Involvement of CaV3.1 T-type calcium channels in cell proliferation in mouse preadipocytes

Abstract

Voltage-gated Ca2+channels (CaV) are ubiquitously expressed in various cell types and play vital roles in regulation of cellular functions including proliferation. However, the molecular identities and function of CaVremained unexplored in preadipocytes. Therefore, whole cell voltage-clamp technique, conventional/quantitative real-time RT-PCR, Western blot, small interfering RNA (siRNA) experiments, and immunohistochemical analysis were applied in mouse primary cultured preadipocytes as well as mouse 3T3-L1 preadipocytes. The effects of CaVblockers on cell proliferation and cell cycle were also investigated. Whole cell recordings of 3T3-L1 preadipocytes showed low-threshold CaV, which could be inhibited by mibefradil, Ni2+(IC50of 200 μM), and NNC55-0396. Dominant expression of α1GmRNA was detected among CaVtranscripts (α1A–α1I), supported by expression of CaV3.1 protein encoded by α1Ggene, with immunohistochemical studies and Western blot analysis. siRNA targeted for α1Gmarkedly inhibited CaV. Dominant expression of α1GmRNA and expression of CaV3.1 protein were also observed in mouse primary cultured preadipocytes. Expression level of α1GmRNA and CaV3.1 protein significantly decreased in differentiated adipocytes. Mibefradil, NNC55-0396, a selective T-type CaVblocker, but not diltiazem, inhibited cell proliferation in response to serum. NNC55-0396 and siRNA targeted for α1Galso prevented cell cycle entry/progression. The present study demonstrates that the CaV3.1 T-type Ca2+channel encoded by α1Gsubtype is the dominant CaVin mouse preadipocytes and may play a role in regulating preadipocyte proliferation, a key step in adipose tissue development.