Korean J Physiol Pharmacol.  2020 Nov;24(6):555-561. 10.4196/kjpp.2020.24.6.555.

Dual regulatory effects of PI(4,5)P₂ on TREK-2 K⁺ channel through antagonizing interaction between the alkaline residues (K³³⁰. and R^355-357 ) in the cytosolic C-terminal helix

• Kim SE
• Kim MH
• Woo J
• Kim SJ

• KMID: 2507740
• DOI: http://doi.org/10.4196/kjpp.2020.24.6.555

Abstract

TWIK-related two-pore domain K⁺ channel-2 (TREK-2) has voltageindependent activity and shows additional activation by acidic intracellular pH (pH i ) via neutralizing the E³³² in the cytoplasmic C terminal (Ct). We reported opposite regulations of TREK-2 by phosphatidylinositol 4,5-bisphosphate (PIP₂ ) via the alkaline K³³⁰ and triple Arg residues (R^355-357) inhibition and activation, respectively. The G³³⁴ between them appeared critical because its mutation (G³³⁴A) endowed hTREK-2 with tonic activity, similar to the mutation of the inhibitory K³³⁰ (K³³⁰ A). To further elucidate the role of putative bent conformation at G³³⁴ ,we compared the dual mutation forms, K³³⁰ A/G³³⁴ A and G³³⁴ A/R ^355-7 A, showing higher and lower basal activity, respectively. The results suggested that the tonic activity of G³³⁴ A owes to a dominant influence from R^355-7. Since there are additional triple Arg residues (R ^377-9 ) distal to R^355-7 , we also examined the triple mutant (G³³⁴A/R^355-7 A/R^377-9 A) that showed tonic inhibition same with G³³⁴ A/R ^355-7 A. Despite the state of tonic inhibition, the activation by acidic pH i was preserved in both G ³³⁴ A/R^355-7 A and G³³⁴ A/R ^355-7A/R^377-9 A, similar to the R^355-7 A. Also, the inhibitory effect of ATP could be commonly demonstrated under the activation by acidic pH i in R^355-7A, G³³⁴ A/R^355-7 A, and G³³⁴ A/R^355-7 A/R^377-9 A. These results suggest that the putative bent conformation at G³³⁴ is important to set the tug-of-war between K³³⁰ and R^355-7 in the PIP₂ -dependent regulation of TREK-2.

Keyword

Amino acids; basic; C-terminal; PIP₂; Tandem pore domain potassium channels; TREK-2

Figure

• 

  Fig. 1 The proposed mechanisms of regulatory interactions between Ct and membrane PIP2 depicted as cartoon models. Schematic drawing of hTREK-2 dimers with TM4 connected to proximal C-terminus (Blue and grey circles for that cationic and anionic residues. Orange circles for the G334). (A) Low activity of hTREK-2 with increased PIP2 level. (B) Applied PIP2 scavenger (pLL) drives hTREK-2 into the inhibited state. (C) High activity state with ATP-free or neutralization of Lys (K330A). (D) Soley mutated of Arg (R355-7A) induces low activity. (E) High activity state with mutated G334, decreased hinge flexibility. (F, G) hTREK-2 is highly activated by acidic pHi and neutralizing mutation, E332A. (H) R355-7A mutation showed high activity in acid pHi. Distal triple cationic residues (R377-9) function were still unknown. (I) The sequence of Ct amino acids in hTREK-2. A pink circle indicates G334 which roles of putative hinge residue. Orange and Red boxes indicate consecutive proximal and distal cationic residues; R355-7 and R377-9. Yellow and blue circles indicate PIP2-dependent inhibitory and pHi sensing site; K330 and E332.

• 

  Fig. 2 Effects of acidic pHi on the activity of hTREK-2 and its various mutations in Ct. Representative traces of inside-out patch clamp recording in wild-type (WT) (A), G334A (B), K330A/G334A (C), G334A/R355-7A (D). The current was measured at symmetrical 145 mM K+ with –60 mV holding voltage. (A) Trace of WT showing spontaneous increase current after excised cell membrane. Activated by acidification (pHi 6.0 and 5.0) and inhibited by 3 mM MgATP. (B, C) G334A and G334A/K330A mutants high tonic activity, partial decrease of acid and slightly inhibition by 3 mM MgATP. (D) G334A/R355-7A showed low basal activity, predominantly activated by acid and slowly activated by 3 mM MgATP. (E, F) Summary of currents at –60 mV normalized to the maximum level measured at pHi 7.0 and pHi 5.0. G334A (6), K330A/G334A (13), WT (32), G334A/R355-7A (28), G334A/R355-7A/R377-9A (16), respectively. *p < 0.05; ***p < 0.005.

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  Fig. 3 Effects of intracellular ATP on the activity of hTREK-2 and its various mutations including the distal R377-9 in Ct. Representative traces of inside-out patch clamp recording in G334A/R355-7A (A), G334A/R355-7A/R377-9A (B), and R355-7A (C). It has to be noted that the panel (A) is same with the trace shown in D. Symmetrical KCl condition with –60 mV of holding voltage was applied. After confirming the stimulatory effects of acidic pHi, 3 mM MgATP was applied in pHi 7.0. Initial current slightly inhibited by 3 mM MgATP then gradually increased (D). Summary of the TREK-2 currents at –60 mV normalized to the amplitude measured at pHi 5.0. Mean ± SEM for G334A/R355-7A (28), G334A/R355-7A/R377-9A (16), and R355-7A (10), respectively.

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