Chloride channels in the plasma membrane of a foetal Drosophila cell line, S2
Chloride channels in the plasma membrane of a foetal Drosophila cell line, S2
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胎儿果蝇细胞系 S2 质膜中的氯离子通道
DOI：
10.1007/s004240000252
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发表时间：
2000
期刊：
影响因子：
--
通讯作者：
N. Willumsen
中科院分区：
文献类型：
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作者：
M. Asmild;N. Willumsen
我们评价了S2胎果蝇细胞系作为脊椎动物阴离子通道蛋白(如囊性纤维化跨膜电导调节器)表达系统在膜片钳研究内源性离子通道中的适用性。在内向外构型(对称的150nmCl-)中，我们发现最常见的是内向整流的氯离子通道，其单通道电导(γ)分别为57，45和17fps，分别为-80，0和80 mV。当[Cl-]还原到40 mM时，翻转电位(Vrev)移至-22.5 mV，表明有明显的氯离子选择性。在外向外结构([Cl-]吸管=40微米，[Cl-]槽=150微米)中，我们观察到一个具有线性单位电流/电压(I/V)关系的氯离子通道，其γ为30微微秒。在这两种构型中，动力学都相当缓慢。在全细胞实验([Cl-]吸管=40 mm)中也观察到了氯离子的选择性，其中Vrev为-43.8 mV，即接近氯离子平衡电位，表明膜电流由氯离子主导。我们得出结论，使S2细胞适合作为异源表达阴离子通道蛋白表达系统的重要特征是：小的总全细胞电流(小于100pA)，单通道电流和全细胞电流，不同于CFTR，不能用Goldman-Hodgkin-Katz机制来描述，以及慢动力学与CFTR明显不同。
We evaluated the suitability of the S2 foetal Drosophila cell line as an expression system for vertebrate anion channel proteins (e.g. cystic fibrosis transmembrane conductance regulator, CFTR) in patch-clamp studies of the endogenous ion channels. In the inside-out configuration (symmetric 150 mM Cl–) we found most frequently an inwardly rectifying Cl– channel with single-channel conductances (γ) of 57, 45 and 17 pS at –80, 0 and 80 mV, respectively. Reduction of bath [Cl–] to 40 mM caused a shift in reversal potential (Vrev) to –22.5 mV indicating pronounced Cl– selectivity. In the outside-out configuration ([Cl–]pipette = 40 mM, [Cl–]bath = 150 mM) we observed a Cl– channel with a linear unitary current/voltage (i/V) relation for which γ was 30 pS. The kinetics were quite slow in both configurations. Cl– selectivity was also observed in whole-cell experiments ([Cl–]pipette = 40 mM) in which a Vrev of –43.8 mV, i.e. close to the Cl– equilibrium potential, demonstrated that the membrane current was dominated by Cl–. We conclude that the important features making S2 cells suitable as an expression system for heterologous expressed anion channel proteins are: small total whole-cell currents (less than 100 pA), single-channel and whole-cell currents that, unlike those of CFTR, cannot be described by the Goldman-Hodgkin-Katz regime, and slow kinetics distinctly different from those of CFTR.