Tubulin binds to the cytoplasmic loop of TRESK background K⁺ channel in vitro.

Péter Enyedi,Irén Veres,Gábor Czirják,Gabriella Braun

doi:10.1371/journal.pone.0097854

Abstract

The cytoplasmic loop between the second and third transmembrane segments is pivotal in the regulation of TRESK (TWIK-related spinal cord K+ channel, K2P18.1, KCNK18). Calcineurin binds to this region and activates the channel by dephosphorylation in response to the calcium signal. Phosphorylation-dependent anchorage of 14-3-3 adaptor protein also modulates TRESK at this location. In the present study, we identified molecular interacting partners of the intracellular loop. By an affinity chromatography approach using the cytoplasmic loop as bait, we have verified the specific association of calcineurin and 14-3-3 to the channel. In addition to these known interacting proteins, we observed substantial binding of tubulin to the intracellular loop. Successive truncation of the polypeptide and pull-down experiments from mouse brain cytosol narrowed down the region sufficient for the binding of tubulin to a 16 amino acid sequence: LVLGRLSYSIISNLDE. The first six residues of this sequence are similar to the previously reported tubulin-binding region of P2X2 purinergic receptor. The tubulin-binding site of TRESK is located close to the protein kinase A (PKA)-dependent 14-3-3-docking motif of the channel. We provide experimental evidence suggesting that 14-3-3 competes with tubulin for the binding to the cytoplasmic loop of TRESK. It is intriguing that the 16 amino acid tubulin-binding sequence includes the serines, which were previously shown to be phosphorylated by microtubule-affinity regulating kinases (MARK kinases) and contribute to channel inhibition. Although tubulin binds to TRESK in vitro, it remains to be established whether the two proteins also interact in the living cell.

Highlights

Two-pore domain (K2P) K+ channels are the widespread molecular correlates of background potassium currents in native cells [1]
TRESK has originally been described as a potassium channel of the human spinal cord [10], afterwards it was found in mouse cerebellum [11], testis, thymus and spleen [12]
The tubulin-binding sequence, identified in the human channel, is not highly conserved in Danio TRESK (Fig. 6). We examined whether this region of the fish channel, analogous to the 16 amino acid human sequence, binds to tubulin or not. (The fragment of Danio TRESK was extended by two amino acids, because of the low homology between the human and fish channels at the C-terminal boundary of the region.) We tested a more closely related homolog of TRESK from Gallus gallus

Summary

Introduction

Two-pore domain (K2P) K+ channels are the widespread molecular correlates of background (leak) potassium currents in native cells [1]. General consensus was reached that TRESK is robustly expressed in pseudounipolar neurons of dorsal root, trigeminal and other sensory ganglia [13,14,15,16,17,18,19] At present, these ganglia are considered as the major location of the channel, some evidence is accumulating that it may be important in ganglia of the autonomic nervous system [20], and in lymphoblastic cell lines [21,22]

Methods

Results

Conclusion