Mindell J A, Silverman J A, Collier R J, Finkelstein A
Department of Physiology and Biophysics, Albert Einstein College of Medicine, Bronx, New York 10461.
J Membr Biol. 1994 Jan;137(1):29-44. doi: 10.1007/BF00234996.
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 alpha-helical hairpin in the recently published crystal structure of DT in solution; the hairpin contains two alpha-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 5.3 cis/7.2 trans (in 1 M KCl), 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 4.0 cis/7.2 trans (with a high concentration of permeant buffer in the cis compartment) imply that residue 352 is on or near the trans side of the membrane. Comparing the conductances of wild-type and DT-D352K channels at large (cis) positive voltages supports this conclusion. The trans location of position 352 severely constrains the number of possible membrane topologies for this region.
由白喉毒素在脂质双分子层中形成的离子传导通道对pH高度敏感。在其他特性中，该通道的单通道电导和选择性取决于膜两侧的质子浓度。我们之前已经表明，白喉毒素的一个61个氨基酸的片段足以形成一个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溶液中白喉毒素晶体结构中的一个α-螺旋发夹结构；该发夹结构包含两个α-螺旋，每个螺旋长到足以跨越膜，由一个约九个残基的环连接。本文报道了改变该环中两个带负电荷残基的突变对单通道的影响。将谷氨酸349变为中性的谷氨酰胺或带正电的赖氨酸对白喉毒素通道的单通道电导或选择性没有影响。相比之下，将天冬氨酸352突变为中性的天冬酰胺（DT-D352N）或带正电的赖氨酸（DT-D352K）会导致在pH 5.3顺式/7.2反式（在1 M KCl中）时单通道电导逐渐降低，这与该基团与通道中的离子发生静电相互作用一致。这些突变通道的阳离子选择性也比野生型通道降低，这一趋势与残基352通过静电力影响渗透离子一致。当膜两侧的pH均为4时，野生型和DT-D352N通道之间的电导差异最小，这表明（野生型中的）天冬氨酸352在此pH下呈中性。在pH 4.0顺式/7.2反式（顺式隔室中有高浓度的渗透缓冲液）条件下，野生型和DT-D352N通道之间观察到的差异意味着残基352位于膜的反式侧或其附近。比较野生型和DT-D352K通道在大的（顺式）正电压下的电导支持了这一结论。352位的跨膜定位严重限制了该区域可能的膜拓扑结构数量。
