Sodium and potassium competition in potassium-selective and non-selective channels

Abstract

Potassium channels selectively conduct K(+), primarily to the exclusion of Na(+), despite the fact that both ions can bind within the selectivity filter. Here we perform crystallographic titration and single-channel electrophysiology to examine the competition of Na(+) and K(+) binding within the filter of two NaK channel mutants; one is the potassium-selective NaK2K mutant and the other is the non-selective NaK2CNG, a CNG channel pore mimic. With high-resolution structures of these engineered NaK channel constructs, we explicitly describe the changes in K(+) occupancy within the filter upon Na(+) competition by anomalous diffraction. Our results demonstrate that the non-selective NaK2CNG still retains a K(+)-selective site at equilibrium, whereas the NaK2K channel filter maintains two high-affinity K(+) sites. A double-barrier mechanism is proposed to explain K(+) channel selectivity at low K(+) concentrations.

Figure 1. Structure of K⁺ selective and non-selective channels.

The overall and filter structure (inset) of the K⁺ selective KcsA and NaK2K channels, and the non-selective NaK and NaK2CNG channels. K⁺ ions within the filter are represented as green spheres.

Figure 2. Electrophysiology of Na⁺–K⁺ competition in NaK2K.

(a) Sample traces (left) and histograms (right) of single-channel currents at −100 mV through NaK2K at various concentrations of K⁺ in bath solution. The pipette and bath solutions contain 150 mM Na⁺ and 150 mM Na⁺–K⁺ mixture, respectively. (b) Magnitude of the inward current (defined as the current from bath to pipette) as a function of potassium concentration. Data were normalized to the Na⁺ current measured in the absence of K⁺ (0 mM K⁺). Data points are mean±s.e.m. from five separate patches.

Figure 3. Crystallographic titration of Na⁺–K⁺ competition in NaK2K.

(a) Anomalous difference Fourier maps (contoured at 0.03 e Å⁻³), (b) 1D anomalous difference density profiles along the central filter axis and (c) estimated occupancies of K⁺ within the filter of NaK2K at various concentrations of K⁺. Dotted line indicates the baseline used for peak integration. (d) Single-channel traces and histograms of K⁺ currents in NaK2K measured with 50 mM symmetrical KCl in the absence (left) and presence (right) of 100 mM Na⁺.

Figure 4. Comparison of Na⁺ and K⁺ binding in NaK2K.

(a) F_o−F_c ion omit maps at the filter region of the NaK2K crystals soaked in 150 mM K⁺ (left), 1 mM K⁺/149 mM Na⁺ (centre) and 0 mM K⁺/150 mM Na⁺ (right) contoured at 6σ. K⁺ and Na⁺ ions are drawn as green and yellow spheres, respectively. Inset depicts the coordination of the Na⁺ bound at site 4 by the Thr63 hydroxyls and a water molecule in the cavity. (b) 1D F_o−F_c electron-density profiles along the central filter axis of NaK2K in 150 mM K⁺ or 150 mM Na⁺ (0 K).

Figure 5. Dependence of K⁺ binding on competing Na⁺ in NaK2K.

Anomalous difference Fourier maps, contoured at 0.03 e Å⁻³, of K⁺ bound within the NaK2K filter in the presence (left) and absence (right) of sodium.

Figure 6. Crystallographic titration of Na⁺–K⁺ competition in NaK2CNG.

(a) Anomalous difference Fourier maps (contoured at 0.03 e Å⁻³), (b) 1D anomalous difference density profiles and (c) estimated occupancies of K⁺ within the NaK2CNG filter from crystals soaked in 100 mM Na⁺–K⁺ mixture with various ratio. Dotted line indicates the baseline used for peak integration. (d) Sample traces and histograms of single-channel currents at −100 mV through NaK2CNG at various concentrations of K⁺. The pipette and bath solutions contain 150 mM Na⁺ and 150 mM Na⁺–K⁺ mixture, respectively. (e) Relative magnitude of the inward current with increasing potassium in bath solution. Data were normalized to the Na⁺ current measured in the absence of K+ (0 mM K⁺). Data points are mean±s.e.m. from three separate patches.

Figure 7. K⁺ and Na⁺ binding in the filter of NaK2CNG.

F_o−F_c ion omit maps of the K⁺ (left) and Na⁺ (right) complexes of NaK2CNG contoured at 6σ. (Inset) Na⁺ binding in plane with the Thr63 side-chain hydroxyls and a water molecule participate in the coordination from the bottom. K⁺ and Na⁺ ions are shown as green and yellow spheres, respectively.

Figure 8. Electrophysiology of ion selectivity in the NaK2CNG_D channel.

Sample single-channel traces (left) and I–V curve (right) of NaK2CNG_D recorded using giant liposome patch clamping with 135 mM Na⁺/15 mM K⁺ in the pipette and 15 mM Na⁺/135 mM K⁺ in the bath.

Figure 9. Mechanism of K⁺ blocking and permeation.

(a) Mechanism of K⁺ blocking of Na⁺ currents in NaK2CNG and NaK2K at low potassium concentrations. (b) K⁺ permeation through NaK2K at high concentration.