McLaughlin J T, Hawrot E, Yellen G
Department of Pharmacology, Tufts University, Boston, MA 02111, USA.
Biochem J. 1995 Sep 15;310 ( Pt 3)(Pt 3):765-9. doi: 10.1042/bj3100765.
We constructed and characterized a series of nicotinic receptor mutants with a cysteine substituted for one of the amino acid residues in the alpha-subunit between positions 183 and 198. This region of the receptor is known to participate in agonist binding and channel activation. The goal of this 'cysteine scanning mutagenesis' is to introduce the reactivity of a free thiol group into functionally important protein domains; modification of the introduced cysteines can then be used to probe the structure and function of the targeted region. Mutants were examined by coexpression with the beta-, gamma- and delta-subunits in Xenopus oocytes using two-microelectrode voltage clamp recording. Twelve of fourteen mutants expressed receptors with properties comparable with the wild-type, including sensitivity to reduction by dithiothreitol (DTT). This indicates that introduction of an additional cysteine within this region of the receptor did not interfere with formation of the native disulphide between alpha Cys-192 and alpha Cys-193. Only one mutation, alpha Y198C, caused dramatic changes in the EC50 for acetylcholine (ACh) and in the sensitivity to DTT. We then examined the effects of the thiol modification and found two mutants, alpha H186C and alpha V188C, that showed significant decreases in responsiveness to ACh after exposure to methylmethanethiosulphonate (MMTS). Dose-response measurements show that exposure of alpha H186C mutants to MMTS causes a shift in apparent agonist affinity without changing the peak response, and this is not reversible by DTT. In contrast, the MMTS-treated alpha V188C mutants show changes in both apparent affinity and peak response which are readily reversed by DTT. Together, our data show that these two nearby residues occupy markedly different environments relative to the contact points for ACh. They also demonstrate that cysteine-substitution mutagenesis can be successfully applied to protein domains that include functionally important disulphides.
我们构建并表征了一系列烟碱型受体突变体，这些突变体在α亚基183位至198位之间的一个氨基酸残基被半胱氨酸取代。已知该受体的这一区域参与激动剂结合和通道激活。这种“半胱氨酸扫描诱变”的目的是将游离巯基的反应性引入功能重要的蛋白质结构域；然后对引入的半胱氨酸进行修饰，以探测目标区域的结构和功能。使用双微电极电压钳记录技术，在非洲爪蟾卵母细胞中与β、γ和δ亚基共表达来检测突变体。14个突变体中有12个表达的受体特性与野生型相当，包括对二硫苏糖醇（DTT）还原的敏感性。这表明在受体的这一区域引入额外的半胱氨酸并不干扰α亚基Cys-192和α亚基Cys-193之间天然二硫键的形成。只有一个突变体αY198C，导致乙酰胆碱（ACh）的半数有效浓度（EC50）和对DTT敏感性发生显著变化。然后我们检测了巯基修饰的影响，发现两个突变体αH186C和αV188C，在暴露于甲硫基甲烷磺酸盐（MMTS）后对ACh的反应性显著降低。剂量反应测量表明，αHl86C突变体暴露于MMTS会导致表观激动剂亲和力发生变化，而峰值反应不变，并且DTT不能使其恢复。相比之下，经MMTS处理的αV188C突变体在表观亲和力和峰值反应上都有变化，DTT很容易使其恢复。总之，我们的数据表明，相对于ACh的接触点，这两个相邻残基所处的环境明显不同。它们还证明，半胱氨酸取代诱变可以成功应用于包含功能重要二硫键的蛋白质结构域。
