doi: 10.1021/acsnano.2c07496. Epub 2022 Oct 12.
DNA-Based Optical Quantification of Ion Transport across Giant Vesicles
Affiliations
- PMID:36222833
- PMCID: PMC9620405
- DOI: 10.1021/acsnano.2c07496
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DNA-Based Optical Quantification of Ion Transport across Giant Vesicles
Marcus Fletcher et al. ACS Nano..
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Abstract
Accurate measurements of ion permeability through cellular membranes remains challenging due to the lack of suitable ion-selective probes. Here we use giant unilamellar vesicles (GUVs) as membrane models for the direct visualization of mass translocation at the single-vesicle level. Ion transport is indicated with a fluorescently adjustable DNA-based sensor that accurately detects sub-millimolar variations in K+ concentration. In combination with microfluidics, we employed our DNA-based K+ sensor for extraction of the permeation coefficient of potassium ions. We measured K+ permeability coefficients at least 1 order of magnitude larger than previously reported values from bulk experiments and show that permeation rates across the lipid bilayer increase in the presence of octanol. In addition, an analysis of the K+ flux in different concentration gradients allows us to estimate the complementary H+ flux that dissipates the charge imbalance across the GUV membrane. Subsequently, we show that our sensor can quantify the K+ transport across prototypical cation-selective ion channels, gramicidin A and OmpF, revealing their relative H+/K+ selectivity. Our results show that gramicidin A is much more selective to protons than OmpF with a H+/K+ permeability ratio of ∼104.
Keywords: G-quadruplex; giant unilamellar vesicles; ion channels; ion sensor; ion transport; microfluidics.
Conflict of interest statement
The authors declare no competing financial interest.
Figures
Design and characterization of G-quadruplex (G4) DNA basedK+ probes.(A) Schematic illustrating the K+ sensing principle ofG4-DNA probes. A human telomeric DNA (HT G4-DNA), modified with afluorophore and quencher at opposing ends (5′ and 3′,respectively), folds in response to K+, thereby bringingthe fluorophore and quencher into closer contact and decreasing thefluorescence intensity of the probe. (B) Variation of FAMQ-G4 fluorescenceintensity with increasing concentration of K+ (blue circles).In the presence of a complementary DNA strand (orange triangles) noreduction in fluorescence is observed for the double-stranded G4-DNA,indicating that folding of the single-stranded G4-DNA in the presenceof K+ causes the fluorescent response. (C) Emission spectraof G4 probes modified with different fluorophores at their 5′end. (D) Fluorescence intensity variation of G4 probes, modified withHEX (green) or Texas Red (red), in response to increasing K+ concentration.
K+ transport measurements across single GUVs using microfluidic-basedapproaches. (A) On-chip production of GUVs (DOPC/DOPG, 3:1 w/w) andencapsulation of FAMQ-G4 (10 μM), using octanol-assisted liposomeassembly. Scale bar: 20 μm. (B) FAMQ-G4 encapsulated GUV immobilizationusing microfluidic hydrodynamic trapping (i). Once trapped, a 1 mMKCl solution, containing 500 nM FAMQ-G4, is perfused into the microfluidicchamber (ii), where GUVs can be visualized for >10 h, allowingoneto capture the transport process of slow-permeating solutes such asK+. Scale bar: 40 μm. (C) (i) Time lapse of lumenalGUV fluorescence during the K+ transport process, showingthe decrease in FAMQ-G4 fluorescence as a result of K+ inducedfolding. Scale bar: 20 μm. (ii) Analysis of K+ permeationacross the lipid bilayer of single GUVs (n = 441)showing the temporal variation of lumenal (black solid line) and background(black dashed line) fluorescence intensity (median). The gray bandsrepresent the lower and upper quartiles of the measured fluorescenceat each time point for each measured vesicle.
Quantificationof K+ permeability across single GUVs.(A) Variation of K+ concentration inside (solid) and outside(dashed) GUVs during the transport process. The distribution of [K+] among GUVs is represented by the upper and lower quartilesof [K+] at each time point. Inset: magnified view of thetransmembrane K+ concentration gradient, Δ[K+], generated in the initial period (typically a few tens ofminutes) of our experiments. (B) Variation of K+ flux intoGUVs over the measurement time course. Inset: flux profile showingthe variation of K+ flux as a function of Δ[K+] during the initial period of Δ[K+] development.The obtained linear flux profile can be represented throughJ =PΔ[K+], thus enablingthe determination of K+ permeability from the slope ofthe curve. (C) Schematic showing the proposed dissipation mechanismof transmembrane potential by a counter flux of protons across theGUV lipid bilayer in our experiments. (D) Distribution of measuredpermeability coefficients for negatively charged OLA DOPC/DOPG (3:1)GUVs (N = 441). Inset: measured permeability distributionof electroformed DOPC/DOPG (3:1) GUVs.
K+ transport kinetics across GUVs with reconstitutedmodel ion channels. (A) Time-resolved variation of lumenal (solidlines) and extravesicular (dashed lines) K+ concentrationfor DOPC/DOPG (3:1) GUVs with (red,n = 46), andwithout (black,n = 38) reconstituted gramicidinA (gA) (see experimental section). The distribution of permeated [K+] over GUVs is represented by the upper and lower quartilesof [K+] at each time point. Inset: schematic illustratingthe two possible transport pathways across gA incorporated GUVs.B. Analysis of lumenal (solid lines) and extravesicular (dashedlines) [K+] across DOPC/DOPG (3:1) GUVs with (blue,n = 83) and without (black,n = 76) reconstitutedOmpF (see Materials and Methods). Inset: schematicillustrating the two possible transport pathways across OmpF-incorporatedGUVs. (C) Flux profiles obtained for GUVs with reconstituted gA (red)and OmpF (blue). The circles are the mean flux values, and the bandsare the lower and upper quartiles for each GUV population. The blackdashed line is the best fit of a linear curve to the mean flux dataat the linear regime (0 < Δ[K+] < 0.13 mM), using linear regression. The red arrow indicatesthe Δ[K+] value at which the measured mean flux (redcircles) is 0.58 of the flux (black dashed line) at the same Δ[K+] in the absence of transmembrane potential development. (D)Schematic demonstrating the suggested origin for the variance in H+/K+ selectivity between gA and OmpF.
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