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

The usefulness of bis-(1,3-dibutylbarbituric acid)-trimethine oxonol (DiBAC(4)(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 alpha beta). The fluorescent intensity of DiBAC(4)(3) was measured at various potentials under voltage-clamp for calibration to estimate the absolute NIP semi-quantitatively. The resting MPs measured with DiBAC(4)(3) were roughly comparable to those recorded with a microelectrode; the NIP in HEKBK alpha beta was 10 - 20 mV more negative than that in native HEK. In HEKBK alpha, the membrane hyperpolarization induced by 10 muM Evans blue, a BK channel opener, was detected with DiBAC(4)(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 alpha beta by application of 10 muM 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 DiBAC(4)(3) in HEY-BK, while the limited temporal resolution and possible artifacts should be taken into account.