PDF(Pubmed)
Abstract：
Neuronal KV7 channels, important regulators of cell excitability, are among the most sensitive proteins to reactive oxygen species. The S2S3 linker of the voltage sensor was reported as a site-mediating redox modulation of the channels. Recent structural insights reveal potential interactions between this linker and the Ca2+-binding loop of the third EF-hand of calmodulin (CaM), which embraces an antiparallel fork formed by the C-terminal helices A and B, constituting the calcium responsive domain (CRD). We found that precluding Ca2+ binding to the EF3 hand, but not to EF1, EF2, or EF4 hands, abolishes oxidation-induced enhancement of KV7.4 currents. Monitoring FRET (Fluorescence Resonance Energy Transfer) between helices A and B using purified CRDs tagged with fluorescent proteins, we observed that S2S3 peptides cause a reversal of the signal in the presence of Ca2+ but have no effect in the absence of this cation or if the peptide is oxidized. The capacity of loading EF3 with Ca2+ is essential for this reversal of the FRET signal, whereas the consequences of obliterating Ca2+ binding to EF1, EF2, or EF4 are negligible. Furthermore, we show that EF3 is critical for translating Ca2+ signals to reorient the AB fork. Our data are consistent with the proposal that oxidation of cysteine residues in the S2S3 loop relieves KV7 channels from a constitutive inhibition imposed by interactions between the EF3 hand of CaM which is crucial for this signaling.
摘要：
神经元KV7通道，细胞兴奋性的重要调节剂，是对活性氧最敏感的蛋白质之一。电压传感器的S2S3连接体被报道为介导通道的氧化还原调制的位点。最近的结构见解揭示了该接头与钙调蛋白（CaM）第三EF手的Ca2结合环之间的潜在相互作用，包含由C末端螺旋A和B形成的反平行叉，构成钙响应域(CRD)。我们发现排除Ca2+与EF3手的结合，但不是EF1，EF2或EF4的手，消除了氧化诱导的KV7.4电流增强。使用用荧光蛋白标记的纯化的CRD结构域监测螺旋A和B之间的FRET，我们观察到S2S3肽在Ca2+的存在下引起信号的逆转，但在没有这种阳离子或肽被氧化的情况下没有效果。用Ca2+加载EF3的能力对于FRET信号的这种逆转是必不可少的，而消除与EF1，EF2或EF4结合的Ca2的后果可以忽略不计。此外，我们证明EF3对于转换Ca2信号以重新定向AB叉至关重要。我们的数据与以下提议一致：S2S3环中半胱氨酸残基的氧化使KV7通道免于由CaM的EF3手之间的相互作用施加的组成型抑制，这对于该信号传导至关重要。
