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

Mammal responses to global changes in human activity vary by trophic group and landscape

Wildlife must adapt to human presence to survive in the Anthropocene, so it is critical to understand species responses to humans in different contexts. We used camera trapping as a lens to view mammal responses to changes in human activity during the COVID-19 pandemic. Across 163 species sampled in 102 projects around the world, changes in the amount and timing of animal activity varied widely. Under higher human activity, mammals were less active in undeveloped areas but unexpectedly more active in developed areas while exhibiting greater nocturnality. Carnivores were most sensitive, showing the strongest decreases in activity and greatest increases in nocturnality. Wildlife managers must consider how habituation and uneven sensitivity across species may cause fundamental differences in human–wildlife interactions along gradients of human influence.Peer reviewe

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)

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

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