Abstract 2312: α1G T-Type Calcium Channels in Mouse Preadipocytes: A Role on Cell Proliferation
Background- 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 CaV remained unexplored in preadipocytes.
Methods and Results- We performed the whole-cell voltage clamp technique using synthetic small interfering RNA (siRNA), conventional/quantitative real-time RT-PCR analysis, Western blots, and immunohistochemical analysis in mouse 3T3-L1 preadipocytes and a primary culture of mouse preadipocytes. The effects of CaV blockers on cell proliferation and cell cycle were also investigated. Whole cell recordings of 3T3-L1 preadipocyte showed low-threshold CaV, which could be inhibited by mibefradil, Ni2+ (IC50 of 200μM) and NNC55– 0396. The dominant expression of α1G mRNA was detected among CaV transcripts (α1A-α1I), supported by the expression of CaV3.1 protein encoded by α1G gene, using immunohistochemical studies and Western blots. siRNA targeted for α1G markedly inhibited CaV. Dominant expression of α1G mRNA and expression of CaV3.1 protein were also observed in a primary culture of mouse preadipocytes. NNC 55– 0396, but not diltiazem, inhibited proliferation, by arresting G0/G1 in cell cycles. Expression level of α1G mRNA and CaV3.1 protein significantly decreased in the well-differentiated adipocytes, and α1G mRNA expression level in inguinal white adipose tissues obtained from obese ob/ob as well as C57BL/6J and lean ob/+ mice was also much less than that of preadipocytes.
Conclusions- The present study demonstrates that the T-type Ca2+ channel encoded by α1G subtype is the dominant CaV in mouse preadipocytes, and may play a role in regulating preadipocyte proliferation.