Abstract

The aim of this study was to establish the mechanisms of action of a novel liposomal nitric oxide (NO) carrier on large-conductance Ca²⁺-activated channels (BK_Ca or Maxi-K) expressed in vascular smooth muscle cells (VSMCs) isolated from the rat main pulmonary artery (MPA). Experimental design comprised of both whole-cell and cell-attached single-channel recordings using the patch-clamp techniques. The liposomal form of NO, Lip(NO), increased whole-cell outward K⁺ currents in a dose dependent manner while shifting the activation curve negatively by about 50 mV with respect to unstimulated cells with the EC₅₀ value of 0.55 ± 0.17 µM. At the single channel level, Lip(NO) increased the probability of the open state (Po) of Maxi-K channels from 0.0020 ± 0.0008 to 0.74 ± 0.02 with half-maximal activation occurring at 4.91 ± 0.01 μM, while sub-maximal activation was achieved at 10⁻⁵ M Lip(NO). Channel activation was mainly due to significant decrease in the mean closed dwell time (about 500-fold), rather than an increase in the mean open dwell time, which was comparatively modest (about twofold). There was also a slight decrease in the amplitude of the elementary Maxi-K currents (approximately 15%) accompanied by an increase in current noise, which might indicate some non-specific effects of Lip(NO) on the plasma membrane itself and/or on the phospholipids environment of the channels. In conclusion, the activating action of Lip(NO) on the Maxi-K channel is due to the destabilization of the closed conformation of the channel protein, which causes its more frequent openings and, accordingly, increases the probability of channel transition to its open state.

摘要

本研究的目的是建立新型脂质体一氧化氮 (NO) 载体对分离的血管平滑肌细胞 (VSMC) 中表达的大电导 Ca ²⁺激活通道（BK _Ca或 Maxi-K）的作用机制。来自大鼠主肺动脉 (MPA)。实验设计包括使用膜片钳技术的全细胞和细胞附着的单通道记录。NO 的脂质体形式 Lip(NO)以剂量依赖的方式增加全细胞外向 K ⁺电流，同时使激活曲线相对于未受刺激的细胞负向移动约 50 mV 与 EC ₅₀0.55 ± 0.17 µM 的值。在单通道水平上，Lip(NO) 将 Maxi-K 通道的开放状态 (Po) 的概率从 0.0020 ± 0.0008 增加到 0.74 ± 0.02，半最大激活发生在 4.91 ± 0.01 μM，而次最大激活达到了 10 ^-5 M 唇（否）。通道激活主要是由于平均关闭停留时间显着减少（约 500 倍），而不是平均开放停留时间增加，后者相对温和（约两倍）。基本 Maxi-K 电流的幅度也略有下降（约 15%），伴随着电流噪声的增加，这可能表明 Lip(NO) 对质膜本身和/或关于通道的磷脂环境。总之，Lip(NO)对Maxi-K通道的激活作用是由于通道蛋白的封闭构象不稳定，导致其更频繁的开放，因此增加了通道过渡到其开放的可能性状态。