Aki Yamada1, Norikazu Gaja1, Susumu Ohya1, Katsuhiko Muraki1, Hiroshi Narita2, Tomohiko Ohwada3 and Yuji Imaizumi1,*
1Department of Molecular and Cellular Pharmacology and 3Department of Organic and Medicinal Chemistry, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
2Discovery Research Laboratories, Tanabe Seiyaku, Co., Ltd., Toda 335-8505, Japan
*Corresponding author. FAX: +81-52-836-3431, E-mail: firstname.lastname@example.org
Abstract: 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 a or a plus b1 subunits of large conductance Ca2+-activated K+ (BK) channels were expressed (HEKBKa and HEKBK ab). 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 HEKBKab was 10 - 20 mV more negative than that in native HEK. In HEKBKa, the membrane hyperpolarization induced by 10 mM Evans blue, a BK channel opener, was detected with DiBAC4(3). NS-1619, another BK channel opener, induced gradual but substantial change in F/FK even in native HEK, while the BK channel opening effect was detected. Oscillatory membrane hyperpolarization was induced in HEKBKab by application of 10 mM 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.
Keywords: Voltage-sensitive dye, DiBAC4(3), Large conductance Ca2+-activated K+ channel, Evans blue, NS-1619
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