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Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1.

ABSTRACT: The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and syntaxin-8 colocalized in the early endosomal compartment, as indicated by the endosomal markers 2xFYVE and rab5. The stimulatory effect of the SNARE protein on the endocytosis of the channel was abolished when both an endocytosis signal in TASK-1 and an endocytosis signal in syntaxin-8 were mutated. A syntaxin-8 mutant that cannot assemble with other SNARE proteins had virtually the same effect as wild-type syntaxin-8. Total internal reflection fluorescence microscopy showed formation and endocytosis of vesicles containing fluorescence-tagged clathrin, TASK-1, and/or syntaxin-8. Our results suggest that the unassembled form of syntaxin-8 and the potassium channel TASK-1 are internalized via clathrin-mediated endocytosis in a cooperative manner. This implies that syntaxin-8 regulates the endocytosis of TASK-1. Our study supports the idea that endosomal SNARE proteins can have functions unrelated to membrane fusion.

SUBMITTER: Renigunta V

PROVIDER: S-EPMC4055267 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1.

Renigunta Vijay V Fischer Thomas T Zuzarte Marylou M Kling Stefan S Zou Xinle X Siebert Kai K Limberg Maren M MM Rinné Susanne S Decher Niels N Schlichthörl Günter G Daut Jürgen J

Molecular biology of the cell 20140417 12

The endosomal SNARE protein syntaxin-8 interacts with the acid-sensitive potassium channel TASK-1. The functional relevance of this interaction was studied by heterologous expression of these proteins (and mutants thereof) in Xenopus oocytes and in mammalian cell lines. Coexpression of syntaxin-8 caused a fourfold reduction in TASK-1 current, a corresponding reduction in the expression of TASK-1 at the cell surface, and a marked increase in the rate of endocytosis of the channel. TASK-1 and synt ...[more]

PMID: 24743596

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Project description:The voltage-gated K+ channel Kv2.1 has been intimately linked with neuronal apoptosis. After ischemic, oxidative, or inflammatory insults, Kv2.1 mediates a pronounced, delayed enhancement of K+ efflux, generating an optimal intracellular environment for caspase and nuclease activity, key components of programmed cell death. This apoptosis-enabling mechanism is initiated via Zn2+-dependent dual phosphorylation of Kv2.1, increasing the interaction between the channel's intracellular C-terminus domain and the SNARE (soluble N-ethylmaleimide-sensitive factor activating protein receptor) protein syntaxin 1A. Subsequently, an upregulation of de novo channel insertion into the plasma membrane leads to the critical enhancement of K+ efflux in damaged neurons. Here, we investigated whether a strategy designed to interfere with the cell death-facilitating properties of Kv2.1, specifically its interaction with syntaxin 1A, could lead to neuroprotection following ischemic injury in vivo The minimal syntaxin 1A-binding sequence of Kv2.1 C terminus (C1aB) was first identified via a far-Western peptide screen and used to create a protherapeutic product by conjugating C1aB to a cell-penetrating domain. The resulting peptide (TAT-C1aB) suppressed enhanced whole-cell K+ currents produced by a mutated form of Kv2.1 mimicking apoptosis in a mammalian expression system, and protected cortical neurons from slow excitotoxic injury in vitro, without influencing NMDA-induced intracellular calcium responses. Importantly, intraperitoneal administration of TAT-C1aB in mice following transient middle cerebral artery occlusion significantly reduced ischemic stroke damage and improved neurological outcome. These results provide strong evidence that targeting the proapoptotic function of Kv2.1 is an effective and highly promising neuroprotective strategy.SIGNIFICANCE STATEMENT Kv2.1 is a critical regulator of apoptosis in central neurons. It has not been determined, however, whether the cell death-enabling function of this K+ channel can be selectively targeted to improve neuronal survival following injury in vivo The experiments presented here demonstrate that the cell death-specific role of Kv2.1 can be uniquely modulated to provide neuroprotection in an animal model of acute ischemic stroke. We thus reveal a novel therapeutic strategy for neurological disorders that are accompanied by Kv2.1-facilitated forms of cell death.

Dataset Information

Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1.

Publications

Cooperative endocytosis of the endosomal SNARE protein syntaxin-8 and the potassium channel TASK-1.

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