Abstract
We herein investigated the effect of low temperature exposure on the expression, degradation, localization and activity of human Kv1.5 (hKv1.5). In hKv1.5-expressing CHO cells, the currents were significantly increased when cultured at a reduced temperature (28°C) compared to those observed at 37°C. Western blot analysis indicated that the protein levels (both immature and mature proteins) of hKv1.5 were significantly elevated under the hypothermic condition. Treatment with a proteasome inhibitor, MG132, significantly increased the immature, but not the mature, hKv1.5 protein at 37°C, however, there were no changes in either the immature or mature hKv1.5 proteins at low temperature following MG132 exposure. These observations suggest that the enhancement of the mature hKv1.5 protein at reduced temperature may not result from the inhibition of proteolysis. Moreover, the hKv1.5 fluorescence signal in the cells increased significantly on the cell surface at 28°C versus those cultured at 37°C. Importantly, the low temperature treatment markedly shifted the subcellular distribution of the mature hKv1.5, which showed considerable overlap with the trans-Golgi component. Experiments using tunicamycin, an inhibitor of N-glycosylation, indicated that the N-glycosylation of hKv1.5 is more effective at 28°C than at 37°C. Finally, the hypothermic treatment also rescued the protein expression and currents of trafficking-defective hKv1.5 mutants. These results indicate that low temperature exposure stabilizes the protein in the cellular organelles or on the plasma membrane, and modulates its maturation and trafficking, thus enhancing the currents of hKv1.5 and its trafficking defect mutants.
Highlights
Previous studies showed that Kv1.5 underlies the cardiac ultrarapid delayed rectifier potassium current (IKur) in humans [1,2]
The mutant N470D of hERG is trafficking-deficient in cells cultured at physiological temperature, whereas the channel protein transports to the plasma membrane to produce functional channels at reduced temperature [21]
Some drugs such as thapsigargin and hERG channel inhibitor E-4031 are reported to promote the functional expression of trafficking-deficient hERG channels [19], suggesting that the common mechanism of rescue effects by low temperature may be utilized in the future to understand the regulatory pathways by which the clinical drugs modulate the trafficking process of the ion channels
Summary
Previous studies showed that Kv1.5 underlies the cardiac ultrarapid delayed rectifier potassium current (IKur) in humans [1,2]. Kv1.5 channels are expressed in many other organs, such as pulmonary arteries, brain, skeletal muscle, and have crucial function in cell cycle regulation [3]. Rapid activation of IKur current following the AP upstroke may reduce Ca2+ influx via L-type Ca2+ channels (ICa,L) and result in less positive plateau phase [6]. Blockade of IKur may facilitate ICa,L activation following a pronounced spike-and-dome configuration and enhance intracellular Ca2+ influx [7]. The normal expression or channel activity of Kv1.5 is important in the regulation of the proper electrical properties of cardiac atrial myocytes. Basic studies have demonstrated that the contribution Kv1.5 can be modified through alterations in the surface density of the channel protein in addition to that of the more classical pore block by drugs [10,11]
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