384通量全自动膜片钳系统IonWorks Barracuda同时检测电压门控和配基门控离子通道

The IonWorks Barracuda™ System Enables 384 Simultaneous Recordings of Ligand- and Voltage-gated Ion Channels
Xin Jiang, Edward Verdonk, Trisha Tutana, Karen Cook, Chuanxiu Yang, Yuri Osipchuk, James Costantin
Molecular Devices, Inc., 1311 Orleans Drive, Sunnyvale, CA 94089

Abstract

Since the introduction of the first automated electrophysiology instrument in 2002,  the maximum number of simultaneous recordings that could be performed on all existing systems has been limited to 48.  Here we report the development and validation of the new IonWorks Barracuda™ system that  allows simultaneous and continuous measurement of both ligand-gated ion channels (LGICs) and voltage-gated ion channels (VGICs) at 384 separate recording sites. To achieve this, IonWorks Barracuda™ system is equipped with 384 individual patch-clamp  amplifiers together with a 384-channel fluidic pipettor.  Similar to its predecessor, IonWorks® Quattro system, IonWorks Barracuda™ system measures cell membrane currents using the perforated patch clamp techniques on a polyimide substrate.  Currents are measured using a single hole (SH) at each recording site or an array of 61 holes at each site (Population Patch Clamp or PPC, Finkel et al. 2006). In this study we examined the performance of IonWorks Barracuda™ system with multiple ion channels.  Data presented here include LGIC recordings of GABA chloride channels, acid sensing ion channels (ASIC), and nicotinic acetylcholine (1 nACh) receptors; as well as VGIC recordings of NaV, KV and hERG channels.  Pharmacological blockade of ion channel activity is also presented to validate the use of this automated, high-throughput system for screening ion channel targets in a drug discovery setting.

Materials

Cells

ASIC1a and human hERG channels stably transfected in CHO cells were generously provided by ChanTest Corporation (Cleveland, Ohio)

GABAA currents were measured in WSS-1 cells obtained from ATCC (Cat # CRL-2029)
1 nACh Receptors were measured in TE671 cells obtained from ATCC
KV1.3 and NaV1.5 channel were stably transfected in CHO cells

Reagents

Currents were measured using standard buffers.  Internal buffer contained (in mM), 140 KCl, 2 MgCl2, 5 EGTA, 10 HEPES, pH 7.25 with KOH. External buffer was D-PBS (Gibco Cat # 14040-117).

Fig 1: IonWorks Barracuda™ system. A) External view of the system; B) Front view of the process deck. 

Fig 2: Recording of ASIC1a currents in both SH and PPC modes. A) Plate view of a SH experiment where current from one cell is recorded per well. ASIC1a currents are elicited by application of pH 5.0 buffer (PBS in columns 12 and 24-highlighted in green); B) A PPC experiment with the same compound map as A; C) Recordings of ASIC1a currents in the presence of different concentrations of amiloride (PPC). 

Fig 3: ASIC1a currents and pharmacology. A) Representative ASIC1a currents recorded in the presence (top) and absence (bottom) of 1mM amiloride (SH),; B) Concentration-dependence of ASIC1a channel inhibition by benzamil and amiloride are determined in two different PPC experiments, with IC50 values matching literature reports.

Fig 4: Recordings of ligand-gated channel currents with different kinetics.   A) 1 nACh receptor current elicited by application of 500 μM of acetylcholine (SH); B) GABA current elicited by application of 500 μM of GABA (SH). Note the two channels activates and desensitizes at different rates.

Fig 5: Recordings of hERG currents in both SH and PPC modes. A) Plate view of a SH experiment with Vh = -70mV; hERG currents were evoked by first stepping to +40mV for 1s and then to -50mV for 1s; B) Plate view of a PPC experiment; C) Recordings of hERG currents in the presence of different concentrations of terfenadine (PPC).

Fig 6: hERG currents and pharmacology. A) Representative hERG currents recorded in the presence (blue) and absence (black) of 200 μM Cisapride (SH); B) Concentration-dependence of hERG channel inhibition by 7 different compounds, with IC50 values matching literature reports.

Fig 7: Recordings of voltage-gated channel currents with different kinetics.   A)  Whole-cell KV1.3 currents elicited by 2s test pulses to potentials from -100 and +90mV in steps of 10mV (Vh = -70mV, sampling rate 1k Hz, SH); B) Whole-cell NaV1.5 currents elicited by 20ms test pulses to potentials from -100mV to +90mV in steps of 10mV (Vh = -90mV, sampling rate 20k Hz, SH). Note the two channels activate and inactivate at different rates.

Summary

►IonWorks Barracuda™ system has 384 individual patch clamp amplifiers together with a 384-channel fluidics pipettor, thereby enabling simultaneous and continuous recordings of both ligand and voltage-gated ion channels on a 384-well PatchPlate substrate.

►Three ligand-gated channels (ASIC1a, GABA and 1 Ach receptor) and three voltage-gated channels (hERG, KV1.3 and NaV1.5) are successfully recorded; in both SH and PPC modes. And the difference in channel kinetics can be sufficiently resolved.

►Results of pharmacology assays obtained from IonWorks Barracuda™ system are comparable to those from IonWorks® Quattro system, or literature reports.

►IonWorks Barracuda™ system is a highly versatile, automated, high-throughput system suitable for screening ion channel targets in a drug discovery setting.