Montal M, Montal M S, Tomich J M
Department of Biology, University of California, San Diego, La Jolla 92093-0319.
Proc Natl Acad Sci U S A. 1990 Sep;87(18):6929-33. doi: 10.1073/pnas.87.18.6929.
A class of proteins that mimic the fundamental pore structure of authentic ionic channels has been designed, synthesized, and characterized. The design is based on our earlier result that a 23-mer peptide with the sequence of the M2 segment of the Torpedo californica acetylcholine receptor delta subunit--Glu-Lys-Met-Ser-Thr-Ala-Ile-Ser-Val-Leu-Leu-Ala-Gln-Ala-Val-Phe -Leu- Leu-Leu-Thr-Ser-Gln-Arg--forms cation-selective channels in lipid bilayers, presumably by self-assembly of conductive oligomers. Accordingly, a tethered parallel tetramer was synthesized with four M2 delta peptides attached to a carrier template--a 9-amino acid backbone with four attachment sites. As expected, the complete 101-residue protein does form channels in lipid bilayers reproducing several features that are characteristic of authentic acetylcholine receptor channels, such as single-channel conductance, cation selectivity, transitions between closed and open states in the millisecond time range, and sensitivity to local anesthetic channel blockers. An analogue protein, in which the serine residue in position 8 is replaced with alanine in each of the four M2 delta 23-mer peptides ([Ala8]M2 delta), also forms channels that, however, exhibit lower single-channel conductance. By contrast, a similar tethered tetramer with M1 delta peptides does not form channels, in accord with expectations. The general validity of this strategy to other channel sequences and oligomer numbers is anticipated. Thus, synporins--a term coined to identify this class of synthetic pore proteins--enrich our armamentarium directed toward the elucidation of structure-function relationships.
一类模拟真实离子通道基本孔结构的蛋白质已被设计、合成并表征。该设计基于我们早期的研究结果,即一种23肽,其序列为加州电鳐乙酰胆碱受体δ亚基的M2片段——Glu-Lys-Met-Ser-Thr-Ala-Ile-Ser-Val-Leu-Leu-Ala-Gln-Ala-Val-Phe-Leu-Leu-Leu-Thr-Ser-Gln-Arg——可能通过导电寡聚体的自组装在脂质双层中形成阳离子选择性通道。因此,合成了一种连接的平行四聚体,其中四个M2δ肽连接到一个载体模板上——一个具有四个连接位点的9氨基酸主链。正如预期的那样,完整的101个残基的蛋白质确实在脂质双层中形成通道,重现了真实乙酰胆碱受体通道的几个特征,如单通道电导、阳离子选择性、在毫秒时间范围内的关闭和开放状态之间的转换以及对局部麻醉通道阻滞剂的敏感性。一种类似物蛋白质,其中四个M2δ23肽中的每一个在第8位的丝氨酸残基被丙氨酸取代([Ala8]M2δ),也形成通道,然而,其单通道电导较低。相比之下,一个带有M1δ肽的类似连接四聚体没有形成通道,这与预期一致。预计该策略对其他通道序列和寡聚体数量具有普遍有效性。因此,合成孔蛋白(synporins)——一个为识别这类合成孔蛋白而创造的术语——丰富了我们用于阐明结构-功能关系的手段。