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Investigation of critical amino acids in c‐terminal regions for the complex sensitivity of TREK‐2 K + channel to membrane PIP 2 and pH i (LB841)
Author(s) -
Woo Joohan,
Kim Hyun Jong,
Park Kyoung Sun,
Shin Dong Hoon,
Zhang YinHua,
Nam Joo Hyun,
Kim Sung Joon
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.lb841
Subject(s) - chemistry , biophysics , patch clamp , mutant , phosphatidylinositol , biochemistry , intracellular , amino acid , alanine , stereochemistry , kinase , biology , receptor , gene
TREK‐2, a member of two‐pore domain K + channel (K2P) family, are activated by various chemical and physical stimuli; arachidonic acid (AA), intracellular acidic pH (pH i ) and membrane stretch. Also, TREK channels show dual sensitivity to plasmalemmal phosphatidylinositol 4, 5‐bisphosphate (PIP 2 ); inhibition by PIP 2 higher than physiological intrinsic level, activation by partial depletion, and complete inhibition by PIP 2 scavenging (e.g. poly‐L lysine). Consistently, in inside‐out (i‐o) patch clamp conditions, application of MgATP (1 mM) inhibits TREK‐2 via PI‐kinase dependent PIP 2 production. To elucidate the regulatory sites interacting with PIP 2 , we made site‐directed mutants of Lys and Arg in TREK‐2 c‐terminal regions that are relatively ‘proximal’ and ‘distal’ to membrane. Whole‐cell patch clamp (w‐c) recordings showed that Arg residues in the ‘distal’ domain (R356, R357) are important for maintaining basal TREK‐2 activity whereas neutralization of ‘proximal’ residues (R323, K327, K328) did not affect the background current. The suppression of basal activity was most prominent by neutralizing (alanine substitution) or deleting three Arg (R355‐7). However, maximum activation by 10 μM AA was not affected. The i‐o patch clamp recording showed that TREK‐2 inhibition by 3 mM MgATP was partly prevented in K330 neutralized mutant (K330A) whereas the neutralization of ‘distal’ cationic residues did not affect the MgATP‐dependent inhibition of TREK‐2. Unexpectedly, the pH i sensitivity of TREK‐2 was also reversed in K330A (inhibition, not activation, by acidic pH i ). Interestingly, neutralization of acidic amino acid (E350) that was previously reported as pH i sensor, also prevented the inhibition by MgATP in addition to reversing the pH i dependence. Taken together, both cationic and anionic amino acids in the ‘proximal’ c‐terminal region of TREK‐2 interact to provide PIP 2 and pH i sensitivity. The distal arginines appear to be important for basal activity of TREK‐2