What Ion Flow along Ion Channels Can Tell us about Their Functional Activity

Abstract

The functional activity of channel-forming peptides and proteins is most directly verified by monitoring the flow of physiologically relevant inorganic ions, such as Na⁺, K⁺ and Cl^-, along the ion channels. Electrical current measurements across bilayer lipid membranes (BLMs) interposed between two aqueous solutions have been widely employed to this end and are still extensively used. However, a major drawback of BLMs is their fragility, high sensitivity toward vibrations and mechanical shocks, and low resistance to electric fields. To overcome this problem, metal-supported tethered BLMs (tBLMs) have been devised, where the BLM is anchored to the metal via a hydrophilic spacer that replaces and mimics the water phase on the metal side. However, only mercury-supported tBLMs can measure and regulate the flow of the above inorganic ions, thanks to mercury liquid state and high hydrogen overpotential. This review summarizes the main results achieved by BLMs incorporating voltage-gated channel-forming peptides, interpreting them on the basis of a kinetic mechanism of nucleation and growth. Hg-supported tBLMs are then described, and their potential for the investigation of voltage-gated and ohmic channels is illustrated by the use of different electrochemical techniques.

Keywords: alamethicin; bilayer lipid membrane; gramicidin; melittin; ohmic ion channel; tethered bilayer lipid membrane; voltage-gated ion channel.

Figure 1

The solid curves are three successive current-time curves on the same bilayer lipid membranes (BLM) following transmembrane potential steps from 0 to −60 mV (a), −55 mV (b) and −50 mV (c), in aqueous 0.1 M KCl containing 0.625 μM monazomycin [29]. The corresponding dashed curves were calculated by the nucleation-and-growth model outlined in the text using the parameters θ₀ = 0.1, n = 2, and k_h,Nv_h,R² = 0.016 s⁻³ for all three curves; p and k_Nk_R² were given the values: (a) 1 and 15 s⁻³; (b) 0.438 and 130 s⁻³; (c) 0.163 and 1250 s⁻³. The three calculated currents were matched to the experimental ones by multiplying them by the same factor 80. (Reprinted with permission from [30]. Copyright 2007, American Chemical Society).

Figure 2

Experimental I-V curve for 0.4 μg/mL melittin in dioleoylphosphatidylcholine (DOPC), taken directly from Figure 7 of Pawlak et al. [20] (black curve), and curve calculated for a = 1 × 10⁻⁴, ∆m = 70 D, θ₀ = 0.1, k_Nk_R² = 1 × 10⁶ s⁻³ and n = 1 (red curve). The height of the calculated curve was normalized to that of the experimental one. (Reprinted with permission from [38]. Copyright 2015, Elsevier).

Figure 3

Plots of ln G vs. V for different values of a and for ∆m = 70 D, θ₀ = 0.1, k_Nk_R² = 1 × 10⁶ s⁻³ and n = 1. The curves shift toward progressively lower ln G values with decreasing a. (Reprinted with permission from [38]. Copyright 2015, Elsevier).

Figure 4

Experimental I-V curve for 0.2 μg/mL alamethicin in bacterial phosphatidylethanolamine, taken from curve 2 in Figure 5 of Vodyanoy et al. [46] (blue curve) and curve calculated for a = 1 × 10⁻², ∆m = 70 D, θ₀ = 0.1, k_Nk_R² = 1 × 10⁵ s⁻³ and n = 1 (red curve). The height of the calculated curve was normalized to that of the experimental one. (Reprinted with permission from [38]. Copyright 2015, Elsevier).

Figure 5

Space filling model and structure of the DPTL thiolipid lipid and of DPhPC, in a tail-to-tail configuration; l, s, and m denote the lipoic acid residue, the tetraethyleneoxy spacer and the hydrocarbon tail region, forming the monomeric unit of a DPTL/DPhPC tethered bilayer lipid membrane. Carbon atoms are in gray, hydrogen atoms in white, oxygen atoms in red, phosphorus atoms in orange and sulfur atoms in yellow.

Figure 6

Charge vs. time curves following potential steps from a fixed initial potential of −0.200 V to progressively more negative final potentials, E_f, varying from −0.525 to −0.925 V by −25 mV increments, at a DPTL/DPhPC tBLM incorporating gramicidin from its 0.1 μM solution in aqueous 0.1 M KCl. (Reprinted with permission from [16]. Copyright 2007, American Chemical Society).

Figure 7

The solid blue curves are three successive charge vs. time curves at a DPTL/DPhPC tBLM following potential steps from the same initial value −0.20 V to the final values −1.05 V (a), −1.00 V (b) and −0.95 V (c) in aqueous 0.1 M KCl containing 0.14 μM melittin. The height of the plateau of the curves is normalized to unity. The corresponding dashed red curves were calculated using the parameters θ₀ = 0.2, n = 2, and k_Nk_R² = 0.05 s⁻³ for all three curves; p was given the value 1 for curve (a), 0.8 for curve (b) and 0.6 for curve (c). (Reprinted with permission from [30]. Copyright 2007, American Chemical Society).

