Activation Of Cloned BK_Ca Channels In Angâ…¡-induced Proliferation Of HEK293 Cells

Large-conductance Ca2+-activated K+ channels (BKCa) are synergistically activated by elevation of the intracellular Ca2+ concentration and membrane depolarization. BKCa channels, which are expressed in most mammalian cells, are involved in the regulation of a variety of physiological functions. BKCa channel is formed by a tetramer of the pore-forming ?-subunits and distinct accessory ?-subunits (?1–?4) which contribute to BKCa channel molecular diversity. The different accessory ?-subunits may be responsible for the tissue-specific characteristics of BKCa channels. For example, ?1-subunit is highly expressed in vascular smooth muscle cells (VSMCs). BKCa?+?1, which are a persistent feature of VSMCs, are considered key regulator in maintaining the membrane potential and normal vascular tone. In addition, a growing number of evidences have demonstrated that BKCa channels play an essential role in the regulation of cell proliferation, particularly in tumor cells. However, whether BKCa channels are involved in AngⅡ-induced cell proliferation and the underlying mechanisms remain to be determined. As we all know, various ion channels including BKCa, CaL and KV, present in arterial VSMCs and the interactions among them are complicated. The Human embryonic kidney cells line (HEK293 cells) has been extensively used as an expression tool for recombinant proteins and ion channels for there are few endogenous ion channels in HEK293 cells. In view of these facts, the present study was designed to establish an expression system that reliably coexpresses BKCa?+?1 subunits in HEK293 cells and to investigate the regulatory role of BKCa channels in AngⅡ-induced cell proliferation.METHODS:1.1 The recombinant plasmid of pIRES-hSlo?+?1/pIRES-hSlo? (with hSlo?+?1 or hSlo? cDNA in the expression vector pIRES) was transformed for replication, purified and identified by restriction digest.1.2 Using Lipofectamine 2000, the recombinant plasmid was transiently transfected into HEK293 cells together with pEGFP plasmid that expresses green fluorescent protein (GFP) as a marker for transfection. GFP expression was detected by fluorescence microscope.1.3 To evaluate the expression of BKCa ?1-subunit, transfected HEK293 cells were immunostained using a rabbit polyclonal antibody raised against BKCa ?1-subunit of human origin. In Western blotting experiment, the protein expressions of BKCa ?- and ?1-subunit were detected with their specific antibodies.1.4 The single-channel kinetics and pharmacological properties of expressed BKCa ?+?1 subunits were investigated by patch clamp technique.2.1 The concentration-dependent and time-dependent cuve of AngⅡ-induced cell proliferation were tested by MTT colorimetry.2.2 The effect of AngⅡ, IbTX, AngⅡ+IbTX, AngⅡ+TEA and NS1619 on the proliferating cell nuclear antigen (PCNA) expression of transfected cells were detected by immunocytochemistry.2.3 The effect of AngⅡ, IbTX, AngⅡ+IbTX, AngⅡ+TEA and NS1619 on cell cycle of transfected cells were detected by flow cytometry (FCM).RESULTS1.1 The sizes of DNA fragments with double-digestion in gel were structurally consistent with their theoretical sizes, which indicated both hSlo? and hSlo?1 cDNA were not lost and stably encoded in the same plasmid by propagation. With successful transfection, the HEK293 cells demonstrated moderate reporter protein (GFP) fluorescence and characteristic morphology and size. Immunolocalization of ?1-subunit indicated well-distributed expression of BKCa?1-subunit on the cell membrane. BKCa ?- and ?1-subunit protein expression were positive in HEK-hSlo?+?1 cells.1.2 The expressed BKCa?+?1 subunits demonstrated to be fully functional for its typical single-channel traces, Ca2+-sensitivity, voltage-dependency, high conductance (151?7 pS) and its pharmacological activation by NS1619 and inhibition by IbTX.2.1 AngⅡinduced proliferation of transfected HEK293-hSlo?+?1/ HEK293-hSlo? cells in a concentration-dependent and time-dependent manner. PCNA expression of transfected cells was enhanced by AngⅡ(100 nmol/L), however, the proliferation effect induced by AngⅡcan be inhibited by IbTX, a selective BKCa channel blocker and TEA.2.3 The percentage of S phase HEK293-hSlo?+?1/ HEK293-hSlo? cells was increased after AngⅡtreatment, however, the effect induced by AngⅡon cell cycle distribution was inhibited by IbTX and TEA. The mechanism by which IbTX inhibit AngⅡ-induced proliferation of transfected cells may involve hold-up of cell mitosis by inhibiting transient from G1 phase to S phase and decreasing the percentage of G2 phase cells.CONCLUSION:Using Lipofectamine, the plasmids pIRES-hSlo?+?1/pIRES-hSlo? was transiently transfected into HEK293 cells. Through immunolocalization, western blotting and electrophysiological method, we successfully established a reliable expression system coexpressing the human BKCa ?-subunit in combination with ?1-subunit in HEK293 cells. For the first time, our results suggested that BKCa channels play an potential role in mediating AngⅡ-induced proliferation of HEK293 cells. These works are supposed to contribute greatly to our further investigation of BKCa channels'role in AngⅡ-induced proliferation of VSMCs.