Die Rolle von SCAMP Proteinen beim intrazellulären Transport des Kaliumkanals TASK-1

TASK-1 (TWIK-related acid-sensitive K+ channel 1) is an acid-sensitive K+ channel. It belongs to the family of two-pore domain K+ (K2P) channels, which are involved in array of physiological and pathophysiological processes. TASK-1 is widely distributed, being particularly abundant in the pancreas and placenta, but it is also found in the brain, heart, lung and kidney. The intracellular traffic of TASK-1 has been studied in some detail but relatively little is known about the endocytosis of TASK-1. The channel expression at the cell surface is regulated by both forward trafficking and endocytosis mechanisms. We have tried here to elucidate the functional relevance of the interaction between the potassium channel and the SCAMPs. We performed a split-ubiquitin based yeast two-hybrid screen with a brain cDNA library using TASK-1 as bait and identified Secretory Carrier Membrane Protein 5 (SCAMP5) as an interaction partner. Structurally, all SCAMPs contain a highly conserved four hydrophobic transmembrane domains (TMDs) with three inter-TMD-flanking sequences. The hydrophilic loop between transmembrane domain 2 and 3, known as the E-peptide, shows the highest degree of sequence conservation. The cytoplasmic N- and C-termini, flanking the SCAMP domain, have variable length and highest diversity. Of all SCAMP isoforms, SCAMP4 and SCAMP5 are the only isoforms that lack the N-terminal NPF-repeat domains. SCAMP5 is mainly expressed in neural tissues, whereas the other members of SCAMP family are ubiquitously expressed. Expression of TASK-1 in Xenopus oocytes gave rise to an acid-sensitive outward current. The amplitude of this current was reduced to about 50 % when TASK-1 was co-expressed with SCAMP5. A similar reduction of current amplitude was observed after co-expression of the channel with SCAMP1 and SCAMP2. Deletion of the (NPF) domains in the amino terminus of SCAMP1 and 2 proteins abolished the effects of SCAMP1 and SCAMP2 on the endocytosis of TASK-1. When we co-expressed the C-terminal part of the adaptor protein AP180 (AP180C) the effect of SCAMPs on the amplitude of TASK-1 currents was abolished. A similar effect was also observed by Dynasore, a specific blocker for dynamin dependant endocytosis. An antibody uptake assay performed in COS7 cells (where only SCAMP1 and 2 are moderately expressed but SCAMP5 is not expressed) showed a substantially reduced surface expression of TASK-1 after co-expression of SCAMP5. Fluorescence microscopy showed a clear co-localisation of TASK-1 and SCAMP5 at the cell surface. Additional biochemical interaction studies like His-tag pull-down assay showed that SCAMP5 can form homo and heteromers with SCAMP1 and SCAMP2. Our data suggests that SCAMP1 and SCAMP2 can exert their effect on TASK-1 via SCAMP5, where SCAMP5 plays a role as an adaptor protein. Taken together, these results strongly indicate that the secretory carrier membrane proteins play a crucial role in clathrin mediated endocytosis of TASK-1 via NPF domains.