Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, PC264
Identification of gating mutations in the TREK-1 K2P potassium channel by functional complementation in K+ uptake deficient yeast
C. Sharma1, M. K. Bollepalli3, M. R. Schmidt2,1, T. Baukrowitz3, S. J. Tucker1
1. Dept Physics, University of Oxford, Oxford, United Kingdom. 2. Dept Biochemistry, University of Oxford, Oxford, United Kingdom. 3. Institute of Physiology, Christian Albrechts University, Kiel, Germany.
TREK-1 is a member of the K2P family of potassium channels. These channels appear to have a unique gating mechanism compared to other types of K+ channel and this may be a reflection of their overall asymmetric structure. In order to address which domains of the channel may be important for channel gating we took advantage of an unbiased random mutagenesis approach which selects for activatory mutations by complementation in a K+ auxotrophic strain of yeast (SGY1528). Wild-type TREK-1 did not complement the growth of this strain on low [K+] media. However, screening a randomly mutated TREK-1 library yielded a number of mutations which robustly complemented growth. One of the mutants has been identified previously through its effects on pHi-gating (Glu-321). However many other novel mutations were also identified. Intriguingly, the majority of these mutations were located in the TMs and/or close to the selectivity filter of TREK-1. Electrophysiological analysis of these mutants expressed in Xenopus oocytes demonstrates dramatic effects on TREK channel gating by intracellular pH. The results also point towards a dominant role for the inactivation gate (i.e. selectivity filter) and TM4 in TREK-1 channel gating.
Where applicable, experiments conform with Society ethical requirements