Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC183
Structural re-arrangement of the BK channel RCK1-RCK2 linker investigated using fluorescence lifetime imaging microscopy
I. Rowe1,2, F. Saleem2, O. Jeffries2, H. McClafferty2, C. McCartney3, E. Rowan3, R. Duncan2, M. Shipston2
1. School of Pharmacy and Life Sciences, Robert Gordon University, Aberdeen, United Kingdom. 2. Centre for Integrative Physiology, University of Edinburgh, Edinburgh, United Kingdom. 3. Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom.
Alternative splicing generates considerable functional diversity in large conductance calcium- and voltage-activated potassium (BK) channels. For example, inclusion of the STREX (STRess-activated EXon) insert into the cytosolic linker between the two regulator of potassium conductance (RCK) domains of the channel generates a cysteine rich domain (CRD) that confers intrinsic hypoxia sensitivity to the channel (McCartney et al., 2005), as well as sites for regulation by phosphorylation and palmitoylation (Tian et al., 2008). The RCK1-RCK2 linker region is considered unstructured yet has clear impact on BK channel function and so we have developed YFP-mCFP fluorescent fusion proteins of the CRD and exploit these to examine conformational rearrangements in the CRD in response to hypoxia using fluorescence lifetime imaging microscopy (FLIM). YFP-mCFP fusion proteins of the CRD encompassing the STREX insert are targeted to the plasma membrane of HEK293 cells. Exposure of intact cells expressing the construct to acute hypoxia (10 minute exposure at <5% oxygen) resulted in a significant shift in fluorescence lifetime distribution (n=9). This suggests that hypoxia induces a conformational rearrangement in the RCK1-RK2 linker. Site directed mutagenesis of the cysteine residues within STREX, which we had previously shown to confer functional hypoxia sensitivity to the channel, abolished the hypoxia-induced shift in lifetimes (n=7). Pre-treatment with either oxidizing or reducing agents did not block the hypoxia-induced shift in fluorescence lifetimes (n=6). The effects of cysteine mutagenesis and REDOX manipulation on hypoxia sensitivity were recapitulated in patch clamp assays of channel activity (n=3 to 9 for each construct) supporting a role for hypoxia-induced shifts in CRD conformation with changes in channel function. These findings suggest that FLIM, in conjunction with patch clamp electrophysiology, will be an important approach to monitor conformational rearrangements and investigate the structure-function relationship of the important unstructured linker region between the channel RCK domains.
Where applicable, experiments conform with Society ethical requirements