Smooth Muscle Research Centre, Dundalk Institute of Technology, Dundalk, Ireland.
Am J Physiol Cell Physiol. 2013 Aug 15;305(4):C427-35. doi: 10.1152/ajpcell.00034.2013. Epub 2013 Jun 19.
A collagenase-proteinase mixture was used to isolate airway smooth muscle cells (ASMC) from rabbit bronchi, and membrane currents were recorded using the whole cell patch-clamp technique. Stepping from -100 mV to a test potential of -40 mV evoked a fast voltage-dependent Na(+) current, sometimes with an amplitude of several nanoamperes. The current disappeared within 15 min of exposure to papain + DTT (n = 6). Comparison of the current in ASMC with current mediated by NaV1.5 α-subunits expressed in human embryonic kidney cells revealed similar voltage dependences of activation (V1/2 = -42 mV for NaV1.5) and sensitivities to TTX (IC50 = 1.1 and 1.2 μM for ASMC and NaV1.5, respectively). The current in ASMC was also blocked by lidocaine (IC50 = 160 μM). Although veratridine, an agonist of voltage-gated Na(+) channels, reduced the peak current by 33%, it slowed inactivation, resulting in a fourfold increase in sustained current (measured at 25 ms after onset). In current-clamp mode, veratridine prolonged evoked action potentials from 37 ± 9 to 1,053 ± 410 ms (n = 8). Primers for NaV1.2-1.9 were used to amplify mRNA from groups of ∼20 isolated ASMC and from whole bronchial tissue by RT-PCR. Transcripts for NaV1.2, NaV1.3, and NaV1.5-1.9 were detected in whole tissue, but only NaV1.2 and NaV1.5 were detected in single cells. We conclude that freshly dispersed rabbit ASMC express a fast voltage-gated Na(+) current that is mediated mainly by the NaV1.5 subtype.
胶原蛋白酶-蛋白酶混合物被用于从兔支气管中分离气道平滑肌细胞（ASMC），并使用全细胞膜片钳技术记录膜电流。从-100 mV 步进到-40 mV 的测试电位引发快速电压依赖性 Na(+)电流，有时幅度可达几个纳安培。暴露于木瓜蛋白酶+DTT 15 分钟内，电流消失（n=6）。将 ASMC 中的电流与在人胚肾细胞中表达的 NaV1.5 α亚基介导的电流进行比较，发现激活的电压依赖性（NaV1.5 的 V1/2 =-42 mV）和对 TTX 的敏感性（ASMC 和 NaV1.5 的 IC50 分别为 1.1 和 1.2 μM）相似。ASMC 中的电流也被利多卡因阻断（IC50=160 μM）。虽然辣椒碱是电压门控 Na(+)通道的激动剂，可将峰值电流减少 33%，但它会减缓失活，导致持续电流增加四倍（在起始后 25 毫秒测量）。在电流钳模式下，辣椒碱将诱发动作电位的持续时间从 37±9 毫秒延长至 1053±410 毫秒（n=8）。使用 RT-PCR 从约 20 个分离的 ASMC 群体和整个支气管组织中扩增 NaV1.2-1.9 的 mRNA。在整个组织中检测到 NaV1.2、NaV1.3 和 NaV1.5-1.9 的转录本，但在单个细胞中仅检测到 NaV1.2 和 NaV1.5。我们得出结论，新分散的兔 ASMC 表达一种快速电压门控 Na(+)电流，主要由 NaV1.5 亚基介导。
