Usefulness and limitation of DiBAC4(3), a voltage-sensitive fluorescent dye, for the measurement of membrane potentials regulated by recombinant large conductance Ca2+-activated K+ channels in HEK293 cells.
Usefulness and limitation of DiBAC4(3), a voltage-sensitive fluorescent dye, for the measurement of membrane potentials regulated by recombinant large conductance Ca2+-activated K+ channels in HEK293 cells.
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DOI：
10.1254/jjp.86.342
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发表时间：
2001-07
期刊：
影响因子：
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通讯作者：
A. Yamada;Norikazu Gaja;Susumu Ohya;K. Muraki;Hiroshi Narita;Tomohiko Ohwada;Yuji Imaizumi
中科院分区：
文献类型：
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作者：
A. Yamada;Norikazu Gaja;Susumu Ohya;K. Muraki;Hiroshi Narita;Tomohiko Ohwada;Yuji Imaizumi
在HEK293细胞中评价了双-（1，3-二丁基巴比妥酸）-三甲川氧杂酚（DiBAC 4（3））（一种电压敏感性荧光染料）用于测量膜电位（MP）的有用性，其中表达了大电导Ca 2+激活K+（BK）通道的α或α + β 1亚基（HEKBK α和HEKBK α）。在用于校准的电压钳下在各种电位下测量DiBAC 4（3）的荧光强度以半定量地估计绝对MP。用DiBAC 4（3）测量的静息MP与用微电极记录的MP大致相当; HEKBK α中的MP比天然HEK中的MP负10 - 20 mV。在HEKBK α中，用DiBAC 4检测到10 μ M Evans蓝（BK通道开放剂）诱导的膜超极化（3）。另一种BK通道开放剂NS-1619即使在天然HEK中也诱导F/F（K）的逐渐但实质性的变化，同时检测到BK通道开放效应。通过增加细胞内Ca2+浓度，应用10 μ M乙酰胆碱在HEKBK alphaeta中诱导振荡膜超极化。然而，振荡超极化仅被检测为DiBAC 4的缓慢超极化（3）。可以得出结论，BK通道调节剂的相对缓慢的作用可以通过在HEKBK中使用DiBAC 4（3）来半定量地测量，同时应考虑有限的时间分辨率和可能的伪影。
The usefulness of bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC4(3)), a voltage-sensitive fluorescent dye, for the measurement of membrane potentials (MPs) was evaluated in HEK293 cells, where alpha or alpha plus beta1 subunits of large conductance Ca2+-activated K+ (BK) channels were expressed (HEKBK alpha and HEKBK alphabeta). The fluorescent intensity of DiBAC4(3) was measured at various potentials under voltage-clamp for calibration to estimate the absolute MP semi-quantitatively. The resting MPs measured with DiBAC4(3) were roughly comparable to those recorded with a microelectrode; the MP in HEKBK alphabeta was 10-20 mV more negative than that in native HEK. In HEKBK alpha, the membrane hyperpolarization induced by 10 microM Evans blue, a BK channel opener, was detected with DiBAC4(3). NS-1619, another BK channel opener, induced gradual but substantial change in F/F(K) even in native HEK, while the BK channel opening effect was detected. Oscillatory membrane hyperpolarization was induced in HEKBK alphabeta by application of 10 microM acetylcholine via increase in intracellular Ca2+ concentration. The oscillatory hyperpolarization was, however, detected only as a slow hyperpolarization with DiBAC4(3). It can be concluded that relatively slow effects of BK channel modulators can be semi-quantitatively measured by use of DiBAC4(3) in HEKBK, while the limited temporal resolution and possible artifacts should be taken into account.