying the potential therapeutic utility of selective nAChR modulation for the treatment of central nervous system disorders.     

Differential role of nicotinic acetylcholine receptor subunits in physical and affective nicotine withdrawal signs

It has been suggested that the negative effects associated with nicotine withdrawal promote continued tobacco use and contribute to the high relapse rate of smoking behaviors. Thus, it is important to understand the receptor-mediated mechanisms underlying nicotine withdrawal to aid in the development of more successful smoking cessation therapies. The effects of nicotine withdrawal are mediated through nicotinic acetylcholine receptors (nAChRs); however, the role of nAChRs in nicotine withdrawal remains unclear. Therefore, we used mecamylamine-precipitated, spontaneous, and conditioned place aversion (CPA) withdrawal models to measure physical and affective signs of nicotine withdrawal in various nAChR knockout (KO) mice. beta2, alpha7, and alpha5 nAChR KO mice were chronically exposed to nicotine through surgically implanted osmotic minipumps. Our results show a loss of anxiety-related behavior and a loss of aversion in the CPA model in beta2 KO mice, whereas alpha7 and alpha5 KO mice displayed a loss of nicotine withdrawal-induced hyperalgesia and a reduction in somatic signs, respectively. These results suggest that beta2-containing nAChRs are involved in the affective signs of nicotine withdrawal, whereas non-beta2-containing nAChRs are more closely associated with physical signs of nicotine withdrawal; thus, the nAChR subtype composition may play an important role in the involvement of specific subtypes in nicotine withdrawal.     

Shortening deactivation of cardiac muscle: physiological mechanisms and clinical implications.

PHYSIOLOGICAL MECHANISM: A rapid change of length applied during isometric contraction of skeletal or cardiac muscle may result in redeveloped tension less than appropriate for the new length because of "deactivation" of the contractile system. The amount of shortening deactivation is directly related to both the time during the contraction when the length change occurs and to the extent of muscle shortening. If the muscle is permitted to shorten early in the contraction, the redeveloped tension will be appropriate to the new length as predicted from the classic Frank-Starling relationship. However, the same length change, which is imposed later in the contraction, results in a redeveloped tension that is less than predicted. Furthermore, a greater change in length results in less tension being redeveloped than if a smaller length decrement is applied at the same time during the contraction. It has been demonstrated that the reduced tension during active muscle shortening is associated with reduced affinity of troponin C for Ca2+. The free Ca2+ is then picked up by the SR, with less Ca2+ available for tension development until the subsequent contraction. CLINICAL SIGNIFICANCE: Although the clinical significance of shortening deactivation remains speculative, it seems likely that in the intact heart deactivation would affect myocardial O2 consumption. The decreased efficiency with which the heart maintains a given stroke work against a high afterload might be related to the lesser degree of fiber shortening and, therefore, less shortening deactivation. Conversely, it is well-known that the same level of stroke work accomplished by an increase in end-diastolic volume requires much less O2. This may be related, at least in part, to the greater degree of shortening with an accompanying increase in deactivation under the latter conditions. For example, in congestive heart failure where ejection fraction and fiber shortening are minimal, the maintenance of the longer fiber lengths could significantly increase the MVO2. Ford has suggested that the deactivating effect of shortening produced by afterload reduction would limit energy expenditure, therefore, exerting a favorable effect on the failing myocardium. It would also seem that an inotropic agent that increased shortening deactivation might compensate for the increased MVO2 caused by the inotrope and have a favorable effect on cardiac work. From most of the studies we have reviewed, it appears likely that shortening deactivation acts as a physiological "feedback" mechanism that affects afterload and in turn, myocardial oxygen consumption. Pathological situations such as acidosis and ischemia have been associated with reduced myofilament Ca2+ sensitivity or affinity and depressed cardiac contractility. Is it then possible that interventions that increase Ca2+ sensitivity might favorably alter ventricular pressure-volume relations during ejection and improve myocardial function by reducing the magnitude of shortening deactivation? Whatever the mechanism and clinical significance, future investigations will help to define the role of shortening deactivation in modifying ventricular function.