Potassium Channel Silencing by Constitutive Endocytosis and Intracellular Sequestration

Sylvain Feliciangeli,Franck C Chatelain,Florian Lesage,Jacques Barhanin,Richard Warth,Saïd Bendahhou,Guillaume Sandoz,Magalie P Tardy

doi:10.1074/jbc.m109.078535

Abstract

Tandem of P domains in a weak inwardly rectifying K(+) channel 1 (TWIK1) is a K(+) channel that produces unusually low levels of current. Replacement of lysine 274 by a glutamic acid (K274E) is associated with stronger currents. This mutation would prevent conjugation of a small ubiquitin modifier peptide to Lys-274, a mechanism proposed to be responsible for channel silencing. However, we found no biochemical evidence of TWIK1 sumoylation, and we showed that the conservative change K274R did not increase current, suggesting that K274E modifies TWIK1 gating through a charge effect. Now we rule out an eventual effect of K274E on TWIK1 trafficking, and we provide convincing evidence that TWIK1 silencing results from its rapid retrieval from the cell surface. TWIK1 is internalized via a dynamin-dependent mechanism and addressed to the recycling endosomal compartment. Mutation of a diisoleucine repeat located in its cytoplasmic C terminus (I293A,I294A) stabilizes TWIK1 at the plasma membrane, resulting in robust currents. The effects of I293A,I294A on channel trafficking and of K274E on channel activity are cumulative, promoting even more currents. Activation of serotoninergic receptor 5-HT(1)R or adrenoreceptor alpha2A-AR stimulates TWIK1 but has no effect on TWIK1I293A,I294A, suggesting that G(i) protein activation is a physiological signal for increasing the number of active channels at the plasma membrane.

Highlights

TWIK12 (KCNK1, K2P1) has been cloned from human kidney [1]
We concluded that the increase of current associated with K274E, and which is absent in K274R, could probably be attributed to a charge effect modifying the TWIK1 gating and that small ubiquitin modifier (SUMO) conjugation at lysine 274 does not underlie TWIK1 silencing [19]
We show that TWIK1 contains a diisoleucine-based motif required for its rapid endocytosis

Summary

Introduction

TWIK12 (KCNK1, K2P1) has been cloned from human kidney [1]. It has four membrane-spanning segments (M1–M4) and two pore-forming loops (P1 and P2) and forms covalent homodimers [2]. Electron microscopy from polarized MDCK cells shows that TWIK1 is present mainly underneath the apical brush border with only a few immunogold particles found at a position corresponding to the plasma membrane (Fig. 1B).

Results

Conclusion