Sun Y M, Favre I, Schild L, Moczydlowski E
Department of Pharmacology, Yale University Medical School, New Haven, Connecticut 06520-8066, USA.
J Gen Physiol. 1997 Dec;110(6):693-715. doi: 10.1085/jgp.110.6.693.
Recent evidence indicates that ionic selectivity in voltage-gated Na+ channels is mediated by a small number of residues in P-region segments that link transmembrane elements S5 and S6 in each of four homologous domains denoted I, II, III, and IV. Important determinants for this function appear to be a set of conserved charged residues in the first three homologous domains, Asp(I), Glu(II), and Lys(III), located in a region of the pore called the DEKA locus. In this study, we examined several Ala-substitution mutations of these residues for alterations in ionic selectivity, inhibition of macroscopic current by external Ca2+ and H+, and molecular sieving behavior using a series of organic cations ranging in size from ammonium to tetraethylammonium. Whole-cell recording of wild-type and mutant channels of the rat muscle micro1 Na+ channel stably expressed in HEK293 cells was used to compare macroscopic current-voltage behavior in the presence of various external cations and an intracellular reference solution containing Cs+ and very low Ca2+. In particular, we tested the hypothesis that the Lys residue in domain III of the DEKA locus is responsible for restricting the permeation of large organic cations. Mutation of Lys(III) to Ala largely eliminated selectivity among the group IA monovalent alkali cations (Li+, Na+, K+, Rb+, Cs+) and permitted inward current of group IIA divalent cations (Mg2+, Ca2+, Sr2+, Ba2+). This same mutation also resulted in the acquisition of permeability to many large organic cations such as methylammonium, tetramethylammonium, and tetraethylammonium, all of which are impermeant in the native channel. The results lead to the conclusion that charged residues of the DEKA locus play an important role in molecular sieving behavior of the Na+ channel pore, a function that has been previously attributed to a hypothetical region of the channel called the "selectivity filter." A detailed examination of individual contributions of the Asp(I), Glu(II), and Lys(III) residues and the dependence on molecular size suggests that relative permeability of organic cations is a complex function of the size, charge, and polarity of these residues and cation substrates. As judged by effects on macroscopic conductance, charged residues of the DEKA locus also appear to play a role in the mechanisms of block by external Ca2+ and H+, but are not essential for the positive shift in activation voltage that is produced by these ions.
最近的证据表明，电压门控Na⁺通道中的离子选择性是由连接四个同源结构域（分别标记为I、II、III和IV）中每个结构域的跨膜元件S5和S6的P区片段中的少数残基介导的。该功能的重要决定因素似乎是前三个同源结构域中的一组保守带电残基，即位于孔道中称为DEKA位点区域的Asp（I）、Glu（II）和Lys（III）。在本研究中，我们检测了这些残基的几个丙氨酸替代突变对离子选择性的改变、外部Ca²⁺和H⁺对宏观电流的抑制作用，以及使用一系列大小从铵到四乙铵的有机阳离子的分子筛分行为。通过全细胞记录稳定表达于HEK293细胞中的大鼠肌肉微小1 Na⁺通道的野生型和突变型通道，来比较在各种外部阳离子存在下以及含有Cs⁺和极低Ca²⁺的细胞内参比溶液中的宏观电流-电压行为。特别地，我们检验了DEKA位点结构域III中的Lys残基负责限制大的有机阳离子通透的假说。将Lys（III）突变为丙氨酸在很大程度上消除了IA族单价碱金属阳离子（Li⁺、Na⁺、K⁺、Rb⁺、Cs⁺）之间的选择性，并允许IIA族二价阳离子（Mg²⁺、Ca²⁺、Sr²⁺、Ba²⁺）的内向电流。同样的突变还导致对许多大的有机阳离子如甲铵、四甲铵和四乙铵具有通透性，而这些阳离子在天然通道中都是不通透的。结果得出结论，DEKA位点的带电残基在Na⁺通道孔道的分子筛分行为中起重要作用，该功能以前被归因于通道的一个假设区域，即“选择性过滤器”。对Asp（I）、Glu（II）和Lys（III）残基的个体贡献以及对分子大小的依赖性的详细研究表明，有机阳离子的相对通透性是这些残基和阳离子底物的大小、电荷和极性的复杂函数。从对宏观电导的影响判断，DEKA位点的带电残基似乎也在外部Ca²⁺和H⁺的阻断机制中起作用，但对于这些离子产生的激活电压的正向偏移不是必需的。
