Screening quality for Ca2+-activated potassium channel in IonWorks Quattro is greatly improved by using BAPTA-AM and ionomycin

摘要

Introduction: IonWorks automated patch clamp systems are being widely used for ion channel drug discovery, but the perforated patch mode of these systems makes it difficult to obtain a steady intracellular Ca2+ concentration ([Ca2+]i). This difficulty prevents obtaining high-quality data regarding Ca2+-activated channels such as BK and SK channels. We examined the methods for stabilizing [Ca2+]i in the IonWorks Quattro automated patch clamp system to evaluate BK channels. Methods: Electrophysiological recordings were performed using the single-hole or population patch clamp mode of IonWorks Quattro. To increase [Ca2+]i, ionomycin was used. The variation in the BK current and the effect of BK channel modulators were examined in the presence and absence of an intracellular Ca2+ chelator, BAPTA-AM (20??M). Results: BK current activated by step pulses to +. 100. mV in the presence of ionomycin exhibited large variation (ranging from 0.086 to 11. nA). In individual cells, oscillation of the current amplitude was observed when five repetitive pulses were applied at 0.1. Hz. Approximately 30% of cells exhibited current variation exceeding 20% when the variation was calculated using the first and third pulses. However, BAPTA-AM treatment before current measurement decreased the number of cells displaying large variation (20%) to 5%. In the presence of BAPTA-AM, the BK channel modulators NS1619 and 12,14-dichlorodehydroabietic acid increased the BK current at concentrations of 10. ??M or more showing clear concentration dependency, whereas in its absence, the effect of both compounds was detected only at 30. ??M. Discussion: The main finding of this study is that the [Ca2+]i variation in the basal condition is very large and hinders the accurate evaluation of compounds in Ca2+-activated ion channels. The application of BAPTA-AM and ionomycin greatly improved the precision of BK channel screening, and this method should be applicable to other Ca2+-activated ion channels such as SK channels. ? 2012 Elsevier Inc.