RESUMEN
The ATP-sensitive potassium (K(ATP)) channel in pancreatic islet beta cells consists of four pore-forming (Kir6.2) subunits and four regulatory sulfonylurea receptor (SUR1) subunits. In beta cells, the K(ATP) channel links intracellular metabolism to the dynamic regulation of the cell membrane potential that triggers insulin secretion. Syntaxin 1A (Syn-1A) is a SNARE protein that not only plays a direct role in exocytosis, but also binds and modulates voltage-gated K(+) and Ca(2+) channels to fine tune exocytosis. We recently reported that wild type Syn-1A inhibits rat islet beta cell K(ATP) channels and binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1. However, wild type Syn-1A inhibition of rat islet beta cell K(ATP) channels seems to be mediated primarily via NBF-1. During exocytosis, Syn-1A undergoes a conformational change from a closed form to an open form, which would fully expose its active domain, the C-terminal H3 domain. Here, we show that the constitutively open form Syn-1A mutant (L165A/E166A) has a similar affinity to NBF-1 and NBF-2 as wild type Syn-1A and was equally effective in inhibiting the K(ATP) channels of rat pancreatic beta cells and a cell line (BA8) stably expressing SUR1/Kir6.2. Although dialysis of NBF-1 into BA8 and islet beta cells effectively blocked wild type and open form Syn-1A inhibition of the K(ATP) current, NBF-2 was also effective in blocking the open form Syn-1A inhibition. This prompted us to examine the specific domains within Syn-1A that would mediate its action on the K(ATP) channels. The C-terminal H3 domain of Syn-1A (Syn-1A-H3), but not the N-terminal H(ABC) domain (Syn-1A-H(ABC)), binds the SUR1 protein of BA8 cells, causing an inhibition of K(ATP) currents, and this inhibition was mediated via both NBF-1 and NBF-2. It therefore appears that the H3 domain of Syn-1A is the putative domain, which binds SUR1, but its distinct actions on the NBFs may depend on the conformation of Syn-1A occurring during exocytosis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Antígenos de Superficie/química , Histonas/química , Proteínas del Tejido Nervioso/química , Canales de Potasio de Rectificación Interna/química , Canales de Potasio/química , Receptores de Droga/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular , Membrana Celular/metabolismo , Relación Dosis-Respuesta a Droga , Exocitosis , Glutatión Transferasa/metabolismo , Humanos , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Cinética , Masculino , Mutación , Nucleótidos/química , Técnicas de Placa-Clamp , Potasio/química , Canales de Potasio/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Ratas Sprague-Dawley , Receptores de Droga/metabolismo , Receptores de Sulfonilureas , Sintaxina 1 , TransfecciónRESUMEN
ATP-sensitive potassium (KATP) channels in neuron and neuroendocrine cells consist of a pore-forming Kir6.2 and regulatory sulfonylurea receptor (SUR1) subunits, which are regulated by ATP and ADP. SNARE protein syntaxin 1A (Syn-1A) is known to mediate exocytic fusion, and more recently, to also bind and modulate membrane-repolarizing voltage-gated K+ channels. Here we show that Syn-1A acts as an endogenous regulator of KATP channels capable of closing these channels when cytosolic ATP concentrations were lowered. Botulinum neurotoxin C1 cleavage of endogenous Syn-1A in insulinoma HIT-T15 cells resulted in the increase in KATP currents, which could be subsequently inhibited by recombinant Syn-1A. Whereas Syn-1A binds both nucleotide-binding folds (NBF-1 and NBF-2) of SUR1, the functional inhibition of KATP channels in rat islet beta-cells by Syn-1A seems to be mediated primarily by its interactions with NBF-1. These inhibitory actions of Syn-1A can be reversed by physiologic concentrations of ADP and by diazoxide. Syn-1A therefore acts to fine-tune the regulation of KATP channels during dynamic changes in cytosolic ATP and ADP concentrations. These actions of Syn-1A on KATP channels contribute to the role of Syn-1A in coordinating the sequence of ionic and exocytic events leading to secretion.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Antígenos de Superficie/metabolismo , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Canales de Potasio de Rectificación Interna/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Antígenos de Superficie/genética , Sitios de Unión , Línea Celular , Cricetinae , Técnicas In Vitro , Islotes Pancreáticos/metabolismo , Masculino , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/química , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas del Tejido Nervioso/genética , Ratas , Ratas Sprague-Dawley , Receptores de Droga , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores de Sulfonilureas , Sintaxina 1 , TransfecciónRESUMEN
Delayed-rectifier K(+) channels (K(DR)) are important regulators of membrane excitability in neurons and neuroendocrine cells. Opening of these voltage-dependent K(+) channels results in membrane repolarization, leading to the closure of the Ca(2+) channels and cessation of insulin secretion in neuroendocrine islet beta cells. Using patch clamp techniques, we have demonstrated that the activity of the K(DR) channel subtype, K(V)1.1, identified by its specific blocker dendrodotoxin-K, is inhibited by SNAP-25 in insulinoma HIT-T15 beta cells. A co-precipitation study of rat brain confirmed that SNAP-25 interacts with the K(V)1.1 protein. Cleavage of SNAP-25 by expression of botulinum neurotoxin A in HIT-T15 cells relieved this SNAP-25-mediated inhibition of K(DR). This inhibitory effect of SNAP-25 is mediated by the N terminus of K(V)1.1, likely by direct interactions with K(Valpha)1.1 and/or K(V)beta subunits, as revealed by co-immunoprecipitation performed in the Xenopus oocyte expression system and in vitro binding. Taken together we have concluded that SNAP-25 mediates secretion not only through its participation in the exocytotic SNARE complex but also by regulating membrane potential and calcium entry through its interaction with K(DR) channels.