On the Structural Basis for Size-selective Permeation of Organic Cations through the Voltage-gated Sodium Channel Effect of Alanine Mutations at the DEKA Locus on Selectivity, Inhibition

Recent evidence indicates that ionic selectivity in voltage-gated Na 1 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 Ca 2 1 and H 1 , 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 m 1 Na 1 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 1 and very low Ca 2 1 . 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 1 , Na 1 , K 1 , Rb 1 , Cs 1 ) and permitted inward current of group IIA divalent cations (Mg 2 1 , Ca 2 1 , Sr 2 1 , Ba 2 1 ). 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 1 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 Ca 2 1 and H 1 , but are not essential for the positive shift in activation voltage that is produced by these ions. key words: Ca 2 1 channel • ionic selectivity • m -conotoxin • Na 1 channel • selectivity filter i n t r o d u c t i o n Channel proteins are generally known for mediating transmembrane currents of the major inorganic ions of physiological solutions. However, many channels also allow permeation of small organic ions and polar nonelectrolytes. In this respect, channel pores exhibit sieving behavior much like a dialysis membrane or gel filtration material designed to exhibit a well-defined molecular weight cutoff. As reviewed by Hille (1992), this latter property has been widely used to estimate the minimum diameter of channel pores as deduced from the size of the largest ions that serve as current carriers in electrophysiological assays. For example, cross-sectional areas corresponding to the molecular cutoff region of various members of the superfamily of voltage-gated ion channels have been estimated as follows: voltagegated K 1 channel, 3.3 3 3.3 Å (Bezanilla and Armstrong, 1972; Hille, 1973); voltage-gated Na 1 channel, 3.2 3 5.2 Å (Hille, 1971, 1972); and voltage-gated Ca 2 1 channel, 5.5 3 5.5 Å (McCleskey and Almers, 1985; Coronado and Smith, 1987). Despite a lack of high resolution structures of channel proteins, there is now much information regarding Address correspondence to Edward Moczydlowski, Department of Pharmacology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520-8066. Fax: 203-785-7670; E-mail: edward. [email protected] on Jne 8, 2017 D ow nladed fom Published December 1, 1997