Improved functional expression of recombinant human ether-a-go-go (hERG) K+ channels by cultivation at reduced temperature

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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°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.</p> <p>Conclusion</p> <p>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.</p

The inwardly rectifying K+ channel KIR7.1 controls uterine excitability throughout pregnancy

Abnormal uterine activity in pregnancy causes a range of important clinical disorders, including preterm birth, dysfunctional labour and post-partum haemorrhage. Uterine contractile patterns are controlled by the generation of complex electrical signals at the myometrial smooth muscle plasma membrane. To identify novel targets to treat conditions associated with uterine dysfunction, we undertook a genome-wide screen of potassium channels that are enriched in myometrial smooth muscle. Computational modelling identified Kir7.1 as potentially important in regulating uterine excitability during pregnancy. We demonstrate Kir7.1 current hyper-polarizes uterine myocytes and promotes quiescence during gestation. Labour is associated with a decline, but not loss, of Kir7.1 expression. Knockdown of Kir7.1 by lentiviral expression of miRNA was sufficient to increase uterine contractile force and duration significantly. Conversely, overexpression of Kir7.1 inhibited uterine contractility. Finally, we demonstrate that the Kir7.1 inhibitor VU590 as well as novel derivative compounds induces profound, long-lasting contractions in mouse and human myometrium; the activity of these inhibitors exceeds that of other uterotonic drugs. We conclude Kir7.1 regulates the transition from quiescence to contractions in the pregnant uterus and may be a target for therapies to control uterine contractility

Small and intermediate conductance Ca2+-activated K+ channels confer distinctive patterns of distribution in human tissues and differential cellular localisation in the colon and corpus cavernosum

The SK/IK family of small and intermediate conductance calcium-activated potassium channels contains four members, SK1, SK2, SK3 and IK1, and is important for the regulation of a variety of neuronal and non-neuronal functions. In this study we have analysed the distribution of these channels in human tissues and their cellular localisation in samples of colon and corpus cavernosum. SKI mRNA was detected almost exclusively in neuronal tissues. SK2 mRNA distribution was restricted but more widespread than SKI, and was detected in adrenal gland, brain, prostate, bladder, liver and heart. SK3 mRNA was detected in almost every tissue examined. It was highly expressed in brain and in smooth muscle-rich tissues including the clitoris and the corpus cavernosum, and expression in the corpus cavernosum was upregulated up to 5-fold in patients undergoing sex-change operations. IK1 mRNA was present in surface-rich, secretory and inflammatory cell-rich tissues, highest in the trachea, prostate, placenta and salivary glands. In detailed immunohistochemical studies of the colon and the corpus cavernosum, SKI-like immunoreactivity was observed in the enteric neurons. SK3-like immunoreactivity was observed strongly in smooth muscle and vascular endothelium. IK1-like immunoreactivity was mainly observed in inflammatory cells and enteric neurons of the colon, but absent in corpus cavernosum. These distinctive patterns of distribution suggest that these channels are likely to have different biological functions and could be specifically targeted for a number of human diseases, such as irritable bowel syndrome, hypertension and erectile dysfunctio

Quantification of total and surface-associated hERG protein in cells maintained at lower temperatures

<p><b>Copyright information:</b></p><p>Taken from "Improved functional expression of recombinant human ether-a-go-go (hERG) Kchannels by cultivation at reduced temperature"</p><p>http://www.biomedcentral.com/1472-6750/7/93</p><p>BMC Biotechnology 2007;7():93-93.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241608.</p><p></p> Cells were cultured for 3 d and analyzed by flow cytometry. Data were averaged from 6 samples, each of 5000 cells (un-gated), and normalized against CHO hERG at 37°C. Normalized fluorescence intensity of fixed and permeablized CHO hERG (open bars) and untransfected control CHO cells (filled bars) at the respective temperatures stained with antibody C20 (which is raised against a C-terminal peptide sequence; A). Representative population fluorescence plots for CHO hERG cells from 37, 30 and 27°C in the same experiment as A (B). Normalized surface fluorescence intensity of non-fixed and non-permeablised CHO hERG (open bars) and control cells (filled bars) maintained at the respective temperatures stained with antibody 2110 (which is raised against a peptide between TM1 and TM2 which is predicted to be located on the surface of the plasma membrane; C)

Comparison of hERG activity measured by patch clamp and H-dofetilide binding in cells maintained at different growth temperatures

<p><b>Copyright information:</b></p><p>Taken from "Improved functional expression of recombinant human ether-a-go-go (hERG) Kchannels by cultivation at reduced temperature"</p><p>http://www.biomedcentral.com/1472-6750/7/93</p><p>BMC Biotechnology 2007;7():93-93.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241608.</p><p></p> CHO hERG cells grown at 37°C were split and kept subconfluent at 37, 30 or 27°C for 3 d (A,B). HEK293 hERG was split and kept at 37 or 30°C for 24 h (C). Mean tail currents recorded by patch clamping (IonWorksHT) from 4 independent experiments, 32–64 cells were patched for each data point in every experiment (A). H-dofetilide binding activity for CHO hERG and HEK hERG membrane preps respectively (B,C). Each data point was from 2 independent experiments, each of 3 repeats. Background (non-specific) binding was measured in the presence of an excess amount of non-radioactive dofetilide. Specific binding (open bars) was calculated as the total counts minus background counts and the total: background ratio (diamonds) was calculated as the total counts divided by the background counts

Ultrastructural characteristics of untransfected control and hERG transfected CHO cells

<p><b>Copyright information:</b></p><p>Taken from "Improved functional expression of recombinant human ether-a-go-go (hERG) Kchannels by cultivation at reduced temperature"</p><p>http://www.biomedcentral.com/1472-6750/7/93</p><p>BMC Biotechnology 2007;7():93-93.</p><p>Published online 20 Dec 2007</p><p>PMCID:PMC2241608.</p><p></p> CHO hERG or untransfected cells from 37°C were split and kept subconfluent for 3 d at 30°C or 27°C. At 30°C, hERG transfected CHO cells showed numerous large vesicle/vacuole-like structures (A,B), which were fewer in number in untransfected 30°C CHO cells (B). At 27°C, the number of vesicle/vacuole-like structures were reduced compared to the 30°C cells (C,D /A,B). Immunolocalized hERG + 5 and 10 nm colloidal gold secondary antibody was present on vesicle/vacuole-type membrane (E,F, respectively). hERG + 10 nm colloidal gold secondary labelling was present adjacent to golgi (F). cm, cell membrane; gl, golgi; nu, nucleus. Bar, A-D,5 μm. E,F, 250 nm. E inset, 100 nm