Steven Hughes

Selective activation of mechanosensitive ion channels using magnetic particles

This study reports the preliminary development of a novel magnetic particle-based technique that permits the application of highly localized mechanical forces directly to specific regions of an ion-channel structure. We demonstrate that this approach can be used to directly and selectively activate a mechanosensitive ion channel of interest, namely TREK-1. It is shown that manipulation of particles targeted against the extended extracellular loop region of TREK-1 leads to changes in whole-cell currents consistent with changes in TREK-1 activity. Responses were absent when particles were coated with RGD (Arg–Gly–Asp) peptide or when magnetic fields were applied in the absence of magnetic particles. It is concluded that changes in whole-cell current are the result of direct force application to the extracellular loop region of TREK-1 and thus these results implicate this region of the channel structure in mechano-gating. It is hypothesized that the extended loop region of TREK-1 may act as a tension spring that acts to regulate sensitivity to mechanical forces, in a nature similar to that described for MscL. The development of a technique that permits the direct manipulation of mechanosensitive ion channels in real time without the need for pharmacological drugs has huge potential benefits not only for basic biological research of ion-channel gating mechanisms, but also potentially as a tool for the treatment of human diseases caused by ion-channel dysfunction.

Expression of the mechanosensitive 2PK+ channel TREK-1 in human osteoblasts.

TREK‐1 is a mechanosensitive member of the two‐pore domain potassium channel family (2PK+) that is also sensitive to lipids, free fatty acids (including arachidonic acid), temperature, intracellular pH, and a range of clinically relevant compounds including volatile anaesthetics. TREK‐1 is known to be expressed at high levels in excitable tissues, such as the nervous system, the heart and smooth muscle, where it is believed to play a prominent role in controlling resting cell membrane potential and electrical excitability. In this report, we use RT‐PCR, Western blotting and immunohistochemistry to confirm that human derived osteoblasts and MG63 cells express TREK‐1 mRNA and protein. In addition, we show gene expression of TREK2c and TRAAK channels. Furthermore, whole cell patch clamp electrophysiology demonstrates that these cells express a spontaneously active, outwardly rectifying potassium “background leak” current that shares many similarities to TREK‐1. The outward current is largely insensitive to TEA and Ba2+, and is sensitive to application of lysophosphatidylcholine (LPC). In addition, blocking TREK‐1 channel activity is shown to upregulate bone cell proliferation. It is concluded that human osteoblasts functionally express TREK‐1 and that these channels contribute, at least in part, to the resting membrane potential of human osteoblast cells. We hypothesise a possible role for TREK‐1 in mechanotransduction, leading to bone remodelling. J. Cell. Physiol.

The influence of static magnetic fields on mechanosensitive ion channel activity in artificial liposomes

The influence of static magnetic fields (SMFs) on the activity of recombinant mechanosensitive ion channels (the bacterial mechanosensitive ion channel of large conductance—MscL) following reconstitution into artificial liposomes has been investigated. Preliminary findings suggest that exposure to 80-mT SMFs does not induce spontaneous MscL activation in the absence of mechanical stimulation. However, SMFs do appear to influence the open probability and single channel kinetics of MscL exposed to negative pipette pressure. Typical responses include an overall reduction in channel activity or an increased likelihood of channels becoming “trapped open” in sub-conducting states following exposure to SMFs. There is a delay in the onset of this effect and it is maintained throughout exposure. Generally, channel activity showed slow or limited recovery following removal of the magnetic field and responses to the magnetic were often reduced or abolished upon subsequent exposures. Pre-exposure of the liposomes to SMFs resulted in reduced sensitivity of MscL to negative pipette pressure, with higher pressures required to activate the channels. Although the mechanisms of this effect are not clear, our initial observations appear to support previous work showing that the effects of SMFs on ion channels may be mediated by changes in membrane properties due to anisotropic diamagnetism of lipid molecules.

Preliminary analysis of magnetic particle techniques for activating mechanotransduction in bone cells

Magnetic beads were coated with RGD and adhered to primary human osteoblasts. During a 21-day culture, the osteoblasts plus adhered magnetic beads underwent a daily exposure to a cylical magnetic field via a permanent magnet thus applying a direct mechanical strain to the cells. After 21 days, preliminary results show that the cells plus magnetic beads were viable and had proliferated. A von-kossa stain showed a small amount of mineralized bone matrix produced at 21 days whereas the control groups showed no mineralized matrix production. RT-PCR at 21 days showed production of collagen 1 and GAPDH from each of the cell samples analysed. These preliminary results indicate that adherence of RGD-coated 4.5 micron ferromagnetic beads to primary human osteoblasts does not initiate cell necrosis up to 21 days in vitro. Also, mechanical stimulation of human osteoblasts by magnetic bead technology appears to have an influence on osteoblastic activity.