Functional ion channels and cell proliferation in 3T3-L1 preadipocytes

Abstract

Mouse 3T3-L1 preadipocytes are widely used for metabolic study of obesity; however, their cellular physiology is not fully understood. The present study investigates functional ion channels and their role in the regulation of cell proliferation using whole-cell patch voltage-clamp, RT-PCR, Western blot, and cell proliferation assay in undifferentiated 3T3-L1 preadipocytes. We found three types of ionic currents present in 3T3-L1 preadipocytes, including an inwardly-rectifying K + current (I Kir, recorded in 15% of cells) inhibited by Ba 2+, a Ca 2+-activated intermediate K + current (IK Ca, recorded in 44% of cells) inhibited by clotrimazole (or TRAM-34) as well as a chloride current (I Cl) inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in 12% of cells, which can be activated in all cells with hypotonic (0.8T) insult, implicating a volume-sensitive I Cl (I Cl.vol). RT-PCR and Western blot analysis revealed the expression of KCa3.1 (for IK Ca), Kir2.1 (for I Kir), and Clcn3 (for I Cl.vol). Blockade of IK Ca with TRAM-34 or I Cl.vol with DIDS inhibited cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific siRNAs also suppressed cell proliferation. Flow cytometry analysis showed that blockade or silencing of KCa3.1 or Clcn3 channels with corresponding blockers or siRNAs caused an accumulation of cells at the G0/G1 phase. These results demonstrate that three functional ion channel currents, I KCa, I Cl.vol, and I Kir, are heterogeneously present in 3T3-L1 preadipocytes. I KCa and I Cl.vol participate in the regulation of cell proliferation. © 2011 Wiley Periodicals, Inc.