Sylvain Feliciangeli

AKAP150, a switch to convert mechano‐, pH‐ and arachidonic acid‐sensitive TREK K+ channels into open leak channels

TREK channels are unique among two‐pore‐domain K+ channels. They are activated by polyunsaturated fatty acids (PUFAs) including arachidonic acid (AA), phospholipids, mechanical stretch and intracellular acidification. They are inhibited by neurotransmitters and hormones. TREK‐1 knockout mice have impaired PUFA‐mediated neuroprotection to ischemia, reduced sensitivity to volatile anesthetics and altered perception of pain. Here, we show that the A‐kinase‐anchoring protein AKAP150 is a constituent of native TREK‐1 channels. Its binding to a key regulatory domain of TREK‐1 transforms low‐activity outwardly rectifying currents into robust leak conductances insensitive to AA, stretch and acidification. Inhibition of the TREK‐1/AKAP150 complex by Gs‐coupled receptors such as serotonin 5HT4sR and noradrenaline β2AR is as extensive as for TREK‐1 alone, but is faster. Inhibition of TREK‐1/AKAP150 by Gq‐coupled receptors such as serotonin 5HT2bR and glutamate mGluR5 is much reduced when compared to TREK‐1 alone. The association of AKAP150 with TREK channels integrates them into a postsynaptic scaffold where both G‐protein‐coupled membrane receptors (as demonstrated here for β2AR) and TREK‐1 dock simultaneously.

Does sumoylation control K2P1/TWIK1 background K+ channels?

A novel model for the regulation of cell excitability has recently been proposed. It originates from the observation that the background K(+) channel K2P1 (TWIK1) may be silenced by sumoylation in Xenopus oocytes and that inactivation of the putative sumoylation site (mutation K274E) gives rise to robust current expression in transfected COS-7 cells. Here, we show that only the mutation K274E, and not K274R, is associated with an increase of K2P1 current density, suggesting a charge effect of K274E. Furthermore, we failed to observe any band shift by western blot analysis that would confirm an eventual sumoylation of K2P1 in COS-7 cells and oocytes.

Potassium Channel Silencing by Constitutive Endocytosis and Intracellular Sequestration

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-HT1R or adrenoreceptor α2A-AR stimulates TWIK1 but has no effect on TWIK1I293A,I294A, suggesting that Gi protein activation is a physiological signal for increasing the number of active channels at the plasma membrane.

TWIK1, a unique background channel with variable ion selectivity

TWIK1 belongs to the family of background K+ channels with two pore domains. In native and transfected cells, TWIK1 is detected mainly in recycling endosomes. In principal cells in the kidney, TWIK1 gene inactivation leads to the loss of a nonselective cationic conductance, an unexpected effect that was attributed to adaptive regulation of other channels. Here, we show that TWIK1 ion selectivity is modulated by extracellular pH. Although TWIK1 is K+ selective at neutral pH, it becomes permeable to Na+ at the acidic pH found in endosomes. Selectivity recovery is slow after restoration of a neutral pH. Such hysteresis makes plausible a role of TWIK1 as a background channel in which selectivity and resulting inhibitory or excitatory influences on cell excitability rely on its recycling rate between internal acidic stores and the plasma membrane. TWIK1−/− pancreatic β cells are more polarized than control cells, confirming a depolarizing role of TWIK1 in kidney and pancreatic cells.

Mtap2 is a constituent of the protein network that regulates twik-related K+ channel expression and trafficking.

Twik-related K+ (TREK) channels produce background currents that regulate cell excitability. In vivo, TREK-1 is involved in neuronal processes including neuroprotection against ischemia, general anesthesia, pain perception, and mood. Recently, we demonstrated that A-kinase anchoring protein AKAP150 binds to a major regulatory domain of TREK-1, promoting drastic changes in channel regulation by polyunsaturated fatty acids, pH, and stretch, and by G-protein-coupled receptors to neurotransmitters and hormones. Here, we show that the microtubule-associated protein Mtap2 is another constituent of native TREK channels in the brain. Mtap2 binding to TREK-1 and TREK-2 does not affect directly channel properties but enhances channel surface expression and current density. This effect relies on Mtap2 binding to microtubules. Mtap2 and AKAP150 interacting sites in TREK-1 are distinct and both proteins can dock simultaneously. Their effects on TREK-1 surface expression and activation are cumulative. In neurons, the three proteins are simultaneously detected in postsynaptic dense bodies. AKAP150 and Mtap2 put TREK channels at the center of a complex protein network that finely tunes channel trafficking, addressing, and regulation.

Silencing of the tandem pore domain halothane-inhibited K+ channel 2 (THIK2) relies on combined intracellular retention and low intrinsic activity at the plasma membrane.

Background: THIK2 does not generate macroscopic currents in heterologous systems. Results: THIK2 produces K+ currents when mutated in its pore region and/or in its cytoplasmic amino terminus. Conclusion: Silencing of THIK2 is due to low intrinsic activity and intracellular retention. Significance: THIK2 is a functional but silent channel. The tandem pore domain halothane-inhibited K+ channel 1 (THIK1) produces background K+ currents. Despite 62% amino acid identity with THIK1, THIK2 is not active upon heterologous expression. Here, we show that this apparent lack of activity is due to a unique combination of retention in the endoplasmic reticulum and low intrinsic channel activity at the plasma membrane. A THIK2 mutant containing a proline residue (THIK2-A155P) in its second inner helix (M2) produces K+-selective currents with properties similar to THIK1, including inhibition by halothane and insensitivity to extracellular pH variations. Another mutation in the M2 helix (I158D) further increases channel activity and affects current kinetics. We also show that the cytoplasmic amino-terminal region of THIK2 (Nt-THIK2) contains an arginine-rich motif (RRSRRR) that acts as a retention/retrieval signal. Mutation of this motif in THIK2 induces a relocation of the channel to the plasma membrane, resulting in measurable currents, even in the absence of mutations in the M2 helix. Cell surface delivery of a Nt-THIK2-CD161 chimera is increased by mutating the arginines of the retention motif but also by converting the serine embedded in this motif to aspartate, suggesting a phosphorylation-dependent regulation of THIK2 trafficking.

Antagonistic Effect of a Cytoplasmic Domain on the Basal Activity of Polymodal Potassium Channels

TREK/TRAAK channels are polymodal K+ channels that convert very diverse stimuli, including bioactive lipids, mechanical stretch and temperature, into electrical signals. The nature of the structural changes that regulate their activity remains an open question. Here, we show that a cytoplasmic domain (the proximal C-ter domain, pCt) exerts antagonistic effects in TREK1 and TRAAK. In basal conditions, pCt favors activity in TREK1 whereas it impairs TRAAK activity. Using the conformation-dependent binding of fluoxetine, we show that TREK1 and TRAAK conformations at rest are different, and under the influence of pCt. Finally, we show that depleting PIP2 in live cells has a more pronounced inhibitory effect on TREK1 than on TRAAK. This differential regulation of TREK1 and TRAAK is related to a previously unrecognized PIP2-binding site (R329, R330, and R331) present within TREK1 pCt, but not in TRAAK pCt. Collectively, these new data point out pCt as a major regulatory domain of these channels and suggest that the binding of PIP2 to the pCt of TREK1 results in the stabilization of the conductive conformation in basal conditions.