Insight into the selectivity and gating functions of Streptomyces lividans KcsA.

Streptomyces lividans KcsA is a 160-aa polypeptide that oligomerizes to form a tetrameric potassium channel. The three-dimensional structure of the polypeptides has been established, but the selectivity and gating functions of the channel remain unclear. It has been shown that the polypeptides copurify with two homopolymers, poly[(R)-3-hydroxybutyrate] (PHB) and inorganic polyphosphate (polyP), which have intrinsic capacities for cation selection and transport. PHB/polyP complexes are highly selective for divalent cations when pH is greater than the pK(2) of polyP ( approximately 6.8), but this preference is lost when pH is < or =pK(2). It is postulated that KcsA polypeptides attenuate the divalent negative charge of the polyP end unit at physiological pH by strategic positioning of two C-terminal arginines. Here we mutate one or both of the C-terminal arginines and observe the effects on channel selectivity in planar lipid bilayers. We find that channels formed by KcsA polypeptides that retain a single C-terminal arginine remain highly selective for K(+) over Mg(2+), independent of medium pH; however, channels formed by KcsA polypeptides in which both C-terminal arginines have been replaced with neutral residues are selective for Mg(2+) when pH is >7 and for K(+) when pH is <7. Channel gating may be triggered by changes in the balance between the K(+) polyP(-) binding energy, the membrane potential, and the gradient force. The results reveal the importance of the C-terminal arginines to K(+) selectivity and argue for a supramolecular structure for KcsA in which the host polypeptides modify the cation preference of a guest PHB/polyP complex.