Intellectual merit. The premise of this research is that complexes of two ubiquitous homopolymers, poly-(R)-3-hydroxybutyrate and polyphosphate (cPHB/polyP), form the conductive cores of protein ion channels. It has previously been shown that cPHB/polyP complexes, isolated from bacterial plasma membranes or prepared from the synthetic polymers, create cation-selective ion channels in planar lipid bilayers. This project will examine the putative modification of intrinsic cPHB/polyP channel selectivity by polypeptides of the Streptomyces lividans potassium channel, KcsA. Phosphates have a divalent negative charge above pH 7.0 and a monovalent negative charge below pH 7.0; accordingly, cPHB/polyP complexes are highly selective for divalent cations above pH 7.0, display no preference at pH 7.0 and become selective for monovalent cations at pH less than 7.0. KcsA demonstrates similar cation selectivity in planar bilayers at room temperatures; the channels prefer divalent cations at pH 7.2, but are highly selective for K+ at pH 6.8. However, at temperatures above 26 C, KcsA is highly selective for K+, regardless of pH. The hypothesis is that KcsA undergoes a thermal transition that results in the placement of basic residues at the ends of the intracellular strands of KcsA polypeptides close to the end units of polyP; the effect of the positive charges on these residues on selectivity of polyP would be comparable to that of a lowered pH. In this project, mutants of these basic residues will be prepared by PCR site-directed mutagenesis; the proteins will be overexpressed in E. coli and purified by affinity chromatography. The cation selectivity of these mutant KcsA channels in planar lipid bilayers will then be compared to that of wild-type KcsA to test the hypothesis.
Broader impact. This proposal offers a novel mechanism for ion transport. Moreover, the cPHB/polyP channels are putative vehicles for DNA transport and they may serve as nanosensors for cations, pH or temperature. Postdoctoral, graduate and undergraduate students from Michigan State University and other institutions are participants in this research. In addition, a web site will be developed to provide information about cPHB and polyP and their complexes at both elementary and advanced levels in order to foster a greater understanding of the important structural and metabolic roles of these fundamental cell constituents among students, scientists and the general public.
