Abstract
BackgroundHERG 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.ResultsWild 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°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°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°C, but not at 30°C.ConclusionOur 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.
Highlights
HERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death
Increase in intracellular and cell surface hERG protein expression levels when cells were maintained at 30°C A number of CHO-K1 stable cell lines over-expressing wild type hERG were established
These results suggest there is a general increase in expression of functional hERG protein on the cell surface of all cells examined at the lower temperature
Summary
HERG potassium channel blockade is the major cause for drug-induced long QT syndrome, which sometimes cause cardiac disrhythmias and sudden death. Loss of function mutations in hERG or the administration of channel blockers cause a delay in membrane potential repolarization of the cardiac action potential, and give rise to a prolongation of the QT interval on the electrocardiograph ([1], for review) This 'long QT syndrome' can trigger polymorphic ventricular dysrhythmias (torsades de pointes), syncope and sudden death. The hERG channel is blocked by a plethora of structurally diverse compounds, including many marketed drugs, some of which, such as terfenadine and cisapride, have been withdrawn from the market due to this unwanted activity [1,2,3] For this reason there is strong interest within the pharmaceutical industry in optimizing the recombinant expression of hERG, in order to improve the quality of medium to high-throughput assays for detecting compounds with potential cardiac liability at the earliest stages of drug development. As low temperature cultivation has been reported to increase the surface expression of some membrane proteins, as well as the production of some non-membrane associated proteins [14,5,13], we looked at the effect of this treatment on wild type hERG expression
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