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

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⁺ channel selectivity filters. Combined with single channel electrophysiology, we show that only the channel with four ion binding sites is K⁺ selective, whereas those with two or three are nonselective and permeate Na⁺ and K⁺ 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⁺ channels is essential for highly selective and efficient permeation of K⁺ ions.