The role of BK channel in cellular proliferation and differentiation in human osteoblast and osteoblast-like cells

Both excitable and non-excitable cells possess plasma membrane ion channels and evidence has accumulated over the last 30 or so years that these channels perhaps play key roles in the cell life and death. This Thesis investigated the characteristics and putative functions of one class of potassium channel, the BK channel in osteoblast-like cells and primary osteoblasts from human, rat and mouse. The properties and functions were defined in vitro using a combination of patch-clamp, reverse transcription-polymerase chain reaction (RT-PCR) and functional assays for cell growth and mineralisation. RT-PCR showed the presence of KCNMA1, KCNMB1, KCNMB2, KCNMB3 and KCNMB4, the gene for BK channel α, β1, β2, β3 and β4 subunits respectively. The channel was voltage-dependent with a mean unitary conductance of 315 pS in cell-attached patches, a conductance of 124 pS in excised outside-out and 151 pS in inside-out patches. The channel was blocked by TEA (0.3 mM), TBuA (1 mM), TPeA (1-10 μM), THeA (1-3 μM), tetrandrine (5-30 μM) and paxilline (10 μM) and was activated by isopimaric acid (20 μM). Notably iberiotoxin (IbTX) (90 nM) only blocked a proportion of the channels tested (2/5). Osteoblast-like MG63 cell number changed in response to BK channel modulators. It increased significantly with TEA and tetrandrine at low concentrations (1 mM, 3 μM respectively), and reduced at high concentrations (>10 mM, >10 μM respectively). It was not affected by IbTX (20-300 nM) or slotoxin (300 nM). The increase in cell number by TEA was blocked by isopimaric acid. In addition, TPeA and THeA caused a decrease of osteoblast-like SaOS2 cell mineralisation at the concentrations (3 and 0.3 μM, respectively) increased MG63 cell numbers. The BK channel has a distinctive pharmacology and represents a new target for therapeutic strategies in modulating osteoblast proliferation