Structural Models of Two-Pore-Domain Potassium Channels Focus on TREK

Abstract

Two-pore-domain background potassium (K2P) channels comprise a distinct gene family of widely distributed, well modulated channels. K2P channels have two similar or identical subunits, each of which has four transmembrane (TM) and two pore-forming (P) segments. Here we focus on mechanosensitive TREK channels. Unfortunately, the only structures available to be used as templates belong to the 2TM channels superfamily. These are distantly related at sequence level with different structural features: four symmetrically arranged subunits, each having two TM segments flanking a P segment. Our model building strategy used two subunits of the template (KcsA) to build one subunit of the target (TREK-1). Our models of the closed channel differ substantially from those of the template, primarily because TM2 of the 2nd repeat is near the axis of the pore whereas TM2 of the 1st repeat is far from the axis. Segments linking the two subunits and immediately following the last TM segment were modeled ab initio as α-helices based on helical periodicities of hydrophobic and hydrophilic residues, highly conserved and poorly conserved residues, and correlated mutations in multiple sequence alignments. The N-terminus segment preceding residue 35, the long loop between first and second TM segments (residues 76-125), and C-terminus past residue 333 were not included in the model due to lack of template. Experimental analysis of the similarly-truncated channel with these loop and C-terminus residues deleted revealed near native-like behavior. The models were further refined by two-fold symmetry-constrained molecular dynamics simulations using a protocol we previously developed. We also built models of the open state and suggest a possible tension-activated gating mechanism in which the inner portion of the TM2 helix of the 2nd repeat swings radially outward. This mechanism will be tested experimentally.