A single P-loop glutamate point mutation to either lysine or arginine switches the cation-anion selectivity of the CNGA2 channel.
A single P-loop glutamate point mutation to either lysine or arginine switches the cation-anion selectivity of the CNGA2 channel.
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DOI：
10.1085/jgp.200509378
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发表时间：
2006-04
期刊：
影响因子：
--
通讯作者：
Barry PH
中科院分区：
文献类型：
--
作者：
Qu W;Moorhouse AJ;Chandra M;Pierce KD;Lewis TM;Barry PH
环核苷酸门控（CNG）通道在嗅觉和视觉转导中起着关键作用。利用位点定向诱变和内向外膜片钳记录技术，研究了HEK293细胞中表达的两个突变型同源大鼠嗅觉CNGA2通道的离子渗透和选择性。带负电荷的孔环（P-loop）谷氨酸（E342）单点突变为带正电荷的赖氨酸或精氨酸，导致功能通道持续响应cGMP，尽管电流通常非常小。赖氨酸突变体通道的浓度-响应曲线与野生型（WT）通道非常相似，表明突变体通道没有发生重大的结构改变。在细胞质NaCl稀释期间的逆转电位测量表明，赖氨酸和精氨酸突变将通道的选择性从阳离子（PCl/PNa = 0.07 [WT]）切换到阴离子（PCl/PNa = 14 [Lys]或10 [Arg]）。用双离子取代法测定的两个突变体通道的相对阴离子渗透性序列为NO3 - > I - > Br - > Cl - > F - > acetate -，与阴离子选择性GABA和甘氨酸通道相同。突变通道似乎也具有极小的单通道电导，使用噪声分析测量约为1-2 pS，而WT值约为29 pS。结果表明，主要是p环中E342残基的电荷，而不是孔螺旋偶极子，控制着该通道的阳离子-阴离子选择性。然而，两个突变通道在对称NaCl溶液中表现出的向外整流表明，孔螺旋偶极子的负端可能在减少Cl -离子通过这些阴离子选择通道的向外运动中起作用。这些结果有潜在的影响阴离子选择性的决定因素在大家族的p环含有通道。
Cyclic nucleotide-gated (CNG) channels play a critical role in olfactory and visual transduction. Site-directed mutagenesis and inside-out patch-clamp recordings were used to investigate ion permeation and selectivity in two mutant homomeric rat olfactory CNGA2 channels expressed in HEK293 cells. A single point mutation of the negatively charged pore loop (P-loop) glutamate (E342) to either a positively charged lysine or arginine resulted in functional channels, which consistently responded to cGMP, although the currents were generally extremely small. The concentration–response curve of the lysine mutant channel was very similar to that of wild-type (WT) channels, suggesting no major structural alteration to the mutant channels. Reversal potential measurements, during cytoplasmic NaCl dilutions, showed that the lysine and the arginine mutations switched the selectivity of the channel from cations (PCl/PNa = 0.07 [WT]) to anions (PCl/PNa = 14 [Lys] or 10 [Arg]). Relative anion permeability sequences for the two mutant channels, measured with bi-ionic substitutions, were NO3 − > I− > Br− > Cl− > F− > acetate−, the same as those obtained for anion-selective GABA and glycine channels. The mutant channels also seem to have an extremely small single-channel conductance, measured using noise analysis of about 1–2 pS, compared to a WT value of about 29 pS. The results showed that it is predominantly the charge of the E342 residue in the P-loop, rather than the pore helix dipoles, which controls the cation–anion selectivity of this channel. However, the outward rectification displayed by both mutant channels in symmetrical NaCl solutions suggests that the negative ends of the pore helix dipoles may play a role in reducing the outward movement of Cl− ions through these anion-selective channels. These results have potential implications for the determinants of anion–cation selectivity in the large family of P-loop–containing channels.