Generation and proliferation assessment of HEK293T cells stably expressing Kv1.5 channel with and without regulatory subunits β1.1 or β1.2

Abstract

Potassium channels, including voltage-gated potassium (Kv) channels, play an important role in regulation of vascular smooth muscle cell (VSMC) contraction and proliferation. Our previous studies indicate that KV1.5 is a major type of shaker-family KV1 channels expressed in human coronary and adipose arterioles. The function of KV1.5 channels is impaired in disease, contributing to reduced KV1.5-dependent VSMC relaxation and leading to vascular dysfunction in subjects with coronary artery disease (CAD). However, it remains largely unknown whether KV1.5, alone and with its regulatory subunits (β1.1 and β1.2), also regulates VSMC proliferation in CAD. This study was designed to examine if expression of KV1.5 with and without β1.1 or β1.2) modulates cell proliferation in cultured HEK293T cells. We created a Kv1.5 cell line with lentiviral transduction at a multiplicity of infection (MOI) of 20. We used this cell line to introduce β1.1 subunit with the same protocol (MOI=20). However, when we attempted to introduce β1.2 subunit to Kv1.5 cell line, transduction efficiency was only 40% prompting us to repeat the experiment with MOI of 40 to obtain transduction efficiency of over 90%. These results suggest that the β1.2 subunit may be responsible for inhibiting cell proliferation. To test our hypothesis, we took images of transduced cultured cells every 24 hours from 24 to 96 hours and analyzed them with ImageJ software. Each cell was outlined to indicate the surface area taken up by the cells as compared to total surface area of the image for estimation of the overall cell density/confluency. ImageJ analysis revealed that the lowest density was observed in Kv1.5+ β1.2 cell line (25±3% at 48 hours, n=15 images from 6 independent experiments P<0.001) as compared with densities of Kv1.5 (72±3%, n=18); Kv1.5+ β1.1 (75±1%, n=15); and GFP (65±2%, n=9). Kv1.5+ β1.2 consistently exhibited the lowest confluency at all time points (24, 72, and 96 hours). Next, we performed cell counting with a trypan blue solution using a hemocytometer to assess the cell density of the entire sample at 48 hours. After we converted the number of cells into percentages relative to GFP control, our data indicated that Kv1.5 was at 97±1%, n=6; Kv1.5+ β1.1 at 96±1%, n=6; and Kv1.5+ β1.2 was at 41±0.5%, n=6 (P<0.001). In conclusion, our results indicate that, expression of Kv1.5 channel + β1.2 regulatory subunit, but not KV1.5 alone or with β1.1 subunit, inhibits cell proliferation in heterologous expression cell systems. Future studies will identify the role and mechanism of KV1.5 and its β1.2 subunit in VSMC proliferation in CAD or other vascular diseases.