RESUMEN
Disturbances in GABA(A) receptor trafficking contribute to several neurological and psychiatric disorders by altering inhibitory neurotransmission. Identifying mechanisms that regulate GABA(A) receptor trafficking could lead to better understanding of disease pathogenesis and treatment. Here, we show that protein kinase Cε (PKCε) regulates the N-ethylmaleimide-sensitive factor (NSF), an ATPase critical for membrane fusion events, and thereby promotes the trafficking of GABA(A) receptors. Activation of PKCε decreased cell surface expression of GABA(A) receptors and attenuated GABA(A) currents. Activated PKCε associated with NSF, phosphorylated NSF at serine 460 and threonine 461, and increased NSF ATPase activity, which was required for GABA(A) receptor downregulation. These findings identify new roles for NSF and PKCε in regulating synaptic inhibition through downregulation of GABA(A) receptors. Reducing NSF activity by inhibiting PKCε could help restore synaptic inhibition in disease states in which it is impaired.
Asunto(s)
Proteínas Sensibles a N-Etilmaleimida/fisiología , Proteína Quinasa C-epsilon/fisiología , Receptores de GABA-A/metabolismo , Adenosina Trifosfatasas/metabolismo , Animales , Biotinilación , Línea Celular , Membrana Celular/metabolismo , Electrofisiología , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Inmunoprecipitación , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Fluorescente , Técnicas de Placa-Clamp , Fosforilación , Proteína Quinasa C-epsilon/genética , Proteína Quinasa C-epsilon/aislamiento & purificación , Receptores de Superficie Celular/metabolismoRESUMEN
Adrenarche is the prepubertal onset of increased adrenal secretion of 19-carbon steroids, especially dehydroepiandrosterone (DHEA). However, while human beings and chimpanzees exhibit adrenarche, other primates such as the baboon and rhesus monkey do not, and the adrenals of most other mammals produce little or no DHEA. Thus, the acquisition of adrenarche is a very recent evolutionary event. DHEA is produced from pregnenolone by the successive 17alpha-hydroxylase and 17,20 lyase activities of a single enzyme, P450c17. To ascertain whether sequence differences in P450c17 contribute to adrenarche, we cloned the rhesus monkey cDNA from adrenal tissue and cloned the chimpanzee and baboon cDNAs from genomic DNA using an exon-trapping strategy. Using microsomes from yeast transformed with rhesus, baboon, chimp, or human P450c17, we measured the Michaelis constant and maximum velocity for the 17alpha-hydroxylase and 17,20 lyase activities. The human and chimp enzymes differ at only two amino acids and baboon and rhesus P450c17 only at a single residue; the human/chimp enzyme differed from the baboon/rhesus enzyme by 25-27 residues (95% identity). Surprisingly, the greatest difference in enzymatic activities was a marked increase in 17alpha-hydroxylase activity of P450c17 in the baboon, which differs from rhesus only at residue 255 [arginine (Arg) in baboon, histine (His) in rhesus]. Residue 255 is also Arg in human and chimp. Wild-type human P450c17 and its Arg255His mutant had similar 17alpha-hydroxylase activities, but the Arg255Ala mutant had decreased 17alpha-hydroxylase activity. These data establish that Arg255 is important for 17alpha-hydroxylase activity and show that the evolution of adrenarche in higher primates is not determined by variations in the sequence of P450c17.
Asunto(s)
Glándulas Suprarrenales/crecimiento & desarrollo , Evolución Molecular , Esteroide 17-alfa-Hidroxilasa/química , Esteroide 17-alfa-Hidroxilasa/genética , Relación Estructura-Actividad , Glándulas Suprarrenales/enzimología , Secuencia de Aminoácidos , Animales , Arginina , Clonación Molecular , Simulación por Computador , ADN Complementario/genética , Humanos , Cinética , Macaca mulatta/genética , Microsomas/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Pan troglodytes/genética , Papio/genética , Alineación de Secuencia , Esteroide 17-alfa-Hidroxilasa/metabolismoRESUMEN
Ethanol enhances gamma-aminobutyrate (GABA) signaling in the brain, but its actions are inconsistent at GABA(A) receptors, especially at low concentrations achieved during social drinking. We postulated that the epsilon isoform of protein kinase C (PKCepsilon) regulates the ethanol sensitivity of GABA(A) receptors, as mice lacking PKCepsilon show an increased behavioral response to ethanol. Here we developed an ATP analog-sensitive PKCepsilon mutant to selectively inhibit the catalytic activity of PKCepsilon. We used this mutant and PKCepsilon(-/-) mice to determine that PKCepsilon phosphorylates gamma2 subunits at serine 327 and that reduced phosphorylation of this site enhances the actions of ethanol and benzodiazepines at alpha1beta2gamma2 receptors, which is the most abundant GABA(A) receptor subtype in the brain. Our findings indicate that PKCepsilon phosphorylation of gamma2 regulates the response of GABA(A) receptors to specific allosteric modulators, and, in particular, PKCepsilon inhibition renders these receptors sensitive to low intoxicating concentrations of ethanol.