Functional evidence for a supramolecular structure for the Streptomyces lividans potassium channel KcsA q E. Zakharian, R.N. Reusch * Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA Received 27 July 2004 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 625 °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 Mg 2+ 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 sup- port a supramolecular structure for KcsA in which influx of ions is prevented by the selectivity pore, whereas efflux of K + is gov- erned by a conductive core of PHB/polyP in partnership with the C-terminal polypeptide strands. Ó 2004 Elsevier Inc. All rights reserved. Keywords: KcsA; Polyhydroxybutyrate; Polyphosphate; Supramolecular; Potassium channel; Selectivity Streptomyces lividans KcsA [1], a tetramer of 160 amino acid polypeptides, is a prokaryotic potassium channel that shares sequence similarity in the pore-form- ing region with eukaryotic potassium channels. The structure of the membrane domain (residues 20–119) of the channel has been well characterized by X-ray crystallography [2]. Less is known about the N-terminal (residues 1–19) and C-terminal strands (residues 120– 160), though their general arrangement has been delin- eated by Cortes et al. [3] using site-directed spin-labeling methods and electron paramagnetic resonance spectros- copy. Of particular interest here was the detection by analytical methods of two ubiquitous polymers, poly- (R)-3-hydroxybutyrate (PHB) [4,5] and inorganic poly- phosphate (polyP) [6–9], in purified KcsA [10], which suggests that KcsA is a supramolecular channel. Functional studies of KcsA have been controversial. The original patch-clamp studies of KcsA in giant pro- toplast–liposomes by Schrempf et al. [1] may afford the most authentic view of the channelÕs in vivo perfor- mance. With asymmetric K + solutions at pH 7.2, the channel displayed a strong preference for K + , slope con- ductances of 20, 40, and 90 pS, and a requirement for intracellular Mg 2+ . Studies by Heginbotham et al. [11] of KcsA in liposomes were also carried out at pH 7.2; how- ever, further investigations indicated that the channel was activated by protons [12,13], thus subsequent planar bilayer studies were performed with solutions of low pH (<5) at the intracellular side [14,15]. The resultant re- ports by different investigators of conductances, open times, and selectivities under these nonphysiological conditions vary significantly. Since it is well-recognized 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.08.027 q Abbreviations: PHB, poly-(R)-3-hydroxybutyrate; polyP, inor- ganic polyphosphate; POPC, 1-palmitoyl, 2-oleoyl, phosphatidylcho- line; POPE, 1-palmitoyl, 2-oleoyl, phosphatidylethanolamine; POPG, 1-palmitoyl, 2-oleoyl, phosphatidylglycerol; TEA, tetraethylammonium. * Corresponding author. Fax: +1 517 353 8957. E-mail address: rnreusch@msu.edu (R.N. Reusch). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 322 (2004) 1059–1065 BBRC