The Role Of Oligo-(R)-3-Hydroxybutyrates in the Streptomyces lividans KcsA Channel

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The Streptomyces lividans potassium channel KcsA is a tetramer of four polypeptides, each covalently modified by oligo-(R)-3-hydroxybutyrate (cOHB), which envelop a core molecule of inorganic polyphosphate (polyP). It has been proposed that the polyanion, polyP, attracts, binds and conducts K+ in response to an electrochemical stimulus whilst the polypeptides govern access to polyP and regulate its selectivity. The function of cOHB, however, has been undefined.Digestion of KcsA with CNBr yields a 6.7 kDa fragment that contains most of the ion pathway (residues 97-154). This fragment was shown to contain cOHB by Western blot and chemical assays. The conjugation sites for cOHB were determined to be on S102 and S129. The effects of the single mutations S102, S129 and the double mutation S102:S129 on channel activity were examined. Wild-type KcsA, incorporated into planar lipid bilayers of POPC:POPE:POPG (3:3:1) between aqueous solution of 200 mM KCl, 5 mM MgCl2, 20 mM Hepes, pH 7.4 cis and 20 mM KCl, 5 mM MgCl2, 20 mM Hepes, pH 7.4 trans forms well-structured channels of 147 pS conductance. Under the same conditions, S102G exhibits irregular conductance with a major conductance state of 75 pS and a minor conductance state of 103 pS, S129G exhibits frequent but unsuccessful attempts to insert into the bilayer, and S102:S129 exhibits no channel activity whatsoever. The results suggest that cOHB-modification of KcsA polypeptides is essential for normal channel function.