Cavendish Laboratory, University of Cambridge, J.J. Thomson Avenue, CambridgeCB3 0HE, U.K.
Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, LondonW12 0BZ, U.K.
ACS Nano. 2022 Oct 25;16(10):17128-17138. doi: 10.1021/acsnano.2c07496. Epub 2022 Oct 12.
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 ∼10.
由于缺乏合适的离子选择性探针，精确测量离子通过细胞膜的渗透性仍然具有挑战性。在这里，我们使用巨大的单层囊泡 (GUV) 作为膜模型，用于在单个囊泡水平上直接可视化质量转移。离子传输用荧光可调 DNA 基传感器指示，该传感器可以准确检测 K 浓度的亚毫摩尔变化。结合微流控技术，我们使用基于 DNA 的 K 传感器提取钾离子的渗透系数。我们测量的 K 渗透率系数比以前从体相实验中报道的值至少大一个数量级，并表明在存在辛醇的情况下，穿过脂质双层的渗透速率增加。此外，对不同浓度梯度中 K 通量的分析使我们能够估计跨 GUV 膜耗散电荷失衡的互补 H 通量。随后，我们表明我们的传感器可以定量测量穿过原型阳离子选择性离子通道，即短杆菌肽 A 和 OmpF 的 K 转运，揭示它们的相对 H/K 选择性。我们的结果表明，与 OmpF 相比，短杆菌肽 A 对质子的选择性高得多，H/K 渗透比约为 10。
