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Comparison of cell expression formats for the characterization of GABA(A) channels using a microfluidic patch clamp system. Assay Drug Dev Technol 2012 Aug;10(4):325-35

Date

05/12/2012

Pubmed ID

22574655

Pubmed Central ID

PMC3419985

DOI

10.1089/adt.2011.415

Scopus ID

2-s2.0-84865449180 (requires institutional sign-in at Scopus site)   7 Citations

Abstract

Ensemble recording and microfluidic perfusion are recently introduced techniques aimed at removing the laborious nature and low recording success rates of manual patch clamp. Here, we present assay characteristics for these features integrated into one automated electrophysiology platform as applied to the study of GABA(A) channels. A variety of cell types and methods of GABA(A) channel expression were successfully studied (defined as I(GABA)>500 pA), including stably transfected human embryonic kidney (HEK) cells expressing α(1)β(3)γ(2) GABA(A) channels, frozen ready-to-assay (RTA) HEK cells expressing α(1)β(3)γ(2) or α(3)β(3)γ(2) GABA(A) channels, transiently transfected HEK293T cells expressing α(1)β(3)γ(2) GABA(A) channels, and immortalized cultures of human airway smooth muscle cells endogenously expressing GABA(A) channels. Current measurements were successfully studied in multiple cell types with multiple modes of channel expression in response to several classic GABA(A) channel agonists, antagonists, and allosteric modulators. We obtained success rates above 95% for transiently or stably transfected HEK cells and frozen RTA HEK cells expressing GABA(A) channels. Tissue-derived immortalized cultures of airway smooth muscle cells exhibited a slightly lower recording success rate of 75% using automated patch, which was much higher than the 5% success rate using manual patch clamp technique by the same research group. Responses to agonists, antagonists, and allosteric modulators compared well to previously reported manual patch results. The data demonstrate that both the biophysics and pharmacologic characterization of GABA(A) channels in a wide variety of cell formats can be performed using this automated patch clamp system.

Author List

Chen Q, Yim PD, Yuan N, Johnson J, Cook JM, Smith S, Ionescu-Zanetti C, Wang ZJ, Arnold LA, Emala CW

Author

Alexander (Leggy) Arnold PhD Professor in the Chemistry & Biochemistry department at University of Wisconsin - Milwaukee




MESH terms used to index this publication - Major topics in bold

Benzodiazepines
Bicuculline
Diazepam
GABA Agents
GABA Agonists
GABA Antagonists
GABA Modulators
Gene Expression
HEK293 Cells
High-Throughput Screening Assays
Humans
Imidazoles
Ion Channel Gating
Microfluidic Analytical Techniques
Muscimol
Patch-Clamp Techniques
Picrotoxin
Receptors, GABA-A
Recombinant Proteins
Transfection