Tuning the ion selectivity of tetrameric cation channels by changing the number of ion binding sites

Abstract

Selective ion conduction across ion channel pores is central to cellular physiology. To understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores based on the nonselective NaK channel and determined their structures to high resolution. These structures showcase an ensemble of selectivity filters with a various number of contiguous ion binding sites ranging from 2 to 4, with each individual site maintaining a geometry and ligand environment virtually identical to that of equivalent sites in K þ channel selectivity filters. Combined with single channel electrophysiology, we show that only the channel with four ion binding sites is K þ selective, whereas those with two or three are nonselective and permeate Na þ and K þ equally well. These observations strongly suggest that the number of contiguous ion binding sites in a single file is the key determinant of the channel's selectivity properties and the presence of four sites in K þ channels is essential for highly selective and efficient permeation of K þ ions.nonselective cation channel | potassium channel T he structure determination of several K þ selective and, more recently, bacterial nonselective cation channels has, over the past decade, vastly increased our knowledge of ion selectivity mechanisms (1-8). In K þ channels, the TVGYG signature sequence has emerged as the fundamental element imparting high K þ over Na þ selectivity (9, 10), forming four contiguous and chemically equivalent K þ binding sites composed of backbone carbonyl oxygen atoms from filter residues together with the hydroxyl oxygen atoms from the conserved threonine. Conversely, the TVGDG filter sequence of the NaK channel from Bacillus cereus, although similar in length and amino acid composition to those of K þ channels, forms a nonselective filter preserving the two most intracellular sites of K þ channel filters along with a wide, water-filled vestibular region on the immediately extracellular side (7) (Fig. 1A). Debate lingers in the field about the underlying factors contributing to these selectivity differences. Although available structural studies of K þ channels seem to favor the classical snug-fit model to account for K þ over Na þ selectivity (11, 12), computational studies on K þ channel selectivity, in some cases using an isolated K þ binding site in their calculations, usually invoke one or more of the following concepts: the coordination number of the ion, chemistry of carbonyl oxygen ligands, intrinsic dynamism of the selectivity filter, solvent exposure of the ion binding sites, or the free energy landscapes of ion entrance and translocation in a multiion configuration (13-21). To further understand the underlying principles of ion selectivity in tetrameric cation channels, we engineered a set of cation channel pores whose selectivity filters contain three (equivalent to sites 2-4 or sites 1-3 of a K þ channel) or four (equivalent to sites 1-4 of a K þ channel) ion binding sites and determined their s...

Role of protein dynamics in ion selectivity and allosteric coupling in the NaK channel

“…These results were entirely unexpected given the available high-resolution crystal structures (20,21). The NaK channel has the same basic pore architecture as K + channels ( Fig.…”

“…In the high-resolution crystal structures of NaK and NaK2K, differences are highly localized to the selectivity filter region only (Fig. 1B) (21). We therefore expected relatively few peak shifts when comparing spectra of NaK and NaK2K bound to K + , localized to the region in and around the selectivity filter.…”

“…The NaK channel has the same basic pore architecture as K + channels ( Fig. 1 B and C) and has become a second model system for investigating ion selectivity and gating due to its distinct selectivity filter sequence ( 63 TVGDGN 68 ) and structure (19)(20)(21)(22)(23). Most strikingly, there are only two ion binding sites in the selectivity filter of the nonselective NaK channel (Fig.…”

“…Most strikingly, there are only two ion binding sites in the selectivity filter of the nonselective NaK channel (Fig. 1A) (21,24). However, mutation of two residues in the selectivity filter sequence converts the NaK selectivity filter to the canonical KcsA sequence ( 63 TVGYGD 68 ; Fig.…”

“…However, mutation of two residues in the selectivity filter sequence converts the NaK selectivity filter to the canonical KcsA sequence ( 63 TVGYGD 68 ; Fig. 1 A and B), leading to K + selectivity and a KcsA-like selectivity filter structure with four ion binding sites (21,23). This K + -selective mutant of NaK is called NaK2K.…”