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J. Gen. Physiol.,
Volume 110, Number 6, December 1, 1997 693-715
From the * Department of Pharmacology, and § Department of Cellular and Molecular Physiology, Yale University Medical School, New Haven, Connecticut 06520-8066; and 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 µ1 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.
Institut de Pharmacologie et Toxicologie, de l'Universite de Lausanne, CH-1005 Lausanne, Switzerland
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