Ion channels in general, and especially Ca2+ permeable channels, such as voltage-gated Ca2+ channels (VGCC) and non-selective cation channels (NSCC), are of central importance for physiological regulation of smooth muscles, e.g. in the control of blood pressure as well as proliferative processes. The importance of NSCC for vascular and visceral smooth muscle function has been appreciated for more than 40 years. In particular, NSCC are responsive to neurotransmitters, circulating and local hormones, endothelial (e.g., nitric oxide), myogenic (e.g., stretch) and various metabolic/environmental (e.g., temperature) influences. However, the molecular composition of these channels remained largely unknown until recently, when important insights started to rapidly emerge indicating connection with mammalian homologues of the Drosophila Transient Receptor Potential (Trp) gene. The research in Prof. Zholos’ laboratory is mainly focused on molecular composition of the diverse NSCC-mediated Ca2+ and Na+ entry pathways, their activation mechanisms, and their specific function and regulation via cross-talk with other cell signalling pathways, such as G protein-coupled receptors, membrane lipids and intracellular Ca2+. Current research is centred on the identification of expression and function of TRP channels in the vasculature. This work has recently revealed novel vascular functions of the “classical” cold and menthol receptor TRPM8, single-channel mechanisms of its voltage- and cold-dependent gating and isoform-specific regulation of receptor-operated TRPC4 channel by phosphatidylinositol 4,5-bisphosphate.
Our research employs a range of molecular/cell biology and functional assessment techniques. These include
- Cell isolation and cell culture with heterologous protein expression
- Gene expression analysis by qRT-PCR and Western blotting
- Analysis of protein sub-cellular localisation and co-localisation with other proteins by immunocytochemistry and confocal imaging
- Patch-clamp recording and analysis of ion channel activity, single-channel modelling
- Laser confocal calcium imaging
- Recordings of vascular contractions (in collaboration with Dr. C. Johnson)
- Pharmacological analysis
Activation of TRPM8 by menthol (green circle appears) causes intracellular Ca2+ rise and strong contraction of a single rat tail artery myocyte, also compared to 10 mM caffeine application as indicated by the red circle.
In addition, our research capabilities are complemented and enhanced by several local, national and international collaborations. Another, more recent interest concerns vascular dysfunction in diabetes mellitus. In this area, we have recently demonstrated Ca2+ sensitisation in diabetic arteries which occurs due to up-regulation of Rho kinase and protein kinase C activities, thus enhancing vascular contractility in diabetes.
Single-channel activity recorded in the cell-attached
configuration in HEK293 cells stably expressing TRPM8.
Examination of 3D sub-cellular localisation of TRPM8
in a rat tail artery myocyte. Bar – 20 µm.