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(+) 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.

Fig. 1.

Ion binding and conductance in NaK. (A) The selectivity filter structure of NaK in complex with K⁺ ions (PDB 3E8H). The 2F_o - F_c map contoured at 1.5σ shows the electron density (blue mesh) of water and K⁺ represented by red and green spheres, respectively. (B) Superimposition of the NaK filter structure (green) with that of KcsA (magenta, PDB 1K4C). Two diagonally opposite subunits are shown for clarity. (C) Single channel traces of NaK at ± 20 and ± 80 mV and its I-V curve. Currents were recorded using giant liposome patch clamping with 150 mM NaCl and 150 mM KCl in the pipette and bath solutions, respectively. The same method was used in the recordings of all NaK mutants.

Fig. 2.

Ion binding and conductance in NaK2CNG-D. (A) The selectivity filter structure of NaK2CNG-D in complex with K⁺ ions. The F_o - F_c ion omit map (1.62 Å) contoured at 5σ shows the density (blue mesh) of three K⁺ ions in the filter. (B) Superimposition of the NaK2CNG-D filter structure (yellow) with that of KcsA (magenta). (C) Single channel traces of NaK2CNG-D at ± 80 mV and its I-V curve.

Fig. 3.

Ion binding and conductance in the K⁺ selective NaK mutant, NaK2K. (A) The selectivity filter structure of NaK2K in complex with K⁺ ions. The F_o - F_c ion omit map (1.55 Å) contoured at 5σ shows the density (blue mesh) of four K⁺ ions in the filter. (B) Superimposition of the NaK2K filter structure (green) with that of KcsA (magenta). (C) Single channel traces of NaK2K at 0 and ± 100 mV, and its I-V curve. For a better assessment of reversal potential, the I-V curve was obtained from the average of 20 current traces recorded using a patch containing multiple channels with voltage pulses ramp from -100 to +100 mV over 400 ms duration.

Fig. 4.

Ion selectivity in NaK2K_T63A and MthK_T59A mutants. Single channel traces at ± 80 mV and their I-V curves of (A) NaK2K_T63A and (B) MthK_T59A recorded using giant liposome patch clamping in biionic conditions (150 mM KCl/150 mM NaCl). (C) Single channel traces of wild-type MthK and I-V curve of wild-type MthK recorded in planar lipid bilayers with 150 mM KCl on one side and 150 mM NaCl on the other. (D) Structure of the MthK ion conduction pore with a Thr59Ala mutation. The 2F_o - F_c ion omit map (1.75 Å) contoured at 2σ shows strong density (blue mesh) of K⁺ ions at the top three sites but much weaker density at site 4.