TTYH family members form tetrameric complexes at the cell membrane

Abstract

The conserved Tweety homolog (TTYH) family consists of three paralogs in vertebrates, displaying a ubiquitous expression pattern. Although considered as ion channels for almost two decades, recent structural and functional analyses refuted this role. Intriguingly, while all paralogs shared a dimeric stoichiometry following detergent solubilization, their structures revealed divergence in their relative subunit orientation. Here, we determined the stoichiometry of intact mouse TTYH (mTTYH) complexes in cells. Using cross-linking and single-molecule fluorescence microscopy, we demonstrate that mTTYH1 and mTTYH3 form tetramers at the plasma membrane, stabilized by interactions between their extracellular domains. Using blue-native PAGE, fluorescence-detection size-exclusion chromatography, and hydrogen/deuterium exchange mass spectrometry (HDX-MS), we reveal that detergent solubilization results in tetramers destabilization, leading to their dissolution into dimers. Moreover, HDX-MS demonstrates that the extracellular domains are stabilized in the context of the tetrameric mTTYH complex. Together, our results expose the innate tetrameric organization of TTYH complexes at the cell membrane. Future structural analyses of these assemblies in native membranes are required to illuminate their long-sought cellular function.

To be or not to be an ion channel: cryo-EM structures have a say

“…In consistence with these structural observations, electrophysiological recordings confirmed that TTYH family proteins do not form ion channels (Dubinet al , 2017;Shin et al , 2020). Interestingly, a most recent study using cross-linking and single-molecule fluorescence microscopy demonstrated that TTYH1/3 assemble as tetramers at the plasma membrane, while detergent disrupt tetramers into dimers (Melvin et al , 2022). This raised the question that whether the cryo-EM structures obtained from purified proteins can represent their native architecture in the cell.…”

Emerging roles of prominin-1 (CD133) in the dynamics of plasma membrane architecture and cell signaling pathways in health and disease

“…Instead, the hypothesis that these proteins might play a role in the dynamics of membrane lipids was proposed [ 320 ]. More interestingly, TTYH1/2 proteins are structurally similar to CD133, including their membrane topology and dimer/tetramer formation [ 210 , 322 ], suggesting that they may all regulate membrane organization leading to fibre formation. Whether this process is directly related to the chloride efflux activity mediated by TTYH1/2 (or CD133) or to their interaction with other molecules remains to be demonstrated [ 178 ].…”

Physiology of the volume-sensitive/regulatory anion channel VSOR/VRAC. Part 1: from its discovery and phenotype characterization to the molecular entity identification

“…Recent cryo-EM studies provided firm evidence against “TTYH = VSOR/VRAC” hypothesis. First, no pore-like structure was observed in the three-dimensional structure of TTYHs [ 151 , 152 ], which were shown to be expressed in the plasma membrane [ 153 ]. The positively charged residues, such as R165 of TTYH1, R164 of TTYH2 as well as R367 and H370 of TTYH3, that were previously proposed to line a pore [ 147 , 149 ], exist remote from the membrane [ 152 ].…”