Regulation of cell proliferation by intermediate-conductance Ca2+-activated potassium and volume-sensitive chloride channels in mouse mesenchymal stem cells

Abstract

Bone marrow mesenchymal stem cells (MSCs) are a promising cell source for regenerative medicine; however, their cellular physiology is not fully understood. The present study aimed at exploring the potential roles of the two dominant functional ion channels, intermediate-conductance Ca²⁺-activated potassium (IK_Ca) and volume-sensitive chloride ( I_Cl.vol) channels, in regulating proliferation of mouse MSCs. We found that inhibition of IK_Ca with clotrimazole and I_Cl.vol with 5-nitro-1-(3-phenylpropylamino) benzoic acid (NPPB) reduced cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific short interference (si)RNAs significantly reduced IK_Ca or I_Cl.vol density and channel protein and produced a remarkable suppression of cell proliferation (by 24.4 ± 9.6% and 29.5 ± 7.2%, respectively, P < 0.05 vs. controls). Flow cytometry analysis showed that mouse MSCs retained at G₀/G₁ phase (control: 51.65 ± 3.43%) by inhibiting IK_Ca or I_Cl.vol using clotrimazole (2 μM: 64.45 ± 2.20%, P < 0.05) or NPPB (200 μM: 82.89 ± 2.49%, P < 0.05) or the specific siRNAs, meanwhile distribution of cells in S phase was decreased. Western blot analysis revealed a reduced expression of the cell cycle regulatory proteins cyclin D1 and cyclin E. Collectively, our results have demonstrated that IK_Ca and I_Cl.vol channels regulate cell cycle progression and proliferation of mouse MSCs by modulating cyclin D1 and cyclin E expression.