RESUMEN
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.
Asunto(s)
Sustitución de Aminoácidos/genética , Cisteína/metabolismo , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismo , Zinc/metabolismo , Zinc/farmacología , Animales , Sitios de Unión , Cisteína/genética , Disulfuros/metabolismo , Relación Dosis-Respuesta a Droga , Electroquímica , Oocitos/efectos de los fármacos , Oocitos/metabolismo , Técnicas de Placa-Clamp , Mutación Puntual/genética , Unión Proteica , Estructura Secundaria de Proteína , Subunidades de Proteína , Ratas , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Termodinámica , XenopusRESUMEN
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.
Asunto(s)
Proteínas Bacterianas , Proteínas de Escherichia coli , Subunidades de Proteína , Receptores de N-Metil-D-Aspartato/metabolismo , Zinc/metabolismo , Animales , Sitios de Unión/genética , Proteínas Portadoras/genética , Células Cultivadas , Secuencia Conservada/genética , Cisteína/metabolismo , Escherichia coli/genética , Ligandos , Modelos Moleculares , Oocitos/citología , Oocitos/metabolismo , Estructura Terciaria de Proteína/efectos de los fármacos , Pseudomonas aeruginosa/genética , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/química , Receptores de N-Metil-D-Aspartato/genética , Reproducibilidad de los Resultados , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Relación Estructura-Actividad , Especificidad por Sustrato/genética , Xenopus , Zinc/farmacologíaRESUMEN
BACKGROUND: Two cases of rubella encephalitis in young adults are reported. CASES REPORTS: 2 patients, 19 and 16-year-old, presented with severe encephalitis. One required mechanical ventilation. Neither were vaccinated against rubella. MRI scan of the brain was normal. The diagnosis was confirmed by serology. Good recovery was noted in both patients. DISCUSSION: Both cases of rubella encephalitis occurring in young adults illustrate the severity of this rare disease. As already shown in Finland, improvement with the French vaccination policy should lead to the prevention of rubella encephalitis.