Novel Mutations in the Extracellular Cap of the Mammalian Mechanosensitive Channel TREK-1

The Twik related potassium channel 1 (TREK-1) is one of the best studied mechanosensitive mammalian channels. TREK-1 is known to play very important roles in depression, ischemia and vasoregulation. TREK-1 belongs to the family of background or "leak" potassium channels are constitutively open at rest and have a central role in the tuning of neuronal resting membrane potential, duration of action potential and regulation of neurotransmitter release. These K+ channels are members of the family of K2P channels subunits containing four transmembrane and two pore domains. The functional channel is formed by two subunits and is predicted to have a two-fold symmetry.Here we show the feasibility of the use of microbial genetics to study the structure-function relationship of this mammalian channel. The advantage of this approach is that we can directly screen for channels with altered phenotypes and correlate this altered function with structural changes. We have successfully expressed a functional TREK-1 channel in bacterial cells, and show that it can partially rescue the slow growth phenotype of an E .coli strain deficient in three mayor potassium transporters. Furthermore, using random mutagenesis and bacterial screens we have isolated five mutants that better remediate the potassium deficiency of this bacterial strain. Because these mutants clustered in a stretch of 20 amino acids in the extracellular cap of the TREK-1, we think that we have found a functional "hot spot" by utilizing the power of bacterial genetics.