Ion channels and their role in cell proliferration of 3T3-L1 preadipocytes

Abstract

BACKGROUND: Mouse 3T3-L1 peradipocytes are widely used for metabolic study; however, cellular physiology (e.g. functional ion channel expression) is not fully understood. The present study was to investigate ion channel expression and functional role of them in regulating cell proliferation using whole cell patch voltage clamp technique, RT-PCR, Western blot, and cell proliferation assay in undifferentiated 3T3-L1 preadipocytes. RESULTS: We found that three types of ionic currents were present in 3T3-L1 preadipocytes, including a Ca2+-activated K+ current (IKCa) in 39% cells, an inwardly-rectifying K+ current (IKir) in 15% cells, and a chloride current (ICl) only in 8% cells under isotonic conditions. Interestingly, ICl was observed in all cells with hypotonic (0.8T) insult, suggesting that it is a volumesensitive ICl (ICl.vol). IKir was inhibited by Ba2+, and IKCa was inhibited by the intermediate conductance IKCa channel blocker clotrimazole. ICl was reduced by the chloride channel blockers DIDS. RT-PCR revealed significant expression of mRNAs: KCa3.1 for IKCa, Kir2.1 for IKir, and Clcn3 for ICl.vol. Proteins of these channels were detected using Western blot analysis. Proliferation assay demonstrated that blockade of IKCa with clotrimazole or ICl.vol with DIDS inhibited cell proliferation in a concentration-dependent manner. Flowcytometry analysis showed that clotrimazole (3 μM) and DIDS (200 μM) accumulated the cells at G0/G1 phase (from control 49.91±2.8% to 57.05±3.6% for clotrimazole, P<0.05; to 61.08±4.3% for DIDS, P<0.05). CONCLUSIONS: These results demonstrate the first information that three types of functional ion channel currents, including intermediate-conductance IKCa, ICl.vol, and IKir, are heterogeneously present in 3T3-L1 preadipocytes. IKCa and ICl.vol participate in the regulation of cell proliferation.