Tyrosine Kinase Phosphorylation of GABAA Receptor α1, β2 and γ2 Subunits Following Chronic Intermittent Ethanol (CIE) Exposure of Cultured Cortical Neurons of Mice

There is evidence that many of the GABA_A receptor subunits contain consensus sequence for tyrosine kinase, and phosphorylation may play a key role in ethanol’s regulation of GABA_A receptors. Recently, we investigated the effect of chronic exposure of ethanol (CE) on tyrosine kinase phosphorylation and reported that there was an up-regulation in tyrosine kinase phosphorylation of the β₂- and γ₂- subunits and no effect on α₁-subunit of the GABA_A receptor in the cultured cortical neurons of mice. In the present study, we have further investigated the effect of chronic intermittent administration of ethanol (CIE) on tyrosine kinase phosphorylation of the GABA_A receptor subunits (α₁, β₂, and γ₂) in the mouse cultured cortical neurons by immunoprecipitation and Western blot techniques. We observed that there was an up-regulation in the tyrosine kinase phosphorylation of the GABA_A receptor β₂- and γ₂-subunits following CIE exposure, and no effect on α₁-subunit in the cultured cortical neurons of mice. These CIE changes, unlike CE, were not reverted back to the control level following ethanol withdrawal even after 7 days. Acute exposure of ethanol did not cause any change in the tyrosine kinase regulation of the GABA_A receptor subunits. In conclusion, the CIE exposure, unlike chronic/acute ethanol exposure, regulates the tyrosine kinase phosphorylation of the selective population of GABA_A receptors in a long lasting manner.     

Evidence for a Reduction of Coupling between GABA<Subscript>A</Subscript> Receptor Agonist and Ionophore Binding Sites by Inorganic Phosphate

[³⁵S]TBPS binding to the GABA_A receptor ionophore binding site is anion dependent. Using autoradiography on rat brain sections, we show that permeabilities of anions through the receptor channel correlate with their efficiencies to promote basal [³⁵S]TBPS binding. Phosphate made an exception as it induced more binding than expected from its permeability. Well-permeable anions (chloride, nitrate, formate) allowed [³⁵S]TBPS binding to be effectively displaced by 1 mM GABA, whereas low-permeable anions (acetate, phosphate, propionate) markedly prevented this GABA effect, especially in the thalamus, the transition from the high to the low GABA effect being between formate and acetate. In the presence of phosphate, GABA enhanced [³H]flunitrazepam binding to benzodiazepine site of recombinant α1β2γ2 receptors with the same efficacy but lower potency as compared to the presence of chloride, whereas [³⁵S]TBPS binding was abnormally modulated by GABA. These results suggest that inorganic phosphate affects coupling between agonist and ionophore sites in GABA_A receptors. Special issue dedicated to Simo S. Oja     

GABA Analogues Derived from 4-Aminocyclopent-1-enecarboxylic Acid

The incorporation of extra binding groups onto known ligands is a powerful tool for the development of more potent and selective agents at target sites such as the GABA receptors. In the present work we have attempted to build on the activity of the know potent GABA_A agonist 4-ACP-3-CA and its cis and trans saturated analogues CACP and TACP. We have investigated reactions to add thiol substituents to the α,β-unsaturated system of 4-ACP-3-CA. The reaction was successful with a limited number of thiols but gave products of mixed stereochemistry. The resultant thioether amino acids were screened for activity at human recombinant α₁β₂ γ_2L GABA_A receptors. The most interesting derivative was the benzylthioether which acted as an antagonist with an IC₅₀ of 42 μM for the inhibition of a GABA EC₅₀ dose (50 μM). This study has shown that GABA analogues derived by thiol addition to 4-aminocyclopent-1-enecarboxylic acid display interesting antagonist activity at the α₁β₂γ_2L GABA_A receptor. Special issue article in honour of Dr. Graham Johnston.     

The γ subunits of the native GABAA/benzodiazepine receptors

Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ₁, γ_2S, γ_L, and γ₃) have been made. The proportion of GABA_A receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ₁, and γ₃ subunits are present in considerable smaller proportion of GABA_A receptor than the γ₂ subunit. Immunocytochemistry shows that γ₁ immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ₁ immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ₃ immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ_2S. In the cerebellu, γ₁ immunolabeling was localized on the Bergmann glia. The γ_2S and γ_2L subunits are differentially expressed in various brain regions. Thus the γ_2S is highly expressed in the olfactory bulb and hippocampus whereas the γ_2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ_2S and γ_2L coexist in some brain areas and cell types. Moreover, the γ_2S and γ_2L subunits can coexist in the same GABA_A receptor pentamer. We have shown that this is the case in some GABA_A receptors expressed in cerebellar granule cells. These GABA_A receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ₁, γ_2S, γ_2L and γ₃ subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ_2S and γ_2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA_A receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA_A receptors in brain. Special issue dedicated to Dr. Kinya Kuriyama     

The Hormonal Control of Uterine Luminal Fluid Secretion and Absorption

GABA_A receptors composed of α, β and γ subunits display a significantly higher single-channel conductance than receptors comprised of only α and β subunits. The pore of GABA_A receptors is lined by the second transmembrane region from each of its five subunits and includes conserved threonines at the 6′, 10′ and 13′ positions. At the 2′ position, however, a polar residue is present in the γ subunit but not the α or β subunits. As residues at the 2′, 6′ and 10′ positions are exposed in the open channel and as such polar channel-lining residues may interact with permeant ions by substituting for water interactions, we compared both the single-channel conductance and the kinetic properties of wild-type α1β1 and α1β1γ2S receptors with two mutant receptors, αβγ(S2′A) and αβγ(S2′V). We found that the single-channel conductance of both mutant αβγ receptors was significantly decreased with respect to wild-type αβγ, with the presence of the larger valine side chain having the greatest effect. However, the conductance of the mutant αβγ receptors remained larger than wild-type αβ channels. This reduction in the conductance of mutant αβγ receptors was observed at depolarized potentials only (E_Cl = −1.8 mV), which revealed an asymmetry in the ion conduction pathway mediated by the γ2′ residue. The substitutions at the γ2′ serine residue also altered the gating properties of the channel in addition to the effects on the conductance with the open probability of the mutant channels being decreased while the mean open time increased. The data presented in this study show that residues at the 2′ position in M2 of the γ subunit affects both single-channel conductance and receptor kinetics.     

Decreased GABA<Subscript>A</Subscript> Receptors Functional Regulation in the Cerebral Cortex and Brainstem of Hypoxic Neonatal Rats: Effect of Glucose and Oxygen Supplementation

Hypoxia in neonates can lead to biochemical and molecular alterations mediated through changes in neurotransmitters resulting in permanent damage to brain. In this study, we evaluated the changes in the receptor status of GABA_A in the cerebral cortex and brainstem of hypoxic neonatal rats and hypoxic rats supplemented with glucose and oxygen using binding assays and gene expression of GABA_Aα1 and GABA_Aγ5. In the cerebral cortex and brainstem of hypoxic neonatal rats, a significant decrease in GABA_A receptors was observed, which accounts for the respiratory inhibition. Hypoxic rats supplemented with glucose alone and with glucose and oxygen showed a reversal of the GABA_A receptors, andGABA_Aα1 and GABA_Aγ5 gene expression to control. Glucose acts as an immediate energy source thereby reducing the ATP-depletion-induced increase in GABA and oxygenation, which helps in encountering anoxia. Resuscitation with oxygen alone was less effective in reversing the receptor alterations. Thus, the results of this study suggest that reduction in the GABA_A receptors functional regulation during hypoxia plays an important role in mediating the brain damage. Glucose alone and glucose and oxygen supplementation to hypoxic neonatal rats helps in protecting the brain from severe hypoxic damage.     

Regulation of GABAergic inhibition by serotonin signaling in prefrontal cortex: molecular mechanisms and functional implications

Serotonergic neurotransmission in prefrontal cortex (PFC) plays a key role in regulating emotion and cognition under normal and pathological conditios. Increasing evidence suggests that serotonin receptors are involved in the complex regulation of GABAergic inhibitory transmission in PFC. Activation of postsynaptic 5-HT₂ receptors in PFC pyramidal neurons inhibits GABA_A-receptor currents via phosphorylation of GABA_A receptor γ2 subunits by RACK1-anchored PKC. In contrast, activation of postsynaptic 5-HT₄ receptors produces an activity-dependent bi-directional regulation of GABA-evoked currents in PFC pyramidal neurons, which is mediated through phosphorylation of GABA_A-receptor β subunits by anchored PKA. On the presynaptic side, GABAergic inhibition is regulated by 5-HT through the activation of 5-HT₂, 5-HT₁, and 5-HT₃ receptors on GABAergic intereneurons. These data provide a molecular and cellular mechanism for serotonin to dynamically regulate synaptic transmission and neuronal excitability in the PFC network, which may underlie the actions of many antidepressant and antipsychotic drugs.     

Cross-talk between NMDA and GABA<Subscript>A</Subscript> receptors in cultured neurons of the rat inferior colliculus

Neuronal ion channels of different types often do not function independently but will inhibit or potentiate the activity of other types of channels, a process called cross-talk. The N-methyl-D-aspartate receptor (NMDA receptor) and the γ-aminobutyric acid type A receptor (GABA_A receptor) are important excitatory and inhibitory receptors in the central nervous system, respectively. Currently, cross-talk between the NMDA receptor and the GABA_A receptor, particularly in the central auditory system, is not well understood. In the present study, we investigated functional interactions between the NMDA receptor and the GABA_A receptor using whole-cell patch-clamp techniques in cultured neurons from the inferior colliculus, which is an important nucleus in the central auditory system. We found that the currents induced by aspartate at 100 μmol L⁻¹ were suppressed by the pre-perfusion of GABA at 100 μmol L⁻¹, indicating cross-inhibition of NMDA receptors by activation of GABA_A receptors. Moreover, we found that the currents induced by GABA at 100 μmol L⁻¹ (I _GABA) were not suppressed by the pre-perfusion of 100 μmol L⁻¹ aspartate, but those induced by GABA at 3 μmol L⁻¹ were suppressed, indicating concentration-dependent cross-inhibition of GABA_A receptors by activation of NMDA receptors. In addition, inhibition of IGABA by aspartate was not affected by blockade of voltage-dependent Ca²⁺ channels with CdCl₂ in a solution that contained Ca²⁺, however, CdCl₂ effectively attenuated the inhibition of I _GABA by aspartate when it was perfused in a solution that contained Ba²⁺ instead of Ca²⁺ or a solution that contained Ca²⁺ and 10 mmol L⁻¹ BAPTA, a membrane-permeable Ca²⁺ chelator, suggesting that this inhibition is mediated by Ca²⁺ influx through NMDA receptors, rather than voltage-dependent Ca²⁺ channels. Finally, KN-62, a potent inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), reduced the inhibition of I _GABA by aspartate, indicating the involvement of CaMKII in this cross-inhibition. Our study demonstrates a functional interaction between NMDA and GABA_A receptors in the inferior colliculus of rats. The presence of cross-talk between these receptors suggests that the mechanisms underlying information processing in the central auditory system may be more complex than previously believed.     

Sex Steroids Effects on the Content of GAD, TH, GABAA, and Glutamate Receptors in the Olfactory Bulb of the Male Rat

Sex steroids exert multiple functions in the central nervous system. They modulate responses to olfactory information in mammals but their participation in the regulation of neurotransmission in the olfactory bulb is unknown. We studied by Western blot the effects of estradiol (E2), progesterone (P4), and allopregnanolone (Allo) on the content of glutamic acid decarboxylase (GAD), γ-aminobutyric acid A receptor α-2 subunit (GABA_AR α-2), glutamate receptor 2/3 (GlutR 2/3), and tyrosine hydroxylase (TH) in the olfactory bulb of gonadectomized male rats. GAD content was increased by all steroids administered alone. Interestingly, progestins reduced E2 effects on GAD content. Steroids increased the content of TH and GABA_AR α-2. In contrast, GlutR 2/3 content was decreased by E2 and P4, whereas Allo did not modify it. These results suggest that estrogens and progestins regulate olfactory bulb functions in the male rat by modulating the expression of key proteins involved in several neurotransmission systems. Special issue article in honor of Dr. Ricardo Tapia.     

GABA A -receptors: Structural requirements and sites of gene expression in mammalian brain

GABA_A-receptors, the major synaptic targets for the neutotransmitter GABA, are gated chloride channels. By their allosteric drug-induced modulation they serve as molecular control elements through which the levels of anxiety, vigilance, muscle tension and epileptiform activity can be regulated. Despite their functional prominence, the structural requirements of fully functional GABA_A-receptors are still elusive. Expression of cDNAs coding for the ₁- and ₁-subunits of rat brain yielded GABA-gated chloride channels which were modulated by barbiturates but displayed only agonistic responses to ligands of the benzodiazepine receptor. GABA_A-receptors with fully functional benzodiazepine receptor sites were formed when the ₁- and ₁-subunits were coexpressed with the ₂-subunit of rat brain. These receptors, however, failed to show cooperativity of GABA in gating the channel. In order to determine the subunit repertoire available for receptor assembly in different neuronal populations in vivo, the sites of subunit gene expression were (₁, ₂, ₃, ₅, ₆, ₁, ₂, ₃, ₂) mapped by in situ hybridization histochemistry in brain sections. The mRNAs of the ₁-, ₁- and ₂-subunits were co-localized e.g. in mitral cells of olfactory bulb, pyramidal cells of hippocampus as well as granule cells of dentate gyrus and cerebellum. The lack of colocalization in various other brain areas points to an extensive receptor heterogeneity. The presence of multiple GABA_A-receptors in brain may contribute to synaptic plasticity, differential responsiveness of neurons to GABA and to variations in drug profiles.Special issue dedicated to Dr. Erminio Costa     

α2A-Adrenoceptor-specific Stimulation of [35S]GTPγS Binding to Membrane Preparations of Rat Frontal Cortex

Functional activation of α_2A adrenergic receptors in the crude membranes from rat frontal cortex was studied by a [³⁵S]-guanosine 5′-O-(γ-thiotriphosphate) ([³⁵S]GTPγS) binding assay. α_2A agonists UK14304 and guanfacine decreased the ability of GDP to compete with [³⁵S]GTPγS binding to the membranes and 0.1 mM GDP was found to be optimal for the following functional experiments. However, even after careful optimization of experimental conditions the specificity of ligands for rat α₂ adrenoceptors were not sufficient, as agonists as well as antagonists became activators of other signal transduction systems before achieving their maximal effect in the α_2A-adrenergic system. Only using compromising concentration of agonist (up to 1 μM UK14304) and antagonist (up to 1 μM RS79948) to inhibit agonist’s effect, allowed us to filtrate out α_2A specific effect for characterization of signal transduction in rat frontal cortex membranes for the comparison efficacies of this system for different animals from behavioral experiments.     

Plasticity of GABA<Subscript>A</Subscript> Receptors in Brains of Rats Treated with Chronic Intermittent Ethanol

The study of alcohol dependence mechanisms has been aided by work in rodents, where regimens of intermittent chronic administration with repeated episodes of intoxication and withdrawal can be coupled with controlled timing of in vitro studies and the possibility of relating them to behavior. The chronic intermittent ethanol (CIE) model in the rat has been found to be a good model of human alcohol dependence, showing persistent signs of withdrawal and self-administration. Studies in CIE rats suggest that plastic changes in GABA-mediated inhibition involving the GABA_A receptor system may be responsible for the behavioral alterations. Here we summarize a combination of evidence that the alcoholic rat CIE model demonstrates changes in GABA_A receptor subunit levels, in receptor localization, and in physiology and pharmacology, leading to alterations in behavior that contribute to the hyperexcitable alcohol withdrawal state (anxiety, insomnia, seizure susceptibility) and alcohol dependence. Special Issue dedicated to Dr. Simo S. Oja     

PA28 subunits of the mouse proteasome: primary structures and chromosomal localization of the genes

 The 20S proteasome is a multi-subunit protease responsible for the production of peptides presented by major histocompatibility complex (MHC) class I molecules. Recent evidence indicates that an interferon-γ (IFN-γ)-inducible PA28 activator complex enhances the generation of class I binding peptides by altering the cleavage pattern of the proteasome. In the present study, we determined the primary structures of the mouse PA28 α- and β-subunits. The deduced amino acid sequences of the α- and β-subunits were 49% identical. We also determined the primary structure of the mouse PA28 γ-subunit (Ki antigen), a protein of unknown function structurally related to the α- and β-subunits. The amino acid sequence identity of the γ-subunit to the α- and β-subunits was 40% and 32%, respectively. Interspecific backcross mapping showed that the mouse genes coding for the α- and β-subunits (designated Psme1 and Psme2, respectively) are tightly linked and map close to the Atp5g1 locus on chromosome 14. Thus, unlike the LMP2 and LMP7 subunits, the IFN-γ-inducible subunits of PA28 are encoded outside the MHC. The gene coding for the γ-subunit (designated Psme3) was mapped to the vicinity of the Brca1 locus on chromosome 11. A computer search of the DNA databases identified a γ-subunit-like protein in ticks and Caenorhabditis elegans, the organisms with no adaptive immune system. It appears that the IFN-γ-inducible α- and β-subunits emerged by gene duplication from a γ-subunit-like precursor. Received: 11 March 1997     

Characterization of neuronal zebra finch GABAA receptors: steroid effects

Songbirds are widely studied to investigate the hormonal control of behavior. However, little is known about the effects of steroids on neurotransmission in these birds. We used electrophysiological and pharmacological techniques to characterize γ-aminobutyric acid (GABA) type A receptors (GABA_A) of primary cultured telencephalic and hippocampal neurons from developing zebra finches. Additionally, their modulation by 17β-estradiol(E₂), 5α- and 5β-dihydrotestosterone (DHT), 5α- and 5β-pregnan-3α-ol-20-one, and corticosterone was examined. Whole-cell GABA-evoked currents were inhibited by picrotoxin (10 μmol l⁻¹) and bicuculline methiodide (10 μmol l⁻¹) and potentiated by pentobarbital (100 μmol l⁻¹) and propofol (3 μmol l⁻¹). Loreclezole (10 μmol l⁻¹) potentiated GABA-evoked currents, suggesting the presence of β₂, β₃ and/or β₄ subunits. Diazepam (1 μmol l⁻¹) potentiated currents, while Zn²⁺ (1 μmol l⁻¹) caused no inhibition, indicating the presence of γ subunits. 5α- and 5β-Pregnan-3α-ol-20-one (100 nmol l⁻¹) potentiated currents, whereas E₂ (1 μmol l⁻¹), 5α- and 5β-DHT (1 μmol l⁻¹), and corticosterone (10 μmol l⁻¹) had no detectable effect. We conclude that zebra finch telencephalic and hippocampal GABA_A receptors include α, β, and γ subunits and are similar to their mammalian counterparts in both their biophysical and pharmacological properties. Additionally, GABA-evoked currents are greatly potentiated by 5α- and 5β-pregnan-3α-ol-20-one but show little or no acute modulation by sex steroids or corticosterone. Accepted: 12 November 1997     

Effects of Mutating Leucine to Threonine in the M2 Segment of α1 and β1 Subunits of GABAAα1β1 Receptors

The conserved leucine residues at the 9′ positions in the M2 segments of α₁ (L264) and β₁ (L259) subunits of the human GABA_A receptor were replaced with threonine. Normal or mutant α₁ subunits were co-expressed with normal or mutant β₁ subunits in Sf9 cells using the baculovirus/Sf9 expression system. Cells in which one or both subunits were mutated had a higher ``resting' chloride conductance than cells expressing wild-type α₁β₁ receptors. This chloride conductance was blocked by 10 mm penicillin, a recognized blocker of GABA_A channels, but not by bicuculline (100 μm) or picrotoxin (100 μm) which normally inhibit the chloride current activated by GABA: nor was it potentiated by pentobarbitone (100 μm). In cells expressing wild-type β₁ with mutated α₁ subunits, an additional chloride current could be elicited by GABA but the rise time and decay were slower than for wild-type α₁β₁ receptors. In cells expressing mutated β₁ subunits with wild-type or mutated α₁ subunits (αβ(L9′T) and α(L9′T)β(L9′T)), no response to GABA could be elicited: this was not due to an absence of GABA_A receptors in the plasmalemma because the cells bound [³H]-muscimol. It was concluded that in GABA_A channels containing the L9′T mutation in the β₁ subunit, GABA-binding does not cause opening of channels, and that the L9′T mutation in either or both subunits gives an open-channel state of the GABA_A receptor in the absence of ligand. Received: 17 April 1996/Revised: 5 July 1996     

GABAA receptor diversity and pharmacology

Because of its control of spike-timing and oscillatory network activity, γ-aminobutyric acid (GABA)-ergic inhibition is a key element in the central regulation of somatic and mental functions. The recognition of GABA_A receptor diversity has provided molecular tags for the analysis of distinct neuronal networks in the control of specific pharmacological and physiological brain functions. Neurons expressing α₁GABA_A receptors have been found to mediate sedation, whereas those expressing α₂GABA_A receptors mediate anxiolysis. Furthermore, associative temporal and spatial memory can be regulated by modulating the activity of hippocampal pyramidal cells via extrasynaptic α₅GABA_A receptors. In addition, neurons expressing α₃GABA_A receptors are instrumental in the processing of sensory motor information related to a schizophrenia endophenotype. Finally, during the postnatal development of the brain, the maturation of GABAergic interneurons seems to provide the trigger for the experience-dependent plasticity of neurons in the visual cortex, with α₁GABA_A receptors setting the time of onset of a critical period of plasticity. Thus, particular neuronal networks defined by respective GABA_A receptor subtypes can now be linked to the regulation of various clearly defined behavioural patterns. These achievements are of obvious relevance for the pharmacotherapy of certain brain disorders, in particular sleep dysfunctions, anxiety disorders, schizophrenia and diseases associated with memory deficits.     

Subunit Composition and Function of GABAA Receptors of Rat Spermatozoa

GABA triggers mammalian sperm acrosome reaction (AR). Here, evidence is presented, showing that rat spermatozoa contain GABA_A receptors, composed of ₅, ₁ and ₃ subunits. The effects of GABA_A receptor agonist and antagonist on the induction of AR in rat spermatozoa were assessed using the chlortetracycline assay. Muscimol, a GABA_A receptor agonist, triggered AR; whereas bicuculline, a GABA_A receptor antagonist and picrotoxin, a GABA_A receptor/Cl^– channel blocker, inhibited the ability of GABA or progesterone to induce AR. In conclusion, GABA_A receptors appear to mediate the action of progesterone in inducing AR in rat spermatozoa.     

Probing functional interfaces of rod PDE γ-subunit using scanning fluorescent labeling

In the dark, the activity of the rod cGMP phosphodiesterase (PDE) catalytic α- and β-subunits (Pαβ) is inhibited by two γ-subunits (Pγ). On light stimulation of the photoreceptor cells, the GTP-bound α-subunit of visual G-protein transducin (G_taGTP) displaces the Pγ-subunits from their inhibitory sites on Pαβ, leading to the effector enzyme activation. We designed a number of Pγ mutants, each with a single cysteine residue evenly distributed at a different position along the Pγ polypeptide chain. These cysteine residues served as sites for the introduction of the environmentally sensitive fluorescent probe, 3-(bromoacetyl)-7-diethyl aminocoumarin (BC). Analysis of the interactions of Pαβ and G_ta with the fluorescently labeled Pγ mutants suggests two distinct functional interfaces of Pγ. The Pαβ/Pγ interface is formed essentially by the C-terminus of Pγ and by the N-terminal portion of the Pγ polycationic region, Pγ-24-45, whereas the Pγ/G_ta interface includes the C-terminal portion of Pγ-24-45 and the region surrounding Pγ Cys68. Such functional organization of Pγ may represent an important element for the PDE activation mechanism during transduction of visual signals.     

Irregular activity oscillations of a rotary molecular motor: A simple kinetic model of F<Subscript>1</Subscript>-ATPase

F₁-ATPase is a catalytic part of the F₁F_o-ATP synthase molecular motor. The cooperative hydrolysis of ATP at three catalytic sites of F₁-ATPase is accompanied by the rotation of the central γ-subunit inside a cylinder formed by three α-subunits and three β-subunits. Experimental works of different authors have shown that the γ-subunit rotates with irregular dwells. A simple kinetic model suggested in this article provides an explanation as to why dwells occur during the rotation of F₁-ATPase. According to this model, rotation dwells happen as a result of deterministic chaos, which in turn occurs at rate constants that are close to those demonstrated experimentally. The time duration of dwells in the model is in agreement with that observed experimentally. Our model explains the known irregular occupancy of catalytic sites of F₁-ATPase by nucleotides.     

Physiological significance of P2X receptor-mediated vasoconstriction in five different types of arteries in rats

P2X₁ receptors, the major subtype of P2X receptors in the vascular smooth muscle, are essential for α,β-methylene adenosine 5′-triphosphate (α,β-MeATP)-induced vasoconstriction. However, relative physiological significance of P2X₁ receptor-regulated vasoconstriction in the different types of arteries in the rat is not clear as compared with α₁-adrenoceptor-regulated vasoconstriction. In the present study, we found that vasoconstrictive responses to noncumulative administration of α,β-MeATP in the rat isolated mesenteric arteries were significantly smaller than those to single concentration administration of α,β-MeATP. Therefore, we firstly reported the characteristic of α,β-MeATP-regulated vasoconstrictions in rat tail, internal carotid, pulmonary, mesenteric arteries, and aorta using single concentration administration of α,β-MeATP. The rank order of maximal vasoconstrictions for α,β-MeATP (E _{max·α,β-MeATP}) was the same as that of maximal vasoconstrictions for noradrenaline (E _max·NA) in the internal carotid, pulmonary, mesenteric arteries, and aorta. Moreover, the value of (E _{max·α,β-MeATP}/E _max·KCl)/(E _max·NA/E _max·KCl) was 0.4 in each of the four arteries, but it was 0.8 in the tail artery. In conclusion, P2X₁ receptor-mediated vasoconstrictions are equally important in rat internal carotid, pulmonary, mesenteric arteries, and aorta, but much greater in the tail artery, suggesting its special role in physiological function.