On the Structural Basis for Size-selective Permeation of Organic Cations through the Voltage-gated Sodium Channel

Abstract

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.

Molecular basis of ion permeability in a voltage‐gated sodium channel

“…Comparisons of the expected‐to‐measured reversal potentials for Na:Li give an estimated relative permeability ratio of 1:0.8. The high values for Na + and Li + over other monovalent and divalent cations determined in this study for NavMs are similar to values that have been measured for eukaryotic sodium channels, such as rat muscle Na v 1.5 [P X / Na  = 1 for Li + and < 0.08 for K + , Cs + , Ca 2+ and Ba 2+ (Sun et al , 1997)].…”

Differences in Apparent Pore Sizes of Low and High Voltage-activated Ca2+ Channels

“…Ammonium and its di-, tri-, and tetramethyl substituents were chosen for two main reasons (49). First of all, given their pK a values, all of these compounds are Ͼ95% ionized at pH 7.4, allowing recordings at physiological pH without complications due to pH-induced changes in the channel itself.…”

“…Second and more importantly, these compounds are expected to offer reliable reagents to determine channel pore size because the progressive addition of methyl groups produces a gradual increase in the size of these molecules without introducing gross modifications in their overall three-dimensional structure. For reference (49), the maximal diameters and the volumes of these ions, respectively, are as follows: NH 4 ϩ , 3.6 Å and 20.1 Å 3 ; methylammonium (MA), 3.8 Å and 35.1 Å 3 ; dimethylammonium (DMA), 4.6 Å and 49.7 Å 3 ; trimethylammonium (TriMA), 6.0 Å and 64.2 Å 3 ; and tetramethylammonium (TMA), 6.0 Å and 78.8 Å 3 . Interesting differences were found in Ca V 1.2-and Ca V 3.2-expressing oocytes when these ions were used as charge carriers for inward currents evoked by depolarizing steps (Fig.…”

“…If this is the case for the T-type channels, one may consider the involvement of a conserved lysine at position Ϫ1 in domain III. Once again, the Na ϩ channel provides precedent for narrowing of a pore by a protruding lysine side chain (49). This hypothesis is worth considering for Ca V 3.1, Ca V 3.2, and Ca V 3.3, but it would not provide an explanation for Ca V 2.3, in which the corresponding position is occupied by a glycine, hardly a bulky residue (41,56).…”

“…One possibility is that the minimal pore diameter is dependent on residues at the EEDD locus itself. This would follow the precedent of the selectivity filter of sodium channels, where alterations of individual amino acid side chains of the DEKA locus have a strong influence on pore size (49). At the EEEE locus of L-type calcium channels (51), like the DEKA locus of sodium channels (52)(53)(54), side chains protrude into the lumen of the pore.…”