Differential ion dehydration energetics explains selectivity in the non-canonical lysosomal K+ channel TMEM175

Abstract

Structures of the human lysosomal K+ channel transmembrane protein 175 (TMEM175) in open and closed states revealed a novel architecture lacking the canonical K+ selectivity filter motif present in previously known K+ channel structures. A hydrophobic constriction composed of four isoleucine residues was resolved in the pore and proposed to serve as the gate in the closed state, and to confer ion selectivity in the open state. Here, we achieve higher-resolution structures of the open and closed states and employ molecular dynamics simulations to analyze the conducting properties of the putative open state, demonstrating that it is permeable to K+ and, to a lesser degree, also Na+. Both cations must dehydrate significantly to penetrate the narrow hydrophobic constriction, but ion flow is assisted by a favorable electrostatic field generated by the protein that spans the length of the pore. The balance of these opposing energetic factors explains why permeation is feasible, and why TMEM175 is selective for K+ over Na+, despite the absence of the canonical selectivity filter. Accordingly, mutagenesis experiments reveal an exquisite sensitivity of the channel to perturbations that mitigate the constriction. Together, these data reveal a novel mechanism for selective permeation of ions by TMEM175 that is unlike that of other K+ channels.

WiChR, a highly potassium-selective channelrhodopsin for low-light one- and two-photon inhibition of excitable cells

“…8). A similar non-canonical mechanism for K + selectivity was recently described for the lysosomal K + channel TMEM175, which also features a hydrophobic restriction in the center of the pore, as required for K + conductance, and which also showed important differences in K + selectivity for different channel subtypes, ranging from P K+ /P Na+ of 2 in bacteria to P K+ / P Na+ of 35 in humans (25). Accordingly, P K+ /P Na+ also varied significantly for different KCRs despite the overall conserved signature motif.…”

Mechanism of 4-aminopyridine inhibition of the lysosomal channel TMEM175

“…S1 ). In contrast to the mixture of open and closed states that were observed for TMEM175 vitrified in the absence of ligand ( 9 , 12 ), only a single conformation could be reconstructed from the particle images of TMEM175 in the presence of 4-AP, despite multiple attempts to identify alternative conformations using 3D classification and 3D variability algorithms ( SI Appendix , Fig. S4 and Table S1 ).…”

“…Near the middle of the ion conduction pore, a ring of highly conserved isoleucine residues (Ile46 and Ile271) forms a narrow hydrophobic constriction site. In the closed conformation of the channel, this isoleucine constriction serves as a physical gate blocking permeation, while in the open conformation it allows permeation of partially dehydrated cations, thereby shaping the ion selectivity of the channel ( 9 , 12 ). Pore-lining residues near the isoleucine constriction—including Ser45, Thr49, and Thr274—contribute to permeation through transient water-mediated interactions with the permeating ions ( 9 , 12 ).…”

“…In the closed conformation of the channel, this isoleucine constriction serves as a physical gate blocking permeation, while in the open conformation it allows permeation of partially dehydrated cations, thereby shaping the ion selectivity of the channel ( 9 , 12 ). Pore-lining residues near the isoleucine constriction—including Ser45, Thr49, and Thr274—contribute to permeation through transient water-mediated interactions with the permeating ions ( 9 , 12 ). Recent studies have indicated that in addition to being selective for K + ions over Na + ions, TMEM175 is permeable to protons ( 13 , 14 ).…”

WiChR, a highly potassium selective channelrhodopsin for low-light two-photon neuronal inhibition

“…5). A similar non-canonical mechanism for K + -selectivity was recently described for the lysosomal K + -channel TMEM175, that also features a hydrophobic restriction in the center of the pore as required for K + -conductance, with important differences in K + -selectivity for different channel subtypes ranging from PK+/PNa+ of 2 in bacteria to PK+/PNa+ of 35 in humans (24). Accordingly, PK+/PNa+ also varied significantly for different KCRs despite an overall conserved signature motif and we observe diverse conformations of the key residues along the pore in the different channels (Fig.…”