Intermediate-conductance Ca 2+-activated potassium and volume-sensitive chloride channels in endothelial progenitor cells from rat bone marrow mononuclear cells

摘要

Aim: Bone marrow endothelial progenitor cells (BMEPCs) are believed to be a promising cell source for regenerative medicine; however, their electrophysiology properties have not been fully clarified, which is important to the clinical application of BMEPCs. The current study was designed to determine the transmembrane ion currents and mRNA expression levels of related ion channel subunits in rat BMEPCs. Methods: Bone marrow mononuclear cells were isolated by density gradient separation and cultured in EPC medium. The transmembrane ion currents were determined using whole-cell patch-voltage clamp technique, and the levels of mRNA and protein expressions of functional ionic channels were measured using RT-PCR and western immunoblot analysis. Results: We observed two types of ionic currents in undifferentiated rat BMEPCs. One was Ca 2+-activated potassium current (I kca), which was seen in approx. 90% of cells when 1 μm Ca 2+ was employed in pipette solution, and it was predominantly inhibited by intermediate-conductance I kca inhibitor clotrimazole. The other one was volume-sensitive chloride current (I cl), which was detected in 85.7% of cells when BMEPCs were subjected to K +-free hypotonic extracellular solution, whose currents could be inhibited by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). The corresponding ion channel genes and proteins, KCNN4 for I kca and Clcn3 for I cl, were confirmed by RT-PCR and western immunoblot analysis of BMEPCs. Conclusion: Our results demonstrated for the first time that rat BMEPCs expressed intermediate-conductance Ca 2+-activated potassium currents and volume-sensitive chloride currents, and corresponding genes and proteins of these two channels are KCNN4 and Clcn3 respectively.