Structure function relationships in diphtheria toxin channels: II. A residue responsible for the channel's dependence on trans pH.
Structure function relationships in diphtheria toxin channels: II. A residue responsible for the channel's dependence on trans pH.
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白喉毒素通道的结构功能关系：II．
DOI：
10.1007/bf00234996
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发表时间：
1994
期刊：
影响因子：
--
通讯作者：
Finkelstein,A
中科院分区：
文献类型：
--
作者：
Mindell,JA;Silverman,JA;Collier,RJ;Finkelstein,A
白喉毒素在脂质双层中形成的离子传导通道具有高度的pH依赖性。在其他性质中，通道的单通道电导和选择性取决于膜两侧的质子浓度。我们先前已经表明，DT的61个氨基酸片段足以形成具有与完整毒素相同的pH依赖性单通道特性的通道。该区域对应于最近发表的DT溶液晶体结构中的α-螺旋发夹;发夹包含两个α-螺旋，每个螺旋足够长以跨越膜，由约9个残基的环连接。本文报道了单通道效应的突变，改变了两个带负电荷的残基在这个环。将谷氨酸349改变为中性谷氨酰胺或正赖氨酸对DT通道的单通道电导或选择性没有影响。相比之下，天冬氨酸352突变为中性天冬酰胺（DT-D352 N）或正赖氨酸（DT-D352 K）导致pH 5.3顺式/7.2反式（在1 mKCl中）单通道电导逐渐降低，这与该基团与通道中的离子静电相互作用一致。这些突变体通道的阳离子选择性也从野生型通道的阳离子选择性降低，这与残基352通过静电力影响渗透离子的方向一致。当膜的两侧都处于pH 4时，野生型和DT-D352 N通道之间的电导差异最小，表明Asp 352在pH4.0顺式/7.2反式下观察到野生型和DT-D352 N通道之间的差异（在隔室中具有高浓度的渗透缓冲液）意味着残留物352在膜的反侧上或附近。比较野生型和DT-D352 K通道在大（顺式）正电压下的电导支持这一结论。位置352的移位严重限制了该区域可能的膜拓扑结构的数量。
Ion-conducting channels formed in lipid bilayers by diphtheria toxin are highly pH dependent. Among other properties, the channel's single channel conductance and selectivity depend on proton concentrations on either side of the membrane. We have previously shown that a 61 amino acid fragment of DT is sufficient to form a channel having the same pH-dependent single channel properties as that of the intact toxin. This region corresponds to an a-helical hairpin in the recently published crystal structure of DT in solution; the hairpin contains two α-helices, each long enough to span a membrane, connected by a loop of about nine residues. This paper reports on the single channel effects of mutations which alter the two negatively charged residues in this loop. Changing Glutamate 349 to neutral glutamine or to positive lysine has no effect on the DT channel's single channel conductance or selectivity. In contrast, mutations of Aspartate 352 to neutral asparagine (DT-D352N) or positive lysine (DT-D352K) cause progressive reductions in single channel conductance at pH 5.3cis/7.2trans(in 1mKCl), consistent with this group interacting electrostatically with ions in the channel. The cation selectivity of these mutant channels is also reduced from that of wild-type channels, a direction consistent with residue 352 influencing permeant ions via electrostatic forces. When both sides of the membrane are at pH 4, the conductance difference between wild-type and DT-D352N channels is minimal, suggesting that Asp 352 (in the wild type) is neutral at this pH. Differences observed between wild-type and DT-D352N channels at pH 4.0cis/7.2 trans (with a high concentration of permeant buffer in theciscompartment) imply that residue 352 is on or near thetransside of the membrane. Comparing the conductances of wild-type and DT-D352K channels at large (cis) positive voltages supports this conclusion. Thetranslocation of position 352 severely constrains the number of possible membrane topologies for this region.