Sun X P, Supplisson S, Mayer E
Department of Medicine, Veteran's Affairs Wadsworth Medical Center, Los Angeles 90073.
Am J Physiol. 1993 Apr;264(4 Pt 1):G774-85. doi: 10.1152/ajpgi.1993.264.4.G774.
Because of the high intracellular Cl- concentration ([Cl-]i) in gastrointestinal smooth muscle, receptor-mediated opening of Cl- channels at the cell resting potential could represent a plausible mechanism for initial receptor-mediated cell depolarization. To test this hypothesis, we characterized activation of large-conductance Cl- channels by the neurokinin-1 (NK-1) receptor agonist [Sar9,Met(O2)11]-substance P, by specific second messengers, and by direct G protein activation in myocytes isolated from the rabbit colon longitudinal muscle layer. In excised inside-out patches, large-conductance ion channels selective for Cl- over Na+ could be induced by holding the patch at pipette potentials values > 60 mV. The channel showed multiple smaller conductance states (< or = 20) but could open and close via a main gate. When the channel was fully open, its slope conductance was 300 pS, with substates as small as 15 pS, comparable to the predominant conductance observed in cell-attached patches. The voltage-activation profile for full conductance was bell-shaped with maximal open probability (Po) for channel opening of approximately 0 mV. In cell-attached patches, addition of the NK-1 agonist to pipette solution activated a channel that corresponded to a subconductance state of the maxi Cl- channel. The voltage-activation profile for this subconductance state showed a maximal Po value for membrane potentials of approximately 0 mV, with rapid inactivation at more positive and partial inactivation at more negative membrane potentials. In excised inside-out patches, both the full and smaller conductance states of the Cl- channel were activated by the nonhydrolyzable guanosine triphosphate analogue guanosine 5'-O-(3-thiotriphosphate) and inhibited by pertussis toxin (PTX), whereas [Ca2+]i increased channel activity only in concentrations > 1 mM. In cell-attached patches, addition of different Ca2+ ionophores resulted in channel activation in 10% of cells, and activators of protein kinase A or protein kinase C had no effect. These findings are consistent with the hypothesis of a possible role of G protein-coupled Cl- channels in receptor-mediated initial cell depolarization in longitudinal colonic smooth muscle.
由于胃肠道平滑肌细胞内氯离子浓度（[Cl-]i）较高，在细胞静息电位时受体介导的氯离子通道开放可能是受体介导的细胞去极化的一种合理机制。为了验证这一假设，我们在从兔结肠纵肌层分离的肌细胞中，通过神经激肽-1（NK-1）受体激动剂[Sar9,Met(O2)11]-P物质、特定的第二信使以及直接激活G蛋白，对大电导氯离子通道的激活进行了表征。在切除的内面向外膜片中，当膜片钳电位值>60 mV时，可诱导出对Cl-选择性高于Na+的大电导离子通道。该通道显示出多个较小的电导状态（≤20），但可通过一个主门打开和关闭。当通道完全打开时，其斜率电导为300 pS，亚状态小至15 pS，与在细胞贴附膜片中观察到的主要电导相当。全电导的电压激活曲线呈钟形，通道开放的最大开放概率（Po）约为0 mV。在细胞贴附膜片中，向膜片钳溶液中添加NK-1激动剂可激活一个与最大氯离子通道的亚电导状态相对应的通道。该亚电导状态的电压激活曲线显示，膜电位约为0 mV时Po值最大，在更正的膜电位下快速失活，在更负的膜电位下部分失活。在切除的内面向外膜片中，氯离子通道的全电导和较小电导状态均被不可水解的鸟苷三磷酸类似物鸟苷5'-O-(3-硫代三磷酸)激活，并被百日咳毒素（PTX）抑制，而[Ca2+]i仅在浓度>1 mM时增加通道活性。在细胞贴附膜片中，添加不同的钙离子载体可使10%的细胞中的通道激活，蛋白激酶A或蛋白激酶C的激活剂则无作用。这些发现与G蛋白偶联氯离子通道在结肠纵肌受体介导的初始细胞去极化中可能发挥作用的假设一致。
