Gating and selectivity mechanisms for the lysosomal K+ channel TMEM175.

Secheol Oh,Richard K Hite,Navid Paknejad

doi:10.7554/elife.53430

Abstract

Transmembrane protein 175 (TMEM175) is a K+-selective ion channel expressed in lysosomal membranes, where it establishes a membrane potential essential for lysosomal function and its dysregulation is associated with the development of Parkinson's Disease. TMEM175 is evolutionarily distinct from all known channels, predicting novel ion-selectivity and gating mechanisms. Here we present cryo-EM structures of human TMEM175 in open and closed conformations, enabled by resolutions up to 2.6 Å. Human TMEM175 adopts a homodimeric architecture with a central ion-conduction pore lined by the side chains of the pore-lining helices. Conserved isoleucine residues in the center of the pore serve as the gate in the closed conformation. In the widened channel in the open conformation, these same residues establish a constriction essential for K+ selectivity. These studies reveal the mechanisms of permeation, selectivity and gating and lay the groundwork for understanding the role of TMEM175 in lysosomal function.

Highlights

Lysosomes are small, acidic organelles that play essential roles in nutrient sensing, signaling, autophagy and degradation of macromolecules (Ballabio and Bonifacino, 2020; Condon and Sabatini, 2019; Lawrence and Zoncu, 2019; Platt et al, 2018)
To measure the ion selectivity of recombinant hTMEM175 channels, we took advantage of the observation that while hTMEM175 is endogenously expressed in the membranes of endosomes and lysosomes, transient overexpression as a GFP-fusion protein in HEK293T cells leads to expression of hTMEM175 at the plasma membrane (Figure 1—figure supplement 1; Lee et al, 2017)
We measured whole-cell currents from non-transfected HEK293T cells, which revealed the presence of non-selective currents whose magnitude varied between 50 and 100 pA at +100 mV (Figure 1—figure supplement 1). Because these endogenous currents are present in the hTMEM175 transfected cells, the ion-selectivity measurements determined using whole-cell patch clamp underrepresent the selectivity of hTMEM175 and the true values are likely closer to those measured in endolysosomal patch clamp (PK/PNa ~36) (Cang et al, 2015)

Summary

Introduction

Acidic organelles that play essential roles in nutrient sensing, signaling, autophagy and degradation of macromolecules (Ballabio and Bonifacino, 2020; Condon and Sabatini, 2019; Lawrence and Zoncu, 2019; Platt et al, 2018) Many of these processes are intricately linked to ion transport across the membrane via numerous channels and transporters and defects in lysosomal transport proteins lead to a variety of diseases (Grimm et al, 2017; Li et al, 2019; van Veen et al, 2020). One of the common K+ channel inhibitors, 4-aminopyridine, can inhibit TMEM175 activity, while others, such as tetraethylammonium, do not alter channel activity (Cang et al, 2015) Together, these features predict unique ion permeation and selectivity mechanisms for TMEM175. To elucidate the mechanisms underlying TMEM175 function in mammalian cells, we determined single-particle cryo-electron microscopic (cryo-EM) structures and analyzed the ion-permeation and selectivity properties of human TMEM175 (hTMEM175)

Results

Discussion

Materials and methods

Funding Funder Searle Scholars Program