GABA - Molecular Devices

PatchXpress assays – protocol for BDZ site agonistsExample of current trace5-pt DRC DiazepamDiazepam (nM)0.1 1 10 100 1000GABA EC20500 pA2 min5s addition of 1 M GABAPre-incubation 2 minutes with each concentration of agonist.V hold= -40 mV

PatchXpress assays - detection of BDZ site agonistsEC 50= 31 6 nM (n = 7)Control current amplitudes240220(number of cells shown in brackets)Percentage increase in GABA response20018016014012010080604020Current (nA)2.52.01.51.00.5(7)(7)01E-4 1E-3 0.01 0.1 1[cf EC 50= 40nM, Rabe et.al. 2007][Diazepam] M0.01 M GABA 1 M DiazepamMean amplitudes of EC20 GABA andhighest concentration of diazepam

PatchXpress assays – protocol for BDZ site antagonistsExample of current trace5-pt DRC FlumazenilFlumazenil (nM)1 10 100 1000 10000Diazepam (1 M)GABA EC201 nA2 min5s addition of 1 M GABAPre-incubation 2 minutes with 1M Diazepam and each concentration of antagonist.V hold= -40 mV

PatchXpress assays - detection of BDZ site antagonists200IC 50= 28 3 nM (n = 5-7)Control current amplitudesPercentage increase in GABA response180160140120100806040200Current (nA)3.53.02.52.01.51.00.5(number of cells shown in brackets)(7)(7)-201E-3 0.01 0.1 1 100.01 M GABA 1 M Diazepam[Flumazenil ] MMean amplitudes of EC20 GABA and1 M diazepam

Use of Ionworks for ligand-gated channel profiling‣ Ligand-gated channels provide technical challenges for high throughputscreening with Ionworks:– rapid activation and desensitisation requires minimal delay between ligand addition and currentrecording - limited by independent electronics and fluidics heads– no compound washout so control responses and different concentrations have to be compared inseparate wells (c.f. PatchXpress, manual patch clamp where same cell can be used, i.e. intra-wellcontrols)‣ Improvements with Iwks Quattro now allow possibility of recording ligand-gatedchannels with slower kinetics– 48 channel fluidics head (shorter delay before E-head can record)– dual compound addition capability (e.g. for measuring antagonists)– greater well-to-well reproducibility of PPC - allows more reliable inter-well comparisonGABA6 sCurrent measured by IonWorks Quattro2 nA5 s(Trace from PatchXpress: V H= -60 mV)

Development of Ionworks protocol for GABA A channelsAEvoked current inthe presence of GABAEvoked current inthe absence of GABACB

GABAa 1 Ionworks assayA) Analysis of GABA-induced current amplitudeSingle cell (HT)PPC (Quattro)B) Concentration-response curves for GABAHTPPC

Comparison of Ionworks and PatchXpress data–GABA A 1 vs. 3IonWorks Quattro (n = 3) PatchXpress (n = 6-15)IW EC 50PX EC 50Published EC 50Reference 3.6 µM 4.8 µM 2.1 µM Williams & Akabas, 2000 9.6 µM 11.4 µM 15 µM Smith et.al. 2001

Ionworks assays - detection of GABA antagonistsIW IC 50Published IC 50ReferenceBicuculline 0.2 µM 0.9 µM Ueno et.al. 1997Picrotoxin 0.5 µM 3.4 µM Dibas et. Al. 2002Bicuculline 0.4 µM -Picrotoxin 0.5 µM -N.B. Experiments carried outas blinded, mock screens

Ionworks assays - detection of BDZ site agonistsIW EC 50Published EC 50ReferenceDiazepam 22.9 nM 40 nM Rabe et.al. 2007Zolpidem 54.8 nM 92 nM Wingrove et.al. 2002Diazepam 5.6 nM 90 nM Rabe et.al. 2007Zolpidem 310 nM 824 nM Wingrove et.al.2002N.B. Experiments carried outas blinded, mock screens

Ionworks assays - detection of subtype selectivemodulatorsN.B. Experiments carried out as blinded, mock screens

Ionworks assays – robustness and resolution65Frequency Count432N.B. Experiments carried outas blinded, mock screens100.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0No GABA EC20EC80 EC100Current (nA)POSITIVE MODULATOR ASSAYEC 20 to 30 M Zolpidem = ~0.3 - 0.5EC 20 to EC 100 = ~ 0.5Z’ VALUES:-ANTAGONIST ASSAYEC 80 to 100 M Bicuculline = 0.6 - 0.7

Summary and Conclusions• Electrophysiological assays for screening compounds against GABA Areceptors have been developed using two automated platforms andMillipore’s and cell lines.• These assays can detect agonists, antagonists and allosteric modulatorswith good resolution and reproducibility and can discriminate subtypeselective compounds• Despite the inability to resolve peak agonist responses, these datademonstrate that assays for improved throughput compound screeningagainst GABA Achannels can be developed on Ionworks• Using a similar approach, assays for other slowly gating LGICs are beingdeveloped, providing the opportunity for information-richelectrophysiological screening of compounds earlier in the screeningcascade

AcknowledgementsMillipore Ion Channel Group, Cambridge, UK:Umesh Patel Ray Helliwell Ed MaughlingLuanda Pym Reena Reehal Jonathan MannToni Cooper Helen Swain Louise WebdaleCelia Holdsworth Andrew CookCell line generation and assay developmentwas partly carried out incollaboration with GSK, Stevenage, UK