Proof for a nonproteinaceous calcium-selective channel in Escherichia coli by total synthesis from (R)-3-hydroxybutanoic acid and inorganic polyphosphate [bacterial calcium channelypolymer electrolyte complexyplanar bilayerypoly(3-hydroxybutyrate)]

Traditionally, the structure and properties of natural products have been determined by total synthesis and comparison with authentic samples. We have now applied this procedure to the first nonproteinaceous ion channel, isolated from bacterial plasma membranes, and consisting of a complex of poly(3-hydroxybutyrate) and calcium polyphosphate. To this end, we have now synthesized the 128-mer of hydroxybutanoic acid and prepared a complex with inorganic calcium polyphosphate (average 65-mer), which was incorporated into a planar lipid bilayer of synthetic phospholipids. We herewith present data that demonstrate unambiguously that the completely synthetic complex forms channels that are indistinguishable in their voltage-dependent conductance, in their selectivity for divalent cations, and in their blocking behavior (by La31) from channels isolated from Escherichia coli. The implications of our finding for prebiotic chemistry, biochemistry, and biology are discussed. Synthetic and naturally occurring ion channels are amphiphilic structures with an outer coat of nonpolar residues and a lining of polar and charged residues (1–7). In this report, we demonstrate the formation of synthetic nonproteinaceous ion channels from two structurally distinct polymers that share the above attributes in a cooperative fashion. The polymers in question, poly(3-hydroxybutyrate) (PHB) and inorganic polyphosphate (polyP), are ubiquitous constituents of biological cells (8–12). PHB is an amphiphilic homopolymer of (R)-3-hydroxybutanoic acid, which in bacteria is synthesized from acetyl-CoA in three steps: dimerization to form acetoacetyl-CoA, reduction by NADPH to form 3-hydroxybutyrylCoA, and polymerization to form PHB (13, 14). Under growth-limiting conditions, some bacteria produce highmolecular-mass PHB (60,000 to .1,000,000 Da) in amounts of up to 90% of the cell dry weight. PolyP, a polyanion composed of phosphate residues linked by anhydride bonds, is formed by repetitive phosphoryl transfer from ATP or other high-energy phosphates (15–17). Both PHB and polyP have molecular characteristics that are consistent with a role in ion conduction. PHB has salt-solvating properties that derive from the recurrence of electrondonating ester carbonyl oxygens at regular and frequent intervals along its f lexible backbone (18, 19). The solvating abilities of PHB are illustrated by studies that demonstrate its capacity to transport cations across methylene chloride layers in U-tubes (20), form ion-conducting complexes with lithium perchlorate (21), or large-conductance nonselective ion channels in planar lipid bilayers (22). PolyP has a flexible backbone and high density of monovalent negative charges that create a large capacity for ion exchange and a stronger affinity for multivalent over monovalent cations (23). Plasma membrane vesicles of Escherichia coli contain complexes of PHB and polyP that function as calcium-selective channels in planar lipid bilayers (24). In vivo, such channels may control the influx of calcium to maintain homeostasis or effect transient increases to trigger certain physiological responses. The importance of calcium regulation in prokaryotes is becoming evident as calcium is increasingly implicated in bacterial functions, including chemotaxis, cell division, differentiation, and pathogenesis (25). The channel complexes can be extracted from the membranes into chloroform and purified by HPLC nonaqueous size-exclusion chromatography (24). Analysis of purified channel extracts indicated that the channels were composed of PHB (130–140 residues) and polyP (55–65 residues). No protein was detected. When the complexes were reconstituted from purified E. coli PHB and synthetic calcium polyphosphate, the same channel activity was observed; however, inasmuch as hydrophobic proteins bind very tightly to PHB, the presence of trace protein or peptide contaminants in PHB of cellular origin could not be ruled out. Consequently, doubt remained as to whether the observed channel activity was entirely attributable to complexes of these two simple polymers. In this study, we prove by total synthesis of the channel complexes that protein is not essential to the observed ion channel activity.