Presenting Author:

Carlos Vanoye, Ph.D.

Principal Investigator:

Alfred George, Jr.

Department:

Pharmacology

Keywords:

KCNQ1, cardiac disease, LQTS, channel variants, high throughput, automated electrophysiology

Location:

Third Floor, Feinberg Pavilion, Northwestern Memorial Hospital

B150 - Basic Science

FUNCTIONAL ANNOTATION OF KCNQ1 VARIANTS BY AUTOMATED ELECTROPHYSIOLOGY

Channelopathies result from ion channel dysfunction caused by mutation in either pore-forming subunits or regulatory proteins. The widespread use of genetic and genomic testing has led to an explosive growth in the number of newly discovered ion channel variants associated with human diseases and in reference populations. For example, in congenital long QT syndrome (LQTS), hundreds of variants have been discovered in KCNQ1 and KCNE1, which encode the pore-forming subunit and accessory protein required to generate the slow delayed cardiac rectifier current (IKs). Functional annotation experiments (e.g., patch-clamp recording) have become the gold standard in assessing the likely pathogenicity of ion channel variants, but the extreme time- and labor-intensity of this approach is insufficient to tackle hundreds, if not thousands, of known variants. To overcome this challenge, we have implemented a work flow that combined high efficiency electroporation to achieve transient co-expression of ion channel subunits in cultured cells with automated planar patch clamp recording performed in a 384-well format. We demonstrated the success of this approach by determining the functional properties of 51 KCNQ1 variants co-expressed with KCNE1 in CHO cells. Channel subunits were expressed from plasmid vectors encoding either EGFP (KCNQ1) or DsRed (KCNE1) enabling quantification of transfection efficiency by flow cytometry. Approximately 2/3 of cells were typically co-transfected with both subunits. Automated patch-clamp recording was performed before and after application of the specific IKs blocker HMR1556 (20 µM), enabling offline subtraction of non-specific currents. Typically, ~80-90% of wells exhibited cell capture, high seal resistance (>0.5Gohms) and low series resistance. Semi-automated data handling routines allowed for rapid analysis of current density, voltage-dependence of activation and gating kinetics. Our results indicate a successful implementation of a robust workflow that enables rapid functional annotation of human ion channel variants.