Shapiro M S, Zagotta W N
Department of Physiology, University of Washington, Seattle, Washington 98195-7290 USA.
Biophys J. 2000 May;78(5):2307-20. doi: 10.1016/S0006-3495(00)76777-4.
In vertebrate olfactory receptors, cAMP produced by odorants opens cyclic nucleotide-gated (CNG) channels, which allow Ca(2+) entry and depolarization of the cell. These CNG channels are composed of alpha subunits and at least two types of beta subunits that are required for increased cAMP selectivity. We studied the molecular basis for the altered cAMP selectivity produced by one of the beta subunits (CNG5, CNCalpha4, OCNC2) using cloned rat olfactory CNG channels expressed in Xenopus oocytes. Compared with alpha subunit homomultimers (alpha channels), channels composed of alpha and beta subunits (alpha+beta channels) were half-activated (K(1/2)) by eightfold less cAMP and fivefold less cIMP, but similar concentrations of cGMP. The K(1/2) values for heteromultimers of the alpha subunit and a chimeric beta subunit with the alpha subunit cyclic nucleotide-binding region (CNBR) (alpha+beta-CNBRalpha channels) were restored to near the values for alpha channels. Furthermore, a single residue in the CNBR could account for the altered ligand selectivity. Mutation of the methionine residue at position 475 in the beta subunit to a glutamic acid as in the alpha subunit (beta-M475E) reverted the K(1/2,cAMP)/K(1/2,cGMP) and K(1/2, cIMP)/K(1/2,cGMP) ratios of alpha+beta-M475E channels to be very similar to those of alpha channels. In addition, comparison of alpha+beta-CNBRalpha channels with alpha+beta-M475E channels suggests that the CNBR of the beta subunit contains amino acid differences at positions other than 475 that produce an increase in the apparent affinity for each ligand. Like the wild-type beta subunit, the chimeric beta/alpha subunits conferred a shallow slope to the dose-response curves, increased voltage dependence, and caused desensitization. In addition, as for alpha+beta channels, block of alpha+betaCNBRalpha channels by internal Mg(2+) was not steeply voltage-dependent (zdelta approximately 1e(-)) as compared to block of alpha channels (zdelta 2.7e(-)). Thus, the ligand-independent effects localize outside of the CNBR. We propose a molecular model to explain how the beta subunit alters ligand selectivity of the heteromeric channels.
在脊椎动物嗅觉受体中，气味剂产生的环磷酸腺苷（cAMP）打开环核苷酸门控（CNG）通道，使钙离子进入并导致细胞去极化。这些CNG通道由α亚基和至少两种类型的β亚基组成，β亚基对于提高cAMP选择性是必需的。我们利用在非洲爪蟾卵母细胞中表达的克隆大鼠嗅觉CNG通道，研究了其中一种β亚基（CNG5、CNCalpha4、OCNC2）导致cAMP选择性改变的分子基础。与α亚基同多聚体（α通道）相比，由α和β亚基组成的通道（α + β通道）被激活一半（K(1/2)）时所需的cAMP浓度低八倍，所需的环磷酸肌苷（cIMP）浓度低五倍，但所需的环磷酸鸟苷（cGMP）浓度相似。α亚基与具有α亚基环核苷酸结合区域（CNBR）的嵌合β亚基形成的异多聚体（α + β - CNBRα通道）的K(1/2)值恢复到接近α通道的值。此外，CNBR中的一个单一残基可以解释配体选择性的改变。将β亚基中第475位的甲硫氨酸残基突变为与α亚基中相同的谷氨酸（β - M475E），可使α + β - M475E通道的K(1/2,cAMP)/K(1/2,cGMP)和K(1/2, cIMP)/K(1/2,cGMP)比值恢复到与α通道非常相似的水平。此外，α + β - CNBRα通道与α + β - M475E通道的比较表明，β亚基的CNBR在475位以外的位置存在氨基酸差异，这些差异导致对每种配体的表观亲和力增加。与野生型β亚基一样，嵌合β/α亚基使剂量 - 反应曲线斜率变浅，增加了电压依赖性，并导致脱敏。此外，与α通道的阻断相比（zdelta 2.7e(-)），α + βCNBRα通道被内部Mg(2+)阻断时的电压依赖性不陡峭（zdelta约为1e(-)）。因此，不依赖配体的效应定位于CNBR之外。我们提出了一个分子模型来解释β亚基如何改变异源通道的配体选择性。
