title: Screening quality for Ca2+-activated potassium channel in IonWorks Quattro is greatly improved by using BAPTA-AM and ionomycin.
abstract: BACKGROUND
  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 Ca(2+) concentration ([Ca(2+)](i)). This difficulty prevents obtaining high-quality data regarding Ca(2+)-activated channels such as BK and SK channels. We examined the methods for stabilizing [Ca(2+)](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 [Ca(2+)](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 Ca(2+) chelator, BAPTA-AM (20μM).
  
  
  RESULTS
  BK current activated by step pulses to +100mV in the presence of ionomycin exhibited large variation (ranging from 0.086 to 11nA). In individual cells, oscillation of the current amplitude was observed when five repetitive pulses were applied at 0.1Hz. 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 10μM or more showing clear concentration dependency, whereas in its absence, the effect of both compounds was detected only at 30μM.
  
  
  CONCLUSIONS
  The main finding of this study is that the [Ca(2+)](i) variation in the basal condition is very large and hinders the accurate evaluation of compounds in Ca(2+)-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 Ca(2+)-activated ion channels such as SK channels.
authors.affiliation: Department of Experimental Pathology, Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan. k-ido@hhc.eisai.co.jp
authors.forename: Katsutoshi
authors.initials: K
authors.lastname: Ido
authors.forename: Tomohiko
authors.initials: T
authors.lastname: Ohwada
authors.forename: Eriko
authors.initials: E
authors.lastname: Yasutomi
authors.forename: Takashi
authors.initials: T
authors.lastname: Yoshinaga
authors.forename: Tohru
authors.initials: T
authors.lastname: Arai
authors.forename: Mitsuyasu
authors.initials: M
authors.lastname: Kato
authors.forename: Kohei
authors.initials: K
authors.lastname: Sawada
mesh_terms: D000818:Animals; D016466:CHO Cells; D061207:Calcium Ionophores / Q000494:pharmacology*; D002460:Cell Line; D002614:Chelating Agents / Q000494:pharmacology*; D006224:Cricetinae; D003412:Cricetulus; D004353:Drug Evaluation, Preclinical / Q000379:methods / Q000592:standards; D004533:Egtazic Acid / Q000031:analogs & derivatives* / Q000494:pharmacology; D015759:Ionomycin / Q000494:pharmacology*; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D018408:Patch-Clamp Techniques / Q000379:methods / Q000592:standards*
chemical_list: D061207:Calcium Ionophores; D002614:Chelating Agents; D051036:Large-Conductance Calcium-Activated Potassium Channels; C070379:1,2-bis(2-aminophenoxy)ethane N,N,N',N'-tetraacetic acid acetoxymethyl ester; D004533:Egtazic Acid; D015759:Ionomycin