Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA

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Abstract

Here we present functional evidence for involvement of poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP) in ion conduction and selection at the intracellular side of the Streptomyces lividans potassium channel, KcsA. At ⩽25 °C, KcsA forms channels in planar bilayers that display signal characteristics of PHB/polyP channels at the intracellular side; i.e., a preference for divalent Mg2+ cations at pH 7.2, and a preference for monovalent K+ cations at pH 6.8. Between 25 and 26 °C, KcsA undergoes a transition to a new conformation in which the channel exhibits high selectivity for K+, regardless of solution pH. This suggests that basic residues of the C-terminal polypeptides have moved closer to the polyP end unit, reducing its negative charge. The data support a supramolecular structure for KcsA in which influx of ions is prevented by the selectivity pore, whereas efflux of K+ is governed by a conductive core of PHB/polyP in partnership with the C-terminal polypeptide strands.

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Materials and methods

Purification and reconstitution of KcsA protein. KcsA, His-tagged at the C-terminal and cloned into pQE60 (courtesy of C. Miller), was transformed into E. coli BL21 (Novagen), overexpressed by addition of isopropyl-β-d-thiogalactopyranoside (IPTG) to a final concentration of 1 mM (Calbiochem), and purified by Ni-affinity chromatography as previously described [16]. The KcsA tetramer was reconstituted into liposomes by incubation at temperatures ranging from 22–30 °C in a micellar solution

Selectivity for anions

KcsA, incorporated into planar bilayers between asymmetric K+ solutions, 200 mM KCl, 5 mM MgCl2, and 20 mM Tris–Hepes, pH 7.2, on the intracellular side and 20 mM KCl, 5 mM MgCl2, and 20 mM Tris–Hepes, pH 7.2, on the extracellular side, forms channels at 22 °C that have a reversal potential of −27 mV [16]. This is far from the Nernst equilibrium potential of −54 mV, and indicates that other ion(s) in the system are permeable.

Potentially permeant ions in the above solutions are Cl and Mg2+. Since Cl is

Discussion

Our single-channel bilayer studies provide persuasive functional evidence in support of the hypothesis that PHB and polyP participate in the selection of ions at the intracellular side of the selectivity pore of KcsA [10]. The extracellular side of KcsA was highly resistant to influx of ions other than K+ and Rb+ under all conditions examined here; however, the primary function of KcsA is to regulate K+ efflux, and discrimination among cations at the intracellular side is strongly dependent on

Acknowledgment

We gratefully acknowledge NIH Grant GM 054090 for partial support of this project.

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    Abbreviations: PHB, poly-(R)-3-hydroxybutyrate; polyP, inorganic polyphosphate; POPC, 1-palmitoyl, 2-oleoyl, phosphatidylcholine; POPE, 1-palmitoyl, 2-oleoyl, phosphatidylethanolamine; POPG, 1-palmitoyl, 2-oleoyl, phosphatidylglycerol; TEA, tetraethylammonium.

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