Department of Physiology and Biophysics, University of California Irvine, Irvine, Calfornia, United States of America.
PLoS One. 2012;7(4):e35090. doi: 10.1371/journal.pone.0035090. Epub 2012 Apr 26.
The impact of synthetic amyloid β (1-42) (Aβ(1-42)) oligomers on biophysical properties of voltage-gated potassium channels Kv 1.3 and lipid bilayer membranes (BLMs) was quantified for protocols using hexafluoroisopropanol (HFIP) or sodium hydroxide (NaOH) as solvents prior to initiating the oligomer formation. Regardless of the solvent used Aβ(1-42) samples contained oligomers that reacted with the conformation-specific antibodies A11 and OC and had similar size distributions as determined by dynamic light scattering. Patch-clamp recordings of the potassium currents showed that synthetic Aβ(1-42) oligomers accelerate the activation and inactivation kinetics of Kv 1.3 current with no significant effect on current amplitude. In contrast to oligomeric samples, freshly prepared, presumably monomeric, Aβ(1-42) solutions had no effect on Kv 1.3 channel properties. Aβ(1-42) oligomers had no effect on the steady-state current (at -80 mV) recorded from Kv 1.3-expressing cells but increased the conductance of artificial BLMs in a dose-dependent fashion. Formation of amyloid channels, however, was not observed due to conditions of the experiments. To exclude the effects of HFIP (used to dissolve lyophilized Aβ(1-42) peptide), and trifluoroacetic acid (TFA) (used during Aβ(1-42) synthesis), we determined concentrations of these fluorinated compounds in the stock Aβ(1-42) solutions by (19)F NMR. After extensive evaporation, the concentration of HFIP in the 100× stock Aβ(1-42) solutions was ∼1.7 μM. The concentration of residual TFA in the 70× stock Aβ(1-42) solutions was ∼20 μM. Even at the stock concentrations neither HFIP nor TFA alone had any effect on potassium currents or BLMs. The Aβ(1-42) oligomers prepared with HFIP as solvent, however, were more potent in the electrophysiological tests, suggesting that fluorinated compounds, such as HFIP or structurally-related inhalational anesthetics, may affect Aβ(1-42) aggregation and potentially enhance ability of oligomers to modulate voltage-gated ion channels and biological membrane properties.
合成淀粉样β（1-42）（Aβ（1-42））低聚物对电压门控钾通道 Kv1.3 和脂质双层膜（BLM）的生物物理性质的影响，使用六氟异丙醇（HFIP）或氢氧化钠（NaOH）作为溶剂进行了定量评估，然后再开始形成低聚物。无论使用哪种溶剂，Aβ（1-42）样品都包含与构象特异性抗体 A11 和 OC 反应的低聚物，并且通过动态光散射确定具有相似的尺寸分布。钾电流的膜片钳记录显示，合成 Aβ（1-42）低聚物加速 Kv1.3 电流的激活和失活动力学，对电流幅度没有显著影响。与低聚物样品相反，新制备的、推测为单体的 Aβ（1-42）溶液对 Kv1.3 通道性质没有影响。Aβ（1-42）低聚物对从表达 Kv1.3 的细胞记录的稳态电流（在-80 mV）没有影响，但以剂量依赖性方式增加人工 BLM 的电导率。然而，由于实验条件的限制，没有观察到淀粉样通道的形成。为了排除 HFIP（用于溶解冻干的 Aβ（1-42）肽）和三氟乙酸（TFA）（用于 Aβ（1-42）合成）的影响，我们通过（19）F NMR 确定了这些含氟化合物在 Aβ（1-42）储备溶液中的浓度。在大量蒸发后，100×储备 Aβ（1-42）溶液中的 HFIP 浓度约为 1.7 μM。70×储备 Aβ（1-42）溶液中残留 TFA 的浓度约为 20 μM。即使在储备浓度下，HFIP 或 TFA 单独使用对钾电流或 BLM 均无影响。然而，用 HFIP 作为溶剂制备的 Aβ（1-42）低聚物在电生理测试中更为有效，这表明氟化化合物（如 HFIP 或结构相关的吸入性麻醉剂）可能影响 Aβ（1-42）聚集，并可能增强低聚物调节电压门控离子通道和生物膜性质的能力。
