Mock-transfected cells underwent the same transfection procedure except no plasmid DNA was added to the transfection mixture or cells were transfected with an empty vector. == Plasmids and construction. not necessary for CCI-006 hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct conversation with ERK1/2 and JNK signaling pathways. Keywords:cell proliferation, intermediate conductance calcium-activated potassium channel, Ca2+influx, mitogen-activated protein kinase cell proliferation is an importantphysiological process for embryonic development, wound healing, and the routine replacement of old cells with new cells as required. Cell proliferation in eukaryotes is a complex process that is controlled by extracellular signals or mitogens, such as growth factors, as well as the state of differentiation of the cell and involves the interaction of numerous regulatory proteins and signaling cascades. Proliferation is tightly regulated by the cell cycle. However, under certain conditions, cells can abnormally proliferate and this underlies the pathophysiology of atherosclerosis, angiogenesis, and tumor growth. Potassium channels have been implicated in proliferation since 1984, when Decoursey and Cahalan demonstrated that voltage-gated K+channels mediate proliferation in T lymphocytes (5). Subsequently, a plethora of K+channels, including the intermediate-conductance calcium-activated potassium (IK1) channel, have been linked to proliferation in various cell types. IK1 channels are widely expressed and have been implicated in proliferation in a variety of nonexcitable tissues, endothelial cells, T lymphocytes, mesenchymal stem cells, vascular smooth muscle cells, and various types of cancer cells (12,13,15,21,23,29,30,3436). The current hypothesis for K+channel-mediated proliferation is that activation of K+channels causes cell hyperpolarization which increases the driving force for Ca2+entry, usually through store-operated Ca2+channels. This leads to enhanced activation of Ca2+-dependent signaling pathways and thus ultimately increases cell proliferation. However, such experiments do not link K+channel activity, Ca2+influx, and Mouse monoclonal to CD31 proliferation per se. This proposed connection has not been rigorously tested in a system where changes in membrane potential, Ca2+influx, and proliferation can be controlled and examined independently. In fact, recent evidence suggests that, in addition to their ability to pass ions across the membrane, ion channels can also have nonconducting functions that enable them to interact with cell signaling pathways to directly regulate biochemical events (18). One such example is the ability of theether–go-go(EAG) K+channel to regulate cell proliferation in fibroblasts via activation of the p38 mitogen-activated protein kinase (MAPK) pathway (14). This study investigated the role of human IK channels (hIK1) in cell proliferation. Many cells that require expression of IK for proliferation, such as endothelial cells or T lymphocytes, express multiple types of ion channels, including a variety of K+channels. Therefore, it would be easier to first dissect the role of hIK1 by means of overexpression of recombinant channels in a heterologous expression system before assessing the role in acutely isolated or primary cultured cells. We have devised a powerful molecular manipulation strategy, using mutant hIK1 channels that either cannot conduct K+ions or cannot traffic to the plasma membrane. This strategy was used to examine the link between K+channel function, Ca2+entry, and cell proliferation. == MATERIALS AND METHODS == == == == Cell culture and transfection of human embryonic CCI-006 kidney CCI-006 293 cells. == Untransfected human embryonic kidney 293 (HEK293) cells and HEK293 cells stably expressing hIK1 channels (HEK293 hIK1 cells) were cultured in minimum essential medium containing Earle’s salts andl-glutamine (Gibco), supplemented with 10% fetal bovine serum (Gibco), 1% nonessential amino acids (Gibco), and 1% antibiotic/antimycotic (Gibco). Selection for HEK293 hIK1 was maintained with 600 g/ml G418 disulfate (Sigma). Transient transfections of HEK293 cells with hemagglutinin (HA)-tagged HA-hIK1 (28), HA-hIK1GYG/AAA(a hIK1 pore mutant), HA-hIK1L18A/L25A[a hIK1 trafficking mutant (17)], or untagged voltage-gated sodium channel Nav1.5 were performed using ExGen 500 in vitro transfection reagent (Fermentas) according to the manufacturer’s instructions. Cells were cotransfected with monomeric red fluorescent protein (mRFP) or green fluorescent protein (GFP) to aid detection of transfected cells for electrophysiology and Ca2+imaging experiments. Mock-transfected cells underwent the same transfection procedure except no plasmid DNA was added to the transfection mixture or cells were transfected with an empty vector. == Plasmids and construction. == All hIK1 constructs contained a HA tagYPYDVPDYAinserted into the second.
