Abstract

Sincetheintroductionofthefirstautomatedelectrophysiologyinstrumentin2002, themaximumnumberofsimultaneousrecordingsthatcouldbeperformedonallexistingsystemshasbeenlimitedto48. HerewereportthedevelopmentandvalidationofthenewIonWorksBarracuda™systemthat allowssimultaneousandcontinuousmeasurementofbothligand-gatedionchannels(LGICs)andvoltage-gatedionchannels(VGICs)at384separaterecordingsites.Toachievethis,IonWorksBarracuda™systemisequippedwith384individualpatch-clamp amplifierstogetherwitha384-channelfluidicpipettor. Similartoitspredecessor,IonWorks®Quattrosystem,IonWorksBarracuda™systemmeasurescellmembranecurrentsusingtheperforatedpatchclamptechniquesonapolyimidesubstrate. Currentsaremeasuredusingasinglehole(SH)ateachrecordingsiteoranarrayof61holesateachsite(PopulationPatchClamporPPC,Finkeletal.2006).InthisstudyweexaminedtheperformanceofIonWorksBarracuda™systemwithmultipleionchannels. DatapresentedhereincludeLGICrecordingsofGABAchloridechannels,acidsensingionchannels(ASIC),andnicotinicacetylcholine(1nACh)receptors;aswellasVGICrecordingsofNaV,KVandhERGchannels. Pharmacologicalblockadeofionchannelactivityisalsopresentedtovalidatetheuseofthisautomated,high-throughputsystemforscreeningionchanneltargetsinadrugdiscoverysetting.

Materials

Cells

ASIC1aandhumanhERGchannelsstablytransfectedinCHOcellsweregenerouslyprovidedbyChanTestCorporation(Cleveland,Ohio)

GABAAcurrentsweremeasuredinWSS-1cellsobtainedfromATCC(Cat#CRL-2029)
1nAChReceptorsweremeasuredinTE671cellsobtainedfromATCC
KV1.3andNaV1.5channelwerestablytransfectedinCHOcells

Reagents

Currentsweremeasuredusingstandardbuffers. Internalbuffercontained(inmM),140KCl,2MgCl2,5EGTA,10HEPES,pH7.25withKOH.ExternalbufferwasD-PBS(GibcoCat#14040-117).

Fig6:hERGcurrentsandpharmacology.A)RepresentativehERGcurrentsrecordedinthepresence(blue)andabsence(black)of200μMCisapride(SH);B)Concentration-dependenceofhERGchannelinhibitionby7differentcompounds,withIC50valuesmatchingliteraturereports.

Summary

►IonWorksBarracuda™systemhas384individualpatchclampamplifierstogetherwitha384-channelfluidicspipettor,therebyenablingsimultaneousandcontinuousrecordingsofbothligandandvoltage-gatedionchannelsona384-wellPatchPlatesubstrate.

►Threeligand-gatedchannels(ASIC1a,GABAand1Achreceptor)andthreevoltage-gatedchannels(hERG,KV1.3andNaV1.5)aresuccessfullyrecorded;inbothSHandPPCmodes.Andthedifferenceinchannelkineticscanbesufficientlyresolved.

►ResultsofpharmacologyassaysobtainedfromIonWorksBarracuda™systemarecomparabletothosefromIonWorks®Quattrosystem,orliteraturereports.

►IonWorksBarracuda™systemisahighlyversatile,automated,high-throughputsystemsuitableforscreeningionchanneltargetsinadrugdiscoverysetting.