Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC109
Modulation of HEK293 proliferation by Kv1.3 channels expression
L. Jiménez-Pérez1,2, D. García-Arribas1,2, E. Miguel-Velado1,2, E. Alonso1,2, J. López-López1,2, M. Pérez-García1,2, P. Cidad1,2
1. Physiology, Universidad de Valladolid, Valladolid, Spain. 2. Instituto de Biolog
The plasticity of vascular smooth muscle cells (VSMCs), that can switch from a contractile to a proliferative phenotype, associates with several cardiovascular diseases. This phenotypic switch involves changes in the expression of membrane receptors and ion channels. We have previously reported that in mouse femoral VSMCs, upregulation of Kv1.3 channels associates with increased proliferation. Furthermore, this pro-proliferative effect of Kv1.3 may be a conserved phenomenon among vascular beds from different species, as Kv1.3 blockade with specific drugs has antiproliferative effects. To explore the mechanism linking Kv1.3 expression to cell proliferation, we examined whether heterologous overexpression of Kv1.3 channels also modulates proliferation of human embryonic kidney cells (HEK293). The complete coding sequence of Kv1.3 was subcloned in different vectors, to obtain Kv1.3 expression associated to reporter genes (dsRed-IRES-Kv1.3) or Kv1.3 fusion proteins (pEGFP-N3-Kv1.3 and Kv1.3-N3-myc). Confocal microscopy images of transfected cells demonstrated the plasma membrane location of the fusion proteins. The effects of Kv1.3 on proliferation were characterized by means EdU incorporation and cell-counting assays in mock-transfected, control cells (transfected with the empty vector) and Kv1.3-transfected cells. Kv1.3 overexpression lead to a significant increase in HEK293 proliferation between 24 and 48 hours after transfection, as a consequence of a decrease of the duplication time of the cell cultures (from 15 hours in control to 10 hours in Kv1.3 expressing cells). Moreover, the effect was inhibited by specific blockade of Kv1.3. Since our previous studies in native VSMCs showed no correlation between changes in membrane potential and cell proliferation1, we generated mutant constructs of Kv1.3 in which either permeation (pEGFP-N3-Kv1.3-W389F) or gating and permeation (pEGFP-N3-Kv1.3-AYA) were removed. Patch-clamp studies showed that both constructs expressed non-conducting Kv1.3 channels. In addition, Kv1.3-AYA failed to produce detectable gating currents, while Kv1.3-W389F showed gating currents similar to wild type channels. Proliferation studies in these two mutants showed that Kv1.3-AYA expression did not affect proliferation rate, but Kv1.3-W389F had the same effect on proliferation than the wild type Kv1.3. Altogether, these results prove that the effects of Kv1.3 on VSMC proliferation can be reproduced in our heterologous system. Moreover, our data suggest that Kv1.3 may be a moonlighting protein that could regulate intracellular signalling pathways leading to cell proliferation by a mechanism that is independent of potassium flux.
Where applicable, experiments conform with Society ethical requirements