Voltage gating and anions, especially phosphate: A model system

The voltage sensor of voltage gated sodium and potassium channels consists of four sets of transmembrane segments, of which one, called S4, contains at least four arginines; these are presumed to each carry positive charges. The channel opening is usually attributed to the outward (i.e., toward the extracellular side of the membrane) motion of S4. The evidence for this motion is based on certain experiments that appear to show differential access to parts of S4 from the intracellular and extracellular sides of the membrane in the open and closed states. A newly available structure [S.B. Long, E.B. Campbell and R. MacKinnon, Crystal structure of a mammalian voltage-dependent Shaker family K+ channel. Science 309 (2005) 897-903; S.B. Long, E.B. Campbell, R. MacKinnon, Voltage sensor of Kv1.2: structural basis of electromechanical coupling. Science 309 (2005) 903-908][1,2] has now been used to argue for a large scale motion, although, as a static structure, it is not conclusive. In this paper, we consider the effect of anions in the surrounding medium. Phosphate is present in the intracellular as well as the extracellular fluid, apparently at hundreds of micromolar concentration, or more. There is evidence in the literature suggesting that phosphate-arginine complexes are rather strong. In a recent calculation one of us [M.E. Green, A possible role for phosphate in complexing the arginines of S4 in voltage gated channels J. Theor. Biol. 233 (2005) 337-341][3] has shown that a model peptide with a 2:1 arg:phosphate complex should have a favorable geometry. Here, we present NMR evidence of the existence of phosphate complexes of a model peptide with two arginines separated by two hydrophobic residues, the same spacing as in S4 segments. The complexes (there are different complexes for HPO42− and for H2PO4− [3]) form with concentrations of peptide in the range of hundreds of micromolar, making it significant in the biological context. NMR spectra provide changes in chemical shift as functions of both phosphate concentration and pH. The resulting curves show titration of the phosphate, with its standard pK. Possible implications for other anion-S4 interactions, including ion pairs rather than complexes, as with Cl−, are also discussed.