title: Comparison of cell expression formats for the characterization of GABA(A) channels using a microfluidic patch clamp system.
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.
authors.affiliation: Fluxion Biosciences, South San Francisco, California, USA.
authors.forename: Qin
authors.initials: Q
authors.lastname: Chen
authors.forename: Peter D
authors.initials: PD
authors.lastname: Yim
authors.forename: Nina
authors.initials: N
authors.lastname: Yuan
authors.forename: Juliette
authors.initials: J
authors.lastname: Johnson
authors.forename: James M
authors.initials: JM
authors.lastname: Cook
authors.forename: Steve
authors.initials: S
authors.lastname: Smith
authors.forename: Cristian
authors.initials: C
authors.lastname: Ionescu-Zanetti
authors.forename: Zhi-Jian
authors.initials: ZJ
authors.lastname: Wang
authors.forename: Leggy A
authors.initials: LA
authors.lastname: Arnold
authors.forename: Charles W
authors.initials: CW
authors.lastname: Emala
mesh_terms: D001569:Benzodiazepines / Q000494:pharmacology; D001640:Bicuculline / Q000494:pharmacology; D003975:Diazepam / Q000494:pharmacology; D018682:GABA Agents / Q000494:pharmacology*; D018755:GABA Agonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D018757:GABA Modulators / Q000494:pharmacology; D015870:Gene Expression; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D015640:Ion Channel Gating / Q000187:drug effects; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D009118:Muscimol / Q000494:pharmacology; D018408:Patch-Clamp Techniques / Q000379:methods*; D010852:Picrotoxin / Q000494:pharmacology; D011963:Receptors, GABA-A / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D011994:Recombinant Proteins; D014162:Transfection
chemical_list: D001569:Benzodiazepines; D001640:Bicuculline; D003975:Diazepam; D018682:GABA Agents; D018755:GABA Agonists; D018756:GABA Antagonists; D018757:GABA Modulators; D007093:Imidazoles; D009118:Muscimol; D010852:Picrotoxin; D011963:Receptors, GABA-A; D011994:Recombinant Proteins; C558116:8-ethynyl-6-(2'-pyridine)-4H-2,5,10b-triazabenzo(e)azulene-3-carboxylic acid ethyl ester