Functional Reconstitution of a Prokaryotic K 1 Channel

SliK, a K 1 channel encoded by the Streptomyces KcsA gene, was expressed, purified, and reconstituted in liposomes. A concentrative 86 Rb 1 flux assay was used to assess the ion transport properties of SliK. SliK-mediated ionic flux shows strong selectivity for K 1 over Na 1 and is inhibited by micromolar concentrations of Ba 2 1 , mirroring the basic permeation characteristic of eukaryotic K 1 channels studied by electrophysiological methods. 86 Rb 1 uptake kinetics and equilibrium measurements also demonstrate that the purified protein is fully active. key words: liposome • conduction • selectivity • flux i n t r o d u c t i o n Mechanistic insights about ion channels have traditionally emanated from physiological antecedents. Recent discoveries of molecular determinants of voltage-dependent gating, ligand binding, pore selectivity, and second messenger modulation connect directly to the essential neurobiological functions of ion channels. The combined use of high-resolution electrical recording techniques and recombinant DNA manipulation of ion channel sequences has yielded a hailstorm of information about the molecular foundations of these functions, but detailed understanding of these proteins will also require high-resolution structures. The K 1 channels of electrically excitable cells, in particular, have been productive targets for functional analysis of mutated proteins (Miller, 1991; Jan and Jan, 1994), but they have proven exceedingly resistant to the biochemical manipulations required for structure determination (Klaiber et al., 1990; Spencer et al., 1997). Prokaryotes provide a plausible route to K 1 channel structure. Examination of eubacterial and archeal genome sequences is uncovering an unexpected class of genes with remarkable resemblance to K 1 channels (Milkman, 1994; Schrempf et al., 1995; MacKinnon and Doyle, 1997). In particular, these genes all display transmembrane a -helical sequences flanking an z 20residue stretch similar to the highly conserved, poreforming “P-region” of eukaryotic K 1 channels. One of these, the KcsA gene from Streptomyces lividans , encodes a 160-residue protein, “SliK.” The predicted transmembrane topology of this protein is reminiscent of the inward rectifier subfamily of K 1 channels—a P-region flanked by two putative membrane-spanning a -helices (Schrempf et al., 1995); moreover, a glutamate residue unique to inward rectifier P-regions is also found in SliK (E71). However, the P-region of SliK actually shows higher overall similarity to the pore-forming sequences of voltage-gated K 1 channels than to inward rectifiers. In stark distinction to the biologically motivated studies on eukaryotic K 1 channels, the physiological roles of these prokaryotic genes are entirely unknown. When overexpressed in Escherichia coli (Schrempf et al., 1995; Cortes and Perozo, 1997; Heginbotham et al., 1997), SliK can be purified readily in quantities (1–5 mg/ liter culture) that make spectroscopic and biochemical studies possible (leCoutre et al., 1998; Tatulian et al., 1998); indeed, the structure of this channel has recently been determined at 3.5-Å resolution (Doyle et al., 1998). We were therefore motivated to verify that the SliK preparations used in such studies are functional. It is known that SliK forms a stable tetramer in detergent micelles (Cortes and Perozo, 1997; Heginbotham et al., 1997), as expected for a K 1 channel (MacKinnon, 1991) and that it shows single-channel activity when reconstituted into planar lipid bilayer membranes (Schrempf et al., 1995; Cuello et al., 1998). However, single-channel recording, because of its extreme sensitivity and intrinsically anecdotal character, cannot by itself argue for the functional competence of a biochemical preparation. Here we describe an assay that permits a quantitative evaluation of SliK ion-transport activity. Reconstitution of SliK into liposomes renders these membrane vesicles selectively permeable to K 1 and its close analogues, as expected of a K 1 channel with properties similar to those of its wellstudied eukaryotic homologues. The results demonstrate the functional competence of SliK protein purified from an E. coli heterologous expression system. Address correspondence to Christopher Miller, Department of Biochemistry, Howard Hughes Medical Institute, Brandeis University, 415 South Street, HHMI, Waltham, Massachusetts 02254. Fax: 781736-2365; E-mail: [email protected] on A uust 9, 2017 jgp.rress.org D ow nladed fom