Streptomyces lividans Potassium Channel Contains Poly-(R)-3-hydroxybutyrate and Inorganic Polyphosphate

Abstract

The Streptomyces lividans KcsA potassium channel, a homotetramer of 17.6 kDa subunits, was found to contain two nonproteinaceous polymers, namely, poly-(R)-3-hydroxybutyrate (PHB) and inorganic polyphosphate (polyP). PHB and polyP are ubiquitous cellular constituents with a demonstrated capacity for cation selection and transport. PHB was detected in both tetramer and monomer species of KcsA by reaction to anti-PHB IgG on Western blots, and estimated as 28 monomer units of PHB per KcsA tetramer by a chemical assay in which PHB is converted to its unique degradation product, crotonic acid. PolyP was detected in KcsA tetramers, but not in monomers, by metachromatic reaction to o-toluidine blue stain on SDS-PAGE gels. A band of free polyP was also visible, suggesting that polyP is released when tetramers dissociate. The exopolyphosphatase of Saccharomyces cerevisiae degraded the free polyP, but tetramer-associated polyP was not affected, indicating it was inaccessible to the enzyme. PolyP in KcsA was estimated as 15 monomer units per tetramer by an enzymatic assay in which polyphosphate kinase is used to transfer phosphates from polyP to [(14)C]ADP, yielding [(14)C]ATP. The experimentally determined isoelectric point of KcsA tetramer was 6.5-7.5, substantially more acidic than the theoretical pI of 10.3, and consistent with the inclusion of a polyanion. The results suggest that PHB is covalently bound to KcsA subunits while polyP is held within tetramers by ionic forces. It is posited that KcsA protein creates an environment in which PHB/polyP is selective for K(+). The basic amino acids attenuate the negative charge density of polyP, thereby transforming the cation binding preference from multivalent to monovalent, and discrimination between K(+) and Na(+) is accomplished by adjusting the ligand geometry in cation binding cavities formed by PHB and polyP.

In vivo monitoring of the potassium channel KcsA in Streptomyces lividans hyphae using immuno-electron microscopy and energy-filtering transmission electron microscopy

“…PHB is a linear polymer of 3-hydroxybutyrate and is an amphiphilic polyester that forms ion-conducting complexes with salts. PolyP is a linear polymer of phosphoryl units and has the capacity for ion exchange and the ability to discriminate among cations by charge (Reusch, 1999). It has recently been suggested (Zakharian & Reusch, 2004) that KcsA may recruit PHB and polyP to form a conductive core that selects and transports K + to the inner face of the selective filter.…”

Insight into the selectivity and gating functions of Streptomyces lividans KcsA

“…pQE60 plasmids were transformed into E. coli BL21 (Novagen), overexpressed by addition of isopropyl ␤-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM (Calbiochem, San Diego, CA) and purified by Ni-affinity chromatography as described (23). The proteins when unheated formed single bands at Ϸ65 kDa on SDS/PAGE gels, corresponding to the tetrameric form, and they were converted to the monomeric form, Ϸ19 kDa, when heated in 2% SDS.…”

“…The presence of PHB in KcsA wild-type and mutants was demonstrated by Western blot analysis using anti-PHB IgG as described (23). The identity of PHB was confirmed by chemical assay as described by Huang and Reusch (27).…”

“…PHB, a flexible, amphiphilic polyester, has solvent properties; polyP, a chain of negatively charged phosphoryl residues, selects, binds, and conducts cations. Studies indicate that PHB is covalently attached, presumably at its CoA ester end, to each KcsA monomer whereas polyP is held within the KcsA tetramer by noncovalent interactions (23). The presence of the polyanion, polyP, within KcsA tetramers was first signaled by the large difference between the theoretical pI of the polypeptides (10.3, EXPASY) and the experimental pI of the tetramer (6.5-7.5).…”

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“…Interestingly, the occurrence of PHA is not limited to the intracellular collection in granules. PHB with lower molecular weight (cPHB; M w < 14,000 Da) was also found in B. subtilis, A. vinelandii and Streptomyces lividans (104)(105)(106) in association with polyphosphate and calcium ions. In addition, non-storage PHAs that are of low molecular weight, PHBs have also been detected in the cytoplasmic membrane and cytoplasm of E. coli (31).…”