Czirják Gábor, Enyedi Péter
From the Department of Physiology, Semmelweis University, Budapest, Hungary
From the Department of Physiology, Semmelweis University, Budapest, Hungary.
J Biol Chem. 2014 Oct 24;289(43):29506-18. doi: 10.1074/jbc.M114.577684. Epub 2014 Sep 8.
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.
人TRESK（TWIK相关脊髓钾通道，K2P18.1）的钙依赖性激活取决于钙调磷酸酶直接靶向PQIIIS基序。在本研究中，我们证明TRESK还包含磷酸酶的另一个功能相关对接位点，即LQLP氨基酸序列。需要对PQIIIS和LQLP基序进行联合突变才能消除通道的钙依赖性调节。与PQIIIS的丙氨酸替代不同，单独将LQLP突变为AQAP并没有显著改变细胞质钙浓度大幅升高所诱发的TRESK激活幅度。然而，AQAP突变减缓了对高钙的反应。此外，有效调节野生型通道的适度[Ca(2+)]升高未能激活TRESK-AQAP。这表明AQAP突变降低了TRESK对钙的敏感性。即使PQIIIS被具有高钙调磷酸酶结合亲和力的PVIVIT序列取代，在这种TRESK-PVIVIT背景下仍能清楚地检测到AQAP突变的影响。用NFAT转录因子的相应片段替代LQLP区域，该片段与先前描述的LXVP钙调磷酸酶结合共有序列完美匹配，增加了TRESK-PVIVIT的钙敏感性。因此，通过掺入PVIVIT增强TRESK对钙调磷酸酶的亲和力并不能补偿或阻止LQLP序列修饰的影响，这表明两个钙调磷酸酶结合区域在调节中发挥不同作用。我们的结果表明，LQLP位点是人类TRESK钙敏感性的基本决定因素。
