Lee Ju-Young, Visser Frank, Lee Jae Sung, Lee Kyu-Hee, Soh Jae-Won, Ho Won-Kyung, Lytton Jonathan, Lee Suk-Ho
National Research Laboratory for Cell Physiology, Department of Physiology, Seoul National University College of Medicine, 28 Yongon-Dong, Chongno-Ku, Seoul 110-799, South Korea.
J Biol Chem. 2006 Dec 22;281(51):39205-16. doi: 10.1074/jbc.M606287200. Epub 2006 Oct 12.
Different members of the Na+/Ca2++K+ exchanger (NCKX) family are present in distinct brain regions, suggesting that they may have cell-specific functions. Many neuronal channels and transporters are regulated via phosphorylation. Regulation of the rat brain NCKXs by protein kinases, however, has not been described. Here, we report an increase in NCKX2 activity in response to protein kinase C (PKC) activation. Outward current of NCKX2 heterologously expressed in HEK293 cells was enhanced by beta-phorbol dibutyrate (PDBu), whereas PDBu had little effect on activity of NCKX3 or NCKX4. The PDBu-induced enhancement (PIE) of NCKX2 activity was abolished by PKC inhibitors and significantly reduced when the dominant negative mutant of PKCepsilon (K437R) was overexpressed. Moreover, PDBu accelerated the decay rate of the Ca2+ transient at the calyx of Held, where NCKX is the major Ca2+-clearance mechanism. Intracellular perfusion with alkaline phosphatase completely inhibited PIE. Consistently, beta-phorbol myristate acetate (PMA), but not 4alpha-PMA, induced a 3-fold stimulation of 32P incorporation into NCKX2 expressed in HEK293 cells. To investigate the sites involved, PIE of wild-type NCKX2 was compared with mutant NCKX2 in which the three putative PKC consensus sites were replaced with alanine, either individually or in combination. Double-site mutation involving Thr-476 (T166A/T476A and T476A/S504A) disrupted PIE, whereas single mutation of Thr-166, Thr-476, or Ser-504 or the double mutant T166A/S504A failed to completely prevent PIE. These findings suggest that PKC-mediated activation of NCKX2 is sensitive to mutation of multiple PKC consensus sites via a mechanism that may involve several phosphorylation events.
钠/钙2+/钾交换体（NCKX）家族的不同成员存在于不同的脑区，这表明它们可能具有细胞特异性功能。许多神经元通道和转运体通过磷酸化进行调节。然而，蛋白激酶对大鼠脑NCKXs的调节尚未见报道。在此，我们报告蛋白激酶C（PKC）激活后NCKX2活性增加。在HEK293细胞中异源表达的NCKX2的外向电流被β-佛波醇二丁酸酯（PDBu）增强，而PDBu对NCKX3或NCKX4的活性影响很小。PKC抑制剂消除了PDBu诱导的NCKX2活性增强（PIE），当PKCε的显性负突变体（K437R）过表达时，PIE显著降低。此外，PDBu加快了Held壶腹处Ca2+瞬变的衰减速率，在那里NCKX是主要的Ca2+清除机制。用碱性磷酸酶进行细胞内灌注完全抑制了PIE。一致地，β-佛波醇肉豆蔻酸酯乙酸酯（PMA）而非4α-PMA诱导了在HEK293细胞中表达的NCKX2的32P掺入量增加3倍。为了研究涉及的位点，将野生型NCKX2的PIE与突变型NCKX2进行了比较，其中三个假定的PKC共有位点被单独或组合地替换为丙氨酸。涉及Thr-476的双位点突变（T166A/T476A和T476A/S504A）破坏了PIE，而Thr-166、Thr-476或Ser-504的单点突变或双突变体T166A/S504A未能完全阻止PIE。这些发现表明，PKC介导的NCKX2激活对多个PKC共有位点的突变敏感，其机制可能涉及多个磷酸化事件。
