Gibbs Gerard M, Scanlon Martin J, Swarbrick James, Curtis Suzanne, Gallant Esther, Dulhunty Angela F, O'Bryan Moira K
Monash Institute of Medical Research, Monash University, Clayton, Melbourne, Victoria, Australia.
J Biol Chem. 2006 Feb 17;281(7):4156-63. doi: 10.1074/jbc.M506849200. Epub 2005 Dec 9.
The cysteine-rich secretory proteins (Crisp) are predominantly found in the mammalian male reproductive tract as well as in the venom of reptiles. Crisps are two domain proteins with a structurally similar yet evolutionary diverse N-terminal domain and a characteristic cysteine-rich C-terminal domain, which we refer to as the Crisp domain. We presented the NMR solution structure of the Crisp domain of mouse Tpx-1, and we showed that it contains two subdomains, one of which has a similar fold to the ion channel regulators BgK and ShK. Furthermore, we have demonstrated for the first time that the ion channel regulatory activity of Crisp proteins is attributed to the Crisp domain. Specifically, the Tpx-1 Crisp domain inhibited cardiac ryanodine receptor (RyR) 2 with an IC(50) between 0.5 and 1.0 microM and activated the skeletal RyR1 with an AC(50) between 1 and 10 microM when added to the cytoplasmic domain of the receptor. This activity was nonvoltage-dependent and weakly voltage-dependent, respectively. Furthermore, the Tpx-1 Crisp domain activated both RyR forms at negative bilayer potentials and showed no effect at positive bilayer potentials when added to the luminal domain of the receptor. These data show that the Tpx-1 Crisp domain on its own can regulate ion channel activity and provide compelling evidence for a role for Tpx-1 in the regulation of Ca(2+) fluxes observed during sperm capacitation.
富含半胱氨酸的分泌蛋白（Crisp）主要存在于哺乳动物雄性生殖道以及爬行动物的毒液中。Crisp是一种双结构域蛋白，其N端结构域在结构上相似但进化上不同，C端结构域富含半胱氨酸，我们将其称为Crisp结构域。我们展示了小鼠Tpx-1的Crisp结构域的核磁共振溶液结构，结果表明它包含两个亚结构域，其中一个亚结构域的折叠方式与离子通道调节剂BgK和ShK相似。此外，我们首次证明Crisp蛋白的离子通道调节活性归因于Crisp结构域。具体而言，当将Tpx-1 Crisp结构域添加到受体的胞质结构域时，它能以0.5至1.0微摩尔的半数抑制浓度（IC50）抑制心肌兰尼碱受体（RyR）2，并以1至10微摩尔的半数激活浓度（AC50）激活骨骼肌RyR1。这种活性分别是非电压依赖性和弱电压依赖性的。此外，当将Tpx-1 Crisp结构域添加到受体的管腔结构域时，它在负双层电位下能激活两种RyR形式，而在正双层电位下则无作用。这些数据表明，Tpx-1 Crisp结构域自身就能调节离子通道活性，并为Tpx-1在精子获能过程中观察到的Ca(2+)通量调节中的作用提供了有力证据。
