Improved Functional Expression of Human Cardiac Kv1.5 Channels and Trafficking-Defective Mutants by Low Temperature Treatment

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.

The function of SUMOylation and its crucial roles in the development of neurological diseases

“…The Kv1.5 channel encoded by the KCNA5 gene is a subtype of voltage‐gated potassium channels that is widely expressed in a variety of cells 144‐147 . The Kv1.5 channel is a unique ion channel expressed in cardiomyocytes 145,148 . The physiological function of Kv1.5 is not limited to the maintenance of myocardial cell membrane potential, but is also related to multiple physiological processes, such as cell proliferation and apoptosis.…”

Interaction of propofol with voltage-gated human Kv1.5 channel through specific amino acids within the pore region

“…The hKv1.5 mutants (H463C, T480A, I502A, I508A, L510A and V516A) were constructed using polymerase chain reaction (PCR)-based site-directed mutagenesis with QuikChange Kit (Stratagene, La Jolla, CA, USA), as previously described (Ding et al, 2014;Wu et al, 2009). All PCR products were fully sequenced (ABI3100x/, Applied Biosystems, Foster City, CA, USA) to confirm the presence of appropriate mutations.…”

“…Electrophysiological recordings and data analysis 5 Whole-cell membrane currents (Hamill et al, 1981) were recorded from green fluorescent protein (GFP)-positive cells 48 h after transfection, using an EPC-8 patch-clamp amplifier (HEKA Elektronik, Lambrecht, Germany). The recording technique used in this study was essentially the same as that described previously (Ding et al, 2014;Wu et al, 2009). The firepolished patch electrodes were fabricated from 1.5-mm capillary glass (Narishige Scientific Instrument Lab., Tokyo, Japan) using a P-97 horizontal puller (Sutter Instrument, Novato, CA, USA).…”

Studies of Conorfamide-Sr3 on Human Voltage-Gated Kv1 Potassium Channel Subtypes

“…Therefore, we decided to examine the effect of CNF-Sr3 on four members of the human Shaker -related subfamily, the Kv1.3, Kv1.4, Kv1.5, and Kv1.6 channels, due to their functional implications in human diseases, particularly for Kv1.3 [ 20 ]. Since CNF-Sr3 has a Kd = 2.7 µM for the Shaker channel and there is evidence that some conotoxins, like κ−PVIIA, bind to Shaker but not to Kv1 channels [ 18 , 19 ], we decided to test CNF-Sr3 at higher final concentrations from 10 to 30 µM. At 10 µM, CNF-Sr3 was capable of abolishing up to 20% and 80% of the current for Kv1.…”

“…To mention just a few examples, changes in Kv1.1 channel may cause episodic ataxia type 1 [ 14 ], whereas the Kv1.3 channel has been associated with multiple sclerosis, type-1 diabetes, and rheumatoid arthritis, but relatively little is known about Kv1.2, Kv1.4, Kv1.5, Kv1.6, Kv1.7, or Kv1.8 channels in human disorders [ 15 ]. However, Martel et al, by electrochemical recordings at rat dorsal striatum, confirmed a role for Kv1.2 and Kv1.6 in dopamine release [ 16 ] and other authors have shown that Kv1.4 and Kv1.5 are related to cardiac effects [ 17 , 18 , 19 ].…”