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1.A.48 The Anion Channel Tweety (Tweety) Family

The Tweety family of anion channel proteins is present in animals and plants. These proteins contain about 500 - 1200 aas and 5 TMSs in an arrangement: 2 + 2 + 1, with an extra N-terminal TMS present in some plant homologues (e.g., NP_178935). They produce large conductance chloride (maxi-Cl-) currents. Calcium-activated chloride currents can be recorded in almost all cells. Tweety, a gene located in the Drosophila flightless locus, possesses five or six TMSs, and a human homologue of tweety (hTTYH3) is a large-conductance Ca2+-activated Cl- channel, while the related gene, hTTYH1, is a swelling-activated Cl- current. hTTYH3 is expressed in excitable tissues, including the heart, brain and skeletal muscle, whereas hTTYH1 is expressed mainly in the brain (Suzuki 2006).  The hTTYH3-induced Cl- current had a linear current-voltage relationship, a large single-channel conductance (260 pS) and the anion permeability sequence I- > Br- > Cl-. The hTTYH3 channel shows complex gating kinetics and voltage-dependent inactivation, dependent on a micromolar intracellular Ca2+ concentration.

'Tweety has three human homologues (hTTYH1-3) which are all reported to be maxi-Cl- channel proteins (Suzuki and Mizuno, 2004). He et al., (2008) provided evidence for a structure for Tweety family proteins which incorporates five membrane-spanning domains with a topology in which the N-terminus is located extracellularly and the C-terminus cytoplasmically. N-glycosylation is important, but not essential, in the processing of members of the Tweety family. Incomplete N-glycosylation gives rise to reduced expression and increased ubiquitination (He et al., 2008).

hTTYH3 mRNA is distributed in excitable tissues. Positively charged amino acyl residues in the putative pore contribute to anion selectivity. The hTTYH3 single channel shows 26 picosiemen linear current voltage, complex kinetics and sensitivity to 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid.  This channel and hTTYH2 encode ionomycin-induced maxi-Cl- channels, but hTTYH1 encodes a Ca2+-independent, swelling-activated maxi-Cl- channel (Suzuki and Mizuno, 2004). TTYHs comprise a conserved family in eukaryotes that are widely expressed in mammals, at high levels in the nervous system. TTYHs have been reported to form Ca2+- and cell volume-regulated anion channels. However, Li et al. 2021 determined cryo-EM structures of Mus musculus TTYH2 and TTYH3 in lipid nanodiscs and showed that they adopt a previously unobserved fold which includes an extended extracellular domain with a partially solvent exposed pocket that may be an interaction site for hydrophobic molecules. In the presence of Ca2+, they form homomeric cis-dimers bridged by extracellularly coordinated Ca2+. In the absence of Ca2+, TTYH2 forms trans-dimers that span opposing membranes across a ~130 Å intermembrane space as well as a monomeric state. These TTYH structures seem to lack ion conducting pathways, and channel activity was not observed. Li et al. 2021 suggested that TTYHs are not pore forming subunits of anion channels, and their functions may involve Ca2+-dependent changes in quaternary structure, interactions with hydrophