RESUMO
In ionotropic glutamate receptors, many channel properties (e.g., selectivity, ion permeation, and ion block) depend on the residue (glutamine, arginine, or asparagine) located at the tip of the pore loop (the Q/R/N site). We substituted a cysteine for the asparagine present at that position in both NR1 and NR2 N-methyl-D-aspartate (NMDA) receptor subunits. Under control conditions, receptors containing mutated NR1 and NR2 subunits show much smaller glutamate responses than wild-type receptors. However, this difference disappears upon addition of heavy metal chelators in the extracellular bath. The presence of cysteines at the Q/R/N site in both subunits of NR1/NR2C receptors results in a 220,000-fold increase in sensitivity of the inhibition by extracellular Zn. In contrast with the high-affinity Zn inhibition of wild-type NR1/NR2A receptors, the high-affinity Zn inhibition of mutated NR1/NR2C receptors shows a voltage dependence, which resembles very much that of the block by extracellular Mg. This indicates that the Zn inhibition of the mutated receptors results from a channel block involving Zn binding to the thiol groups introduced into the selectivity filter. Taking advantage of the slow kinetics of the Zn block, we show that both blocking and unblocking reactions require prior opening of the channel.
Assuntos
Substituição de Aminoácidos/genética , Cisteína/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Zinco/metabolismo , Zinco/farmacologia , Animais , Sítios de Ligação , Cisteína/genética , Dissulfetos/metabolismo , Relação Dose-Resposta a Droga , Eletroquímica , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Mutação Puntual/genética , Ligação Proteica , Estrutura Secundária de Proteína , Subunidades Proteicas , Ratos , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Termodinâmica , XenopusRESUMO
Ionotropic glutamate receptors (iGluRs) bind agonists in a domain that has been crystallized and shown to have a bilobed structure. Eukaryotic iGluRs also possess a second extracellular N-terminal domain related to the bacterial periplasmic binding protein LIVBP. In NMDA receptors, the high-affinity Zn inhibition is eliminated by mutations in the LIVBP-like domain of the NR2A subunit. Using LIVBP structure, we have modeled this domain as two lobes connected by a hinge and show that six residues controlling Zn inhibition form two clusters facing each other across a central cleft. Upon Zn binding the two lobes close tightly around the divalent cation. Thus, the extracellular region of NR2A consists of a tandem of Venus flytrap domains, one binding the agonist and the other a modulatory ligand. Such a functional organization may apply to other eukaryotic iGluRs.
Assuntos
Proteínas de Bactérias , Proteínas de Escherichia coli , Subunidades Proteicas , Receptores de N-Metil-D-Aspartato/metabolismo , Zinco/metabolismo , Animais , Sítios de Ligação/genética , Proteínas de Transporte/genética , Células Cultivadas , Sequência Conservada/genética , Cisteína/metabolismo , Escherichia coli/genética , Ligantes , Modelos Moleculares , Oócitos/citologia , Oócitos/metabolismo , Estrutura Terciária de Proteína/efeitos dos fármacos , Pseudomonas aeruginosa/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Reprodutibilidade dos Testes , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Especificidade por Substrato/genética , Xenopus , Zinco/farmacologiaRESUMO
Scorpion toxins are miniglobular proteins containing a common structural motif formed by an alpha-helix on one face, an antiparallel beta-sheet on the opposite face, and three disulfide bonds making up most of its internal volume. We have investigated the role of these evolutionary conserved bonds by replacing each couple of bridged cysteine residues of the scorpion charybdotoxin by a pair of nonbridging L-alpha-aminobutyric acid (Aba) residues. Three analogues were obtained by solid-phase synthesis, Chab I, Chab II, and Chab III, containing the Aba residues in positions 7 and 28, 13 and 33, 17 and 35, respectively. Circular dichroism analysis showed that the purified Chab II acquired a conformation similar to that of charybdotoxin, while the Chab I and Chab III possess decreased nativelike characteristics. All analogues block single high-conductance Ca(2+)-activated K+ channels from rat skeletal muscle inserted into planar lipid bilayers, but with different potencies. Chab II is the most active analogue (KD = 8.0 x 10(-8) M), with a 9-fold lower affinity as compared to native charybdotoxin. Chab I and Chab III have, respectively, 180- and 580-fold lower affinity. Therefore, the removal of evolutionary conserved disulfide bridges does not prevent the toxin to adopt a functional and presumably nativelike structure. However, removal of one disulfide bond affects the yields of formation of correct pairing between the remaining cysteine residues, and only Chab I preserves the ability to form the native disulfide pairings with high efficiency. This is the only analogue to preserve particular spacings of three and one residue between the cysteines, which have been described to thermodynamically disfavor disulfide bond formation between the cysteines [Zhang R., and Snyder, G. H. (1989) J. Biol. Chem. 264, 18472-18479]. Therefore, we conclude that the position of the cysteine residues in the sequence of charybdotoxin, by disfavoring specific pairings and favoring others, may govern selective formation of specific disulfide bonds, thus, explaining the efficient folding properties of Chab I and of native charybdotoxin. The structural properties of the Chab analogues and the discovered role of the cysteine spacings have interesting implications in protein design and engineering.
Assuntos
Charibdotoxina/síntese química , Charibdotoxina/farmacologia , Sequência Conservada , Cisteína/química , Dissulfetos/química , Evolução Molecular , Sequência de Aminoácidos , Animais , Charibdotoxina/isolamento & purificação , Dicroísmo Circular , Cristalografia por Raios X , Cisteína/fisiologia , Dissulfetos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Músculo Esquelético/efeitos dos fármacos , Bloqueadores dos Canais de Potássio , Conformação Proteica , Dobramento de Proteína , Ratos , Relação Estrutura-AtividadeRESUMO
In many tissues cells communicate directly through arrays of intercellular channels which are organized to form gap junctions. These channels are permeant to inorganic ions as well as to small hydrophilic molecules up to Mr 2000. The electrical and chemical coupling provided by such junctions is under the control of intracellular and, in many cases, extracellular substances. The latter (hormones or neurotransmitters) function via the activation of intracellular second messengers. These can rapidly affect the state of opening of the junctions, or induce long-term modulation of the coupling. What are the second messengers and how do they control the functional state of the junctions? These questions' remain largely unanswered, although several internal molecules are thought to be involved in these modulations (e.g. Ca2+, H+ or cyclic AMP). The double patch-clamp technique which enables control of both the intracellular milieu and high resolution measurement of transjunctional currents, has recently been applied to study these problems. In particular, it is now possible to examine at the single channel level how junctional conductance is modulated in terms, for example, of the number of open channels or channel elementary properties.
Assuntos
Permeabilidade da Membrana Celular , Junções Intercelulares/fisiologia , Animais , AMP Cíclico/fisiologia , Condutividade Elétrica , Feminino , Glândula de Harder/fisiologia , Técnicas In Vitro , Canais Iônicos/fisiologia , Potenciais da Membrana , Folículo Ovariano/fisiologia , Gravidez , Útero/fisiologiaRESUMO
The axon terminals of the H1 horizontal cells of the turtle retina are electrically coupled by extensive gap junctions. Dopamine (10 nM to 10 microM) induces a narrowing of the receptive field profile of the H1 horizontal cell axon terminals, increases the coupling resistance between them, and decreases the diffusion of the dye Lucifer Yellow in the network formed by the coupled axon terminals. These actions of dopamine involve the activation of D1 receptors located on the membrane of the H1 horizontal cell axon terminals proper. Increases of the intracellular cyclic AMP concentration induced by either stimulating the adenylate cyclase activity with forskolin or inhibiting the phosphodiesterase activity with isobutylmethylxanthine, theophylline, aminophylline, or compound RO 20-1724 elicit effects similar to those of dopamine on the receptive field profile of the H1 horizontal cell axon terminals, on their coupling resistance, and on the diffusion of Lucifer Yellow in the axon terminal network. It is concluded that dopamine decreases the permeability of the gap junctions between the axon terminals of the H1 horizontal cells of the turtle retina and that this action probably involves cyclic AMP as a second messenger.