Synthetic multifunctional pores: deletion and inversion of anion/cation selectivity using pM and pH

Abstract

We report the characterization of multifunctional rigid-rod β-barrel ion channels with either internal aspartates or arginine-histidine dyads by planar bilayer conductance experiments. Barrels with internal aspartates form cation selective, large, unstable and ohmic barrel-stave (rather than toroidal) pores; addition of magnesium cations nearly deletes cation selectivity and increases single-channel stability. Barrels with internal arginine-histidine dyads form cation selective (P K ϩ/P Cl Ϫ = 2.1), small and ohmic ion channels with superb stability (single-channel lifetime > 20 seconds). Addition of "protons" results in inversion of anion/cation selectivity (P Cl Ϫ/P K ϩ = 3.8); addition of an anionic guest (HPTS) results in the blockage of anion selective but not cation selective channels. These results suggest that specific, internal counterion immobilization, here magnesium (but not sodium or potassium) cations by internal aspartates and inorganic phosphates by internal arginines (but not histidines), provides access to synthetic multifunctional pores with attractive properties.

The determination of the ion selectivity of synthetic ion channels and pores in vesicles

“…7c, dotted vs solid). 13 BLM experiments confirmed that Mg 2þ -binding reduced the permeability ratio from P K þ =P Cl À ¼ 5:6 to P K þ =P Cl À ¼ 1:4 15 and, together with circular dichroism spectroscopy measurements, that this functional change is not caused by a change in pore structure. The CF assay is also sensitive to pH and can only be used above pH $ 6.5.…”

“…1,[11][12][13][14] Conductance measurements in BLMs confirmed that the cation selectivity of pore 6 was as high as expected for an inner surface covered with carboxylates (P K þ =P Cl À ¼ 5:6). 15 This high permeability ratio may also serve as instructive example to correct the frequent misconception that the large inner diameter of pores is incompatible with anion/ cation selectivity: It is the chemical nature rather than the size of the pore that matters, absence of anion/cation selectivity does not identify pores (and vice versa). The inactivity of pore 6 observed in the CF assay, therefore, is a direct consequence of the incompatibility of the ion selectivity of pore and assay.…”

“…10, 15 The puzzling ability of the polycationic pore 5 to attract Figure 2. Determination of ion selectivity in planar bilayers (BLMs).…”

“…Note that this graph is highly schematic: IV relations, for example, follow the GHK current equation and are therefore often curved when a salt concentration gradient is applied cations rather than anions, that is, P K þ =P Cl À ¼ 2:0 at pH 6, was found to originate from internal charge inversion by counteranion scavenging. 15 This surprising result of a routine determination of the ion selectivity of a synthetic ion channel marked the beginning of a new and fruitful research program on the general contributions of counteranions to what is commonly referred to 'arginine magic' in biomembranes. 38,39 The selectivity sequence describes the preference of an anion channel between different anions or that of a cation channel between different cations.…”

“…For example, the synthetic pore 5 was found to undergo a pH-gated inversion of ion selectivity from P Cl À =P K þ ¼ 0:5 at pH 6.0 to P Cl À =P K þ ¼ 3:8 at pH 4.0. 15 Several lines of evidence demonstrated that the puzzling cation selectivity of the cationic pore 5 near physiological pH originates from internal charge inversion by scavenged and immobilized phosphate counteranions. As mentioned previously, the study of the contributions of counteranions to the biological functions of oligoarginines in biomembranes in general corroborated the importance of this key finding.…”