Improved functional expression of recombinant human ether-a-go-go (hERG) K+ channels by cultivation at reduced temperature.
BACKGROUND
HERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death. There is a strong interest in the pharmaceutical industry to develop high quality medium to high-throughput assays for detecting compounds with potential cardiac liability at the earliest stages of drug development. Cultivation of cells at lower temperature has been used to improve the folding and membrane localization of trafficking defective hERG mutant proteins. The objective of this study was to investigate the effect of lower temperature maintenance on wild type hERG expression and assay performance.


RESULTS
Wild type hERG was stably expressed in CHO-K1 cells, with the majority of channel protein being located in the cytoplasm, but relatively little on the cell surface. Expression at both locations was increased several-fold by cultivation at lower growth temperatures. Intracellular hERG protein levels were highest at 27 degrees C and this correlated with maximal 3H-dofetilide binding activity. In contrast, the expression of functionally active cell surface-associated hERG measured by patch clamp electrophysiology was optimal at 30 degrees C. The majority of the cytoplasmic hERG protein was associated with the membranes of cytoplasmic vesicles, which markedly increased in quantity and size at lower temperatures or in the presence of the Ca2+-ATPase inhibitor, thapsigargin. Incubation with the endocytic trafficking blocker, nocodazole, led to an increase in hERG activity at 37 degrees C, but not at 30 degrees C.


CONCLUSIONS
Our results are consistent with the concept that maintenance of cells at reduced temperature can be used to boost the functional expression of difficult-to-express membrane proteins and improve the quality of assays for medium to high-throughput compound screening. In addition, these results shed some light on the trafficking of hERG protein under these growth conditions.
Chen
Mao Xiang
MX
BR&AD, GlaxoSmithKline R&D, Stevenage, UK. mark.x.chen@gsk.com
Sandow
Shaun L
SL
Doceul
Virginie
V
Chen
Yu Hua
YH
Harper
Heather
H
Hamilton
Bruce
B
Meadows
Helen J
HJ
Trezise
Derek J
DJ
Clare
Jeff J
JJ
D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D016466:CHO Cells; D017301:Ca(2+) Mg(2+)-ATPase / Q000187:drug effects; D018929:Cell Culture Techniques / Q000379:methods*; D006224:Cricetinae; D003412:Cricetulus; D051638:Ether-A-Go-Go Potassium Channels / Q000096:biosynthesis* / Q000187:drug effects; D006801:Humans; D008565:Membrane Proteins; D015739:Nocodazole / Q000494:pharmacology; D010627:Phenethylamines / Q000494:pharmacology; D026902:Potassium Channel Blockers; D021381:Protein Transport; D011994:Recombinant Proteins; D013449:Sulfonamides / Q000494:pharmacology; D013696:Temperature; D019284:Thapsigargin / Q000494:pharmacology; D022161:Transport Vesicles / Q000187:drug effects; D015854:Up-Regulation
D051638:Ether-A-Go-Go Potassium Channels; D008565:Membrane Proteins; D010627:Phenethylamines; D026902:Potassium Channel Blockers; D011994:Recombinant Proteins; D013449:Sulfonamides; D019284:Thapsigargin; D017301:Ca(2+) Mg(2+)-ATPase; C063533:dofetilide; D015739:Nocodazole