Synthesis of Transmembrane Segment of A Single Spanning Protein, Isk, Forming Potassium Channel and Its Interaction with Phospholipid Bilayr

Isk是一种膜蛋白，由130个氨基酸残基组成，只有一个跨膜结构域，可以在哺乳动物肾脏、子宫和心脏中形成电压门控钾通道，该通道可以通过膜电位的变化快速打开或关闭。单个跨膜结构域被认为通过与生物膜相互作用形成α -螺旋，并作为Isk蛋白单体在膜内组装形成的孔的组成部分发挥作用，从而形成导电孔。通过组装只有一个由疏水氨基酸残基组成的跨膜结构域的Isk蛋白来明确钾离子选择性通道的形成机制是一个很有意义的问题。因此，对这部分的构象和组装在膜上的行为的研究将导致对这种蛋白质性质的基本理解。因此，我们合成了一个包含40个残基的肽，包含与38-77残基对应的跨膜结构域（Isk-O）及其类似物，该类似物将Isk-0中的Ala替换为三个Gly （Isk-A）。在本研究中，Isk-0在中性和酸性脂质体中均呈β结构，且未形成通道。另一方面，风险- a导致电导逐渐增加，然后是恒定的电导，但没有表现出通道状的打开和关闭尖峰。最近关于通道形成肽和蛋白质的出版物表明-链似乎是膜跨越域的一部分。跨Isk结构域的膜被假设为螺旋结构，在生物膜中可能采用β -结构或α -螺旋和β -结构的混合物。Isk-0H-Arg-Asp-Asp-Ser-Lys-Leu-Glu-Ala-Leu-Tyr-Ile-Leu-Met-Val-Leu-Gly-Phe-Phe-Gly-Phe-Phe-Thr-Leu-Gly-Ile-Met-Leu-Ser-Tyr-Ile-Arg-Ser-Lys-Lys-Leu-Glu-His-Ser-His-Asp-0H

A membrane protein, Isk, consisting of 130 amino acid residues with only a single transmembrane domain can form a voltage-gated potassium channel which can be opened or closed rapidly by changes in membrane potential in kidney, womb and heart of mammals. The single transmembrane domain is considered to form an alpha-helix by interacting with biomembrane and play a role as a constituent of the pore formed in the assembly of Isk protein monomers within membranes, resulting in the formation of a conducting pore. It is a matter of great interest to make clear the mechanism of the potassium ion selective channel formation by assembling of Isk proteins which have only one transmembrane domain consisting of hydrophobic amino acid residues. Thus, an investigation on the conformation of this portion and behavior of the assembly in membranes will lead to fundamental understanding of the nature of this protein. Therefore we synthesized a peptide of 40 residues containing the transmembrane domain corresponding to residues 38-77 (Isk-O) and its analog which is substituted Ala in Isk-0 for three Gly (Isk-A).In the present study, Isk-0 took beta-structure in neutral and acidic liposomes and showed no channel formation. On the other hand, Isk-A resulted in a gradual increase in conductance followed by a constant conductance but did not exhibit channel-like opening and closing spikes.. Recent publications on the channel forming peptides and proteins have shown that beta-strands seem to be a part of the membrane spanning domain. The membrane spanning Isk domain hypothesized as an alpha-helical structure may adopt beta-structure or a mixture of alpha-helical and beta-structures in biological membranes.Isk-0H-Arg-Asp-Asp-Ser-Lys-Leu-Glu-Ala-Leu-Tyr-Ile-Leu-Met-Val-Leu-Gly-Phe-Phe-Gly-Phe-Phe-Thr-Leu-Gly-Ile-Met-Leu-Ser-Tyr-Ile-Arg-Ser-Lys-Lys-Leu-Glu-His-Ser-His-Asp-0H