RESUMO
ATP-sensitive potassium (K(ATP)) channels play important roles in many cellular functions such as hormone secretion and excitability of muscles and neurons. Classical ATP-sensitive potassium (K(ATP)) channels are heteromultimeric membrane proteins comprising the pore-forming Kir6.2 subunits and the sulfonylurea receptor subunits (SUR1 or SUR2). The molecular mechanism by which hormones and neurotransmitters modulate K(ATP) channels via protein kinase A (PKA) is poorly understood. We mutated the PKA consensus sequences of the human SUR1 and Kir6.2 subunits and tested their phosphorylation capacities in Xenopus oocyte homogenates and in intact cells. We identified the sites responsible for PKA phosphorylation in the C-terminus of Kir6.2 (S372) and SUR1 (S1571). Kir6.2 can be phosphorylated at its PKA phosphorylation site in intact cells after G-protein (Gs)-coupled receptor or direct PKA stimulation. While the phosphorylation of Kir6.2 increases channel activity, the phosphorylation of SUR1 contributes to the basal channel properties by decreasing burst duration, interburst interval and open probability, and also increasing the number of functional channels at the cell surface. Moreover, the effect of PKA could be mimicked by introducing negative charges in the PKA phosphorylation sites. These data demonstrate direct phosphorylation by PKA of the K(ATP) channel, and may explain the mechanism by which Gs-coupled receptors stimulate channel activity. Importantly, they also describe a model of heteromultimeric ion channels in which there are functionally distinct roles of the phosphorylation of the different subunits.
Assuntos
Transportadores de Cassetes de Ligação de ATP , Trifosfato de Adenosina/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Fosfatase Alcalina/metabolismo , Animais , Células COS , Linhagem Celular , Humanos , Ilhotas Pancreáticas/metabolismo , Cinética , Microssomos/metabolismo , Modelos Biológicos , Mutagênese Sítio-Dirigida , Técnicas de Patch-Clamp , Fosforilação , RNA Complementar/metabolismo , Ratos , Receptores de Droga/metabolismo , Receptores de Sulfonilureias , Fatores de Tempo , Xenopus/embriologiaRESUMO
Assembly of alpha- and beta-subunits in the endoplasmic reticulum is a prerequisite for the structural and functional maturation of oligomeric P-type ATPases. In Xenopus oocytes, overexpressed, unassembled alpha- and beta-subunits of Xenopus Na,K-ATPase are retained in the endoplasmic reticulum (ER) and are degraded with different kinetics, while unassembled beta-subunits of gastric H, K-ATPase leave the ER. In this study, we have investigated the role of the immunoglobulin-binding protein, BiP, in the folding, assembly, and ER retention of ATPase subunits. We determined the primary sequence of Xenopus BiP and used polyclonal antibodies to examine the interaction with BiP of various wild type and mutant alpha- and beta-subunits overexpressed in Xenopus oocytes. Our results show that ER-retained, unassembled Na,K-ATPase beta-subunits, but not transport-competent H,K-ATPase beta-subunits, efficiently associate with BiP until assembly with alpha-subunits occurs. Furthermore, the kinetics of BiP interaction with unassembled wild type and with mutant Na,K-ATPase beta-subunits parallels their respective stability against cellular degradation. Finally, alpha-subunits that are overexpressed in oocytes and are rapidly degraded and endogenous oocyte alpha-subunits that are stably expressed as individual assembly-competent proteins also interact with oocyte or exogenous BiP, and the interaction time correlates with the protein's stability. These data demonstrate for the first time that BiP might be involved in a long term maturation arrest and/or in the ER quality control of a multimembrane-spanning protein and lend support for a universal chaperone function of BiP.
Assuntos
Proteínas de Transporte/metabolismo , Retículo Endoplasmático Rugoso/metabolismo , ATPase Trocadora de Hidrogênio-Potássio/metabolismo , Proteínas de Choque Térmico , Chaperonas Moleculares/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Sequência de Bases , Transporte Biológico , Compartimento Celular , Clonagem Molecular , Primers do DNA/química , DNA Complementar/genética , Chaperona BiP do Retículo Endoplasmático , Substâncias Macromoleculares , Dados de Sequência Molecular , Oócitos , Testes de Precipitina , Ligação Proteica , Dobramento de Proteína , Ratos , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , ATPase Trocadora de Sódio-Potássio/química , Xenopus laevisRESUMO
The current knowledge on the biosynthetic mechanisms of three hypothalamic neuropeptides, neurophysins, vasopressin and somatostatin, is reviewed. Both neurophysin and vasopressin appear to be first synthesized as higher molecular weight precursors. The methodology elaborated allows to characterize essentially two main forms with apparent Mr similar to or approximately 25 000 and 80 000 respectively. It can be demonstrated that both neurophysin and vasopressin are derived from common precursors.