Figure 8

The solid curves are charge vs. time curves at a DPTL/DPhPC tBLM following potential steps from −0.20 to −1.05 V in aqueous 0.1 M KCl containing 0.14 μM melittin. Curve (a) was obtained after keeping the potential at −0.20 V for 15 min; curve (b) was obtained after keeping the potential at −1.05 V for 150 s, stepping it back to −0.20 V and carrying out the recorded potential jump to −1.05 V immediately after. The dashed curve (a) was calculated using the parameters θ₀ = 0.2, n = 2, k_h,Nv_h,R² = 0.08 s⁻³, k_Nk_R² = 0.05 s⁻³ and p = 1; the dashed curve (b) was calculated using the parameters θ₀ = 0.2, n = 2, k_Nk_R² = 40 s⁻³ and p = 1. (Reprinted with permission from [30]. Copyright 2007, American Chemical Society).

Figure 9

Charge transients following potential steps from E_i = −0.30 V to two different final potentials E_f at a DPTL/DOPS tBLM in a pH 3 solution of 0.1 M KCl and 0.8 μM SR-E. (a) Pristine potential step to E_f = −0.90; (b) potential step to E_f = −0.75 V recorded immediately after a potential step from −0.30 to −1.00 V and a rest time of 30 s at E_i. The curves a′ and b′ are the corresponding current transients. Curve c is the charge transient from −0.30 V to −1.00 V in the absence of SR-E under otherwise identical conditions. (Reprinted and minimally adapted with permission from [57]. Copyright 2015, Elsevier).

Figure 10

Charge transients at a DPTL/DOPC tBLM in a pH 6.8 buffer solution of 0.1 M KCl and 1 μg/mL SP25A, obtained by jumping from E_i = −0.30 V to final potentials varying from −0.50 to −1.00 V by −100 mV increments. (Reprinted with permission from [18]. Copyright 2015, Elsevier).

Figure 11

Charge transients at a DPTL/DOPC tBLM in a pH 6.8 buffer solution of 0.1 M KCl and 0.4 μg/mL SP25A, obtained by jumping from E_i = −0.30 V to final potentials varying from −0.50 to −1.00 V by −100 mV increments. (Reprinted with permission from [18]. Copyright 2015, Elsevier).

Figure 12

Plot of ωZ′ (black circles) against –ωZ″ for a mercury-supported DPTL/DPhPC bilayer immersed in aqueous 0.1 M KCl at −0.41 V vs. SCE. The black curve is the best fit of the impedance spectrum by the equivalent circuit shown in the inset, with R_l = 17 MΩ·cm², C_l = 1.6 μF·cm⁻², R_s = 0.18 MΩ·cm², C_s = 5.5 μF·cm⁻², R_m = 4.2 MΩ·cm², C_m = 1.1 μF·cm⁻², R_Ω = 3.6 Ω·cm², C_Ω = 27 nF·cm⁻². The colored curves are contributions to ωZ′ from the single slabs.

Figure 13

Plot of ωZ′ (solid circles) against −ωZ″ for a mercury-supported DPTL/DPhPC bilayer incorporating valinomicin from its 0.15 μM solution in aqueous 0.1 M KCl at −0.41 V vs. saturated calomel electrode (SCE). The solid black curve is the best fit of the impedance spectrum by the equivalent circuit shown in the inset of Figure 12, with R_l = 2.8 MΩ·cm², C_l = 4.2 μF·cm⁻², R_s = 48 kΩ·cm², C_s = 3.3 μF·cm⁻², R_m = 3.4 kΩ·cm², C_m = 0.9 μF·cm⁻², R_Ω = 3.7 Ω·cm², C_Ω = 43 nF·cm⁻². The colored curves are contributions to ωZ′ from the single slabs. (Adapted with permission from [61]. Copyright 2005 American Chemical Society).

Figure 14

Plot of ωZ′ (solid circles) against −ωZ″ at a Hg-supported DPTL/DPhPC tBLM incorporating gramicidin from its 0.1 μM solution in aqueous 0.1 KCl at −0.675 V vs. SCE. The solid black curve is the best fit of this plot by an equivalent circuit consisting of a series of four RC meshes. The purple, red, blue and green curves are calculated contributions to ωZ′ from the spacer moiety, the “inner layer” moiety, the lipid bilayer moiety and the aqueous solution adjacent to the BLM. The inset shows plots of conductance g (blue curve) and capacitance C (red curve) of the inner layer against E at the same tBLM. (Reproduced from [53] with permission from the Royal Society of Chemistry).

Figure 15

Cyclic voltammograms (CVs) at a DPTL/DOPC tBLM in a pH 3 solution of 0.1 M KCl, 1 × 10⁻³ M HCl and 0.8 μg/mL melittin, recorded between −0.20 and −1.20 V at a scan rate of 50 mV/s. Pristine CV (purple curve) and three further CVs (blue, green and red curves) after a few potential cycles. The grey curve is the CV in the absence of melittin. (Reprinted from [19]. Copyright 2016, with permission from Elsevier).

Figure 16

The solid blue curves are experimental cyclic voltammograms at a DPTL/DOPC tBLM in aqueous solutions of 0.1 M KCl at pH 6.8, 5.4 and 3, in the presence of either 0.1 μM gramicidin (left column) or 1 μg/mL SP25A (right column). Scan rate = 50 mV/s. The corresponding dashed curves were calculated as outlined in the text. (Reprinted from [66], Copyright 2015 and from [19], Copyright 2016, with permission from Elsevier).