The improved effect of hypothermic cultivation on thefunctional expression of human cardiac Kv1.5 channels

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We herein investigated the effect of low temperature exposure in the channel activity, expression, degradation and localization of human Kv1.5 (hKv1.5). In hKv1.5-expressing CHO cells, the currents significantly increased at a reduced temperature cultivation (28℃) in comparison to those observed at 37℃. A Western blot analysis confirmed that the protein levels (immature and mature proteins) of hKv1.5 were significantly elevated under hypothermic conditions. The treatment with a proteasome inhibitor, MG132, significantly increased the immature (but not the mature) hKv1.5 protein at 37℃; there were no changes in either the immature or mature hKv1.5 proteins at low temperature conditions after MG132 exposure, thus indicating 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 HEK cells increased significantly on the cell surface at 28℃ versus those cultured at 37℃. Importantly, the low temperature treatment markedly shifted the subcellular distribution of the mature hKv1.5 lighter fractions, which showed considerable overlap with the trans-Golgi component. Finally, the hypothermic treatment also rescued the protein expression and functional currents of trafficking-defective hKv1.5 mutants (I502A, I508A, located in the pore region). These results indicate that low temperature exposure stabilizes the protein in the cellular organs, modulates its recycling trafficking, thus enhancing functional hKv1.5 currents.