title: The LQLP calcineurin docking site is a major determinant of the calcium-dependent activation of human TRESK background K+ channel.
abstract: Calcium-dependent activation of human TRESK (TWIK-related spinal cord K(+) channel, K2P18.1) depends on direct targeting of calcineurin to the PQIIIS motif. In the present study we demonstrate that TRESK also contains another functionally relevant docking site for the phosphatase, the LQLP amino acid sequence. Combined mutations of the PQIIIS and LQLP motifs were required to eliminate the calcium-dependent regulation of the channel. In contrast to the alanine substitutions of PQIIIS, the mutation of LQLP to AQAP alone did not significantly change the amplitude of TRESK activation evoked by the substantial elevation of cytoplasmic calcium concentration. However, the AQAP mutation slowed down the response to high calcium. In addition, modest elevation of [Ca(2+)], which effectively regulated the wild type channel, failed to activate TRESK-AQAP. This indicates that the AQAP mutation diminished the sensitivity of TRESK to calcium. Even if PQIIIS was replaced by the PVIVIT sequence of high calcineurin binding affinity, the effect of the AQAP mutation was clearly detected in this TRESK-PVIVIT context. Substitution of the LQLP region with the corresponding fragment of NFAT transcription factor, perfectly matching the previously described LXVP calcineurin-binding consensus sequence, increased the calcium-sensitivity of TRESK-PVIVIT. Thus the enhancement of the affinity of TRESK for calcineurin by the incorporation of PVIVIT could not compensate for or prevent the effects of LQLP sequence modifications, suggesting that the two calcineurin-binding regions play distinct roles in the regulation. Our results indicate that the LQLP site is a fundamental determinant of the calcium-sensitivity of human TRESK.
authors.affiliation: From the Department of Physiology, Semmelweis University, Budapest, Hungary czirjak.gabor@med.semmelweis-univ.hu.
authors.forename: Gábor
authors.initials: G
authors.lastname: Czirják
authors.affiliation: From the Department of Physiology, Semmelweis University, Budapest, Hungary.
authors.forename: Péter
authors.initials: P
authors.lastname: Enyedi
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D019703:Calcineurin / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects*; D051379:Mice; D008954:Models, Biological; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D009154:Mutation / Q000235:genetics; D010766:Phosphorylation / Q000187:drug effects; D015221:Potassium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D013329:Structure-Activity Relationship
chemical_list: C476574:KCNK18 protein, human; D050505:Mutant Proteins; D015221:Potassium Channels; D019703:Calcineurin; D002118:Calcium
keywords: Calcineurin; Calcium; Ion Channel; K2P; KCNK18; PXIXIT; Potassium Channel; Protein Phosphatase 2B (PP2B); Two-pore Domain