Functional reconstitution of a prokaryotic K+ channel

Abstract

SliK, a K+ channel encoded by the Streptomyces KcsA gene, was expressed, purified, and reconstituted in liposomes. A concentrative 86Rb+ flux assay was used to assess the ion transport properties of SliK. SliK-mediated ionic flux shows strong selectivity for K+ over Na+ and is inhibited by micromolar concentrations of Ba2+, mirroring the basic permeation characteristic of eukaryotic K+ channels studied by electrophysiological methods. 86Rb+ uptake kinetics and equilibrium measurements also demonstrate that the purified protein is fully active.

Figure 5

Lipid dependence of SliK-mediated ⁸⁶Rb⁺ uptake. Liposomes were made from the indicated lipid mixtures, incorporating SliK at a concentration of 2.5 μg/mg lipid. Values of 3.5-min ⁸⁶Rb⁺ uptake were normalized to the equilibrium valinomycin- mediated uptake in the same vesicles. Bars reflect means ± SEM of three measurements. In mixtures of defined lipids, PE made up 75% of the lipid mass. For the PE:PG:CL mixture, the lipid composition was 75% PE, 15% PG, and 10% CL.

Figure 1

Concentrative uptake of ⁸⁶Rb⁺. (A) Time course of ⁸⁶Rb⁺ uptake into liposomes reconstituted with 0.9 μg SliK/mg lipid (circles) or into control liposomes containing no protein (squares). Hollow points refer to samples taken after addition of valinomycin (arrows). Each point represents a single 60-ml sample containing 0.15 mg lipid, removed from the reaction mixture. External ⁸⁶Rb⁺ concentration was ∼0.2 μCi/ml (104 cpm per 60-ml sample). (B) Liposomes were formed in the presence of SliK as in A, but were loaded with 450 mM NaCl instead of KCl. Where indicated, valinomycin was added, and at 3 min the sample was split into two parts: a control that was allowed to proceed without further addition (▪, at 9 min), and a test sample to which gramicidin A (10 μg/ml) was added (□).

Figure 2

Selectivity of SliK- dependent uptake. (A) Variation of internal cation. Liposomes were formed as in materials and methods except that the indicated cation was substituted for K⁺. Samples (60 μl, 0.12 mg lipid) were taken after 5 min of influx. Each bar represents samples reconstituted in triplicate. (B) Variation of external cation. 5-min uptake was determined in flux buffers to which test cations had been added at the indicated concentrations (on a background of 50 μM K⁺). Sephadex G-50 columns were used to terminate the uptake reaction, as in materials and methods. Samples were normalized to duplicate control samples containing 50 μM KCl but no added test ion. Points and error bars represent means and ranges of two separate runs.

Figure 3

Ba²⁺ block of SliK-mediated flux. Liposomes were reconstituted with SliK at 1 μg/mg. (A) Time courses of ⁸⁶Rb⁺ influx were followed in the presence of 50 μM of the indicated divalent cations. Samples were normalized to 10-min control samples with no added divalent cation. (B) Concentration dependence of Ba²⁺ block. The indicated concentration of Ba²⁺ was added externally, and initial rate of ⁸⁶Rb⁺ uptake was measured by taking samples at 20 s. Points and error bars represent means ± SEM of three separate runs.

Figure 4

⁸⁶Rb⁺ uptake at different SliK concentrations. (A) Time course of ⁸⁶Rb⁺ uptake into vesicles containing 0, 0.5, or 2.5 μg SliK/mg lipid. Samples were removed from a common reaction mixture at the indicated times and normalized to valinomycin- induced uptake in the same vesicles. Solid curves are single exponentials with time constants of 58 and 29 s for 0.5 and 2.5 μg/mg SliK, respectively. (B) ⁸⁶Rb⁺ uptake as a function of SliK concentration. Equilibrium ⁸⁶Rb⁺ accumulation was measured after 10 min in liposomes containing SliK at the indicated concentrations. The solid curve is drawn according to Eq. 4, where r = 460 Å, φ = 1, θ = 0.20. Each point reflects mean ± SEM of three independent measurements.