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首页> 外文期刊>Gene expression >Dominant-negative suppression of big brain ion channel activity by mutation of a conserved glutamate in the first transmembrane domain.
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Dominant-negative suppression of big brain ion channel activity by mutation of a conserved glutamate in the first transmembrane domain.

机译：通过在第一个跨膜结构域中保守的谷氨酸的突变显着阴性抑制大脑离子通道活性。

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摘要

The neurogenic protein Drosophila big brain (BIB), which is involved in the process of neuroblast determination, and the water channel aquaporin-1 (AQP1) are among a subset of the major intrinsic protein (MIP) channels that have been found to show gated monovalent cation channel activity. A glutamate residue in the first transmembrane (M1) domain is conserved throughout the MIP family. Mutation of this residue to asparagine in BIB (E71N) knocks out ion channel activity, and when coexpressed with BIB wild-type as shown here generates a dominant-negative effect on ion channel function, measured in the Xenopus oocyte expression system using two-electrode voltage clamp. cRNAs for wild-type and mutant BIB or AQP1 channels were injected individually or as mixtures. The magnitude of the BIB ionic conductance response was greatly reduced by coexpression of the mutant E71N subunit, suggesting a dominant-negative mechanism of action. The analogous mutation in AQP1 (E17N) did not impair ion channel activation by cGMP, but did knock out water channel function, although not via a dominant-negative effect. This contrast in sensitivity between BIB and AQP1 to mutation of the M1 glutamate suggests the possibility of interesting structural differences in the molecular basis of the ion permeation between these two classes of channels. The dominant-negative construct of BIB could be a tool for testing a role for BIB ion channels during nervous system development in Drosophila. The neurogenic protein Drosophila big brain (BIB), which is involved in the process of neuroblast determination, and the water channel aquaporin-1 (AQP1) are among a subset of the major intrinsic protein (MIP) channels that have been found to show gated monovalent cation channel activity. A glutamate residue in the first transmembrane (M1) domain is conserved throughout the MIP family. Mutation of this residue to asparagine in BIB (E71N) knocks out ion channel activity, and when coexpressed with BIB wild-type as shown here generates a dominant-negativeeffect on ion channel function, measured in the Xenopus oocyte expression system using two-electrode voltage clamp. cRNAs for wild-type and mutant BIB or AQP1 channels were injected individually or as mixtures. The magnitude of the BIB ionic conductance response was greatly reduced by coexpression of the mutant E71N subunit, suggesting a dominant-negative mechanism of action. The analogous mutation in AQP1 (E17N) did not impair ion channel activation by cGMP, but did knock out water channel function, although not via a dominant-negative effect. This contrast in sensitivity between BIB and AQP1 to mutation of the M1 glutamate suggests the possibility of interesting structural differences in the molecular basis of the ion permeation between these two classes of channels. The dominant-negative construct of BIB could be a tool for testing a role for BIB ion channels during nervous system development in Drosophila.
机译：神经元蛋白果蝇大脑（BIB），参与成神经细胞的确定过程，水通道aquaporin-1（AQP1）是主要内在蛋白（MIP）通道的一部分，这些通道已显示出门控单价阳离子通道活性。在整个MIP家族中，第一个跨膜（M1）域中的谷氨酸残基是保守的。在BIB（E71N）中将此残基突变为天冬酰胺会敲除离子通道的活性，当与BIB野生型共表达时，如此处所示，它对离子通道功能产生显性负效应，这是在非洲爪蟾卵母细胞表达系统中使用两电极测量的电压钳。野生型和突变型BIB或AQP1通道的cRNA分别或混合注射。通过共表达突变体E71N亚基，BIB离子电导响应的幅度大大降低，表明存在显性负作用机制。 AQP1（E17N）中的类似突变不会损害cGMP激活离子通道，但确实消除了水通道功能，尽管不是通过显性负效应。 BIB和AQP1对M1谷氨酸突变的敏感性之间的这种对比表明，这两类通道之间离子渗透的分子基础存在有趣的结构差异。 BIB的显性负构建体可能是测试果蝇神经系统发育过程中BIB离子通道作用的工具。神经元蛋白果蝇大脑（BIB），参与神经母细胞的确定过程，水通道aquaporin-1（AQP1）是主要内在蛋白（MIP）通道中的一个子集，这些通道已显示出门控单价阳离子通道活性。在整个MIP家族中，第一个跨膜（M1）域中的谷氨酸残基是保守的。在BIB（E71N）中将此残基突变为天冬酰胺会敲除离子通道活性，当与BIB野生型共表达时，如此处所示，该离子通道功能在Xenopus卵母细胞表达系统中使用两电极电压测量时产生显性负效应。钳。野生型和突变型BIB或AQP1通道的cRNA分别或混合注射。通过共表达突变体E71N亚基，BIB离子电导响应的幅度大大降低，表明存在显性负作用机制。 AQP1（E17N）中的类似突变不会损害cGMP激活离子通道，但确实消除了水通道功能，尽管不是通过显性负效应。 BIB和AQP1对M1谷氨酸突变的敏感性之间的这种对比表明，这两类通道之间离子渗透的分子基础存在有趣的结构差异。 BIB的显性负构建体可能是测试果蝇神经系统发育过程中BIB离子通道作用的工具。

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