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.
Highlights
Calcium-dependent activation of TRESK is mediated by calcineurin
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
Mutations of the LQLP Site Are Effective in TRESK-PVIVIT Channel—We examined the calcium-dependent regulation, when PQIIIS was replaced by the PVIVIT sequence of high calcineurin binding affinity [14, 15]
Summary
Calcium-dependent activation of TRESK is mediated by calcineurin. Results: The sensitivity of human TRESK to calcium is determined by the LQLP calcineurin-docking site. Calcium-dependent activation of human TRESK (TWIK-related spinal cord K؉ channel, K2P18.1) depends on direct targeting of calcineurin to the PQIIIS motif. Modest elevation of [Ca2؉], which effectively regulated the wild type channel, failed to activate TRESKAQAP This indicates that the AQAP mutation diminished the sensitivity of TRESK to calcium. In addition to the enzymatic interaction, calcineurin is directly anchored to a PXIXIT-like site in TRESK (where X may be any amino acid) [12] This mechanism for the targeting of the phosphatase is unparalleled within the ion channel superfamily. Mutation of PQIVID to PQAVAD in mouse TRESK completely prevented the calcium-dependent activation of the Kϩ current, indicating that the docking of calcineurin to the channel is a prerequisite of the regulation [12]. No other protein has been known to contain leucine instead of valine in the third position of the motif, we demonstrate that LQLP interacts with calcineurin and critically determines the calcium sensitivity of the channel
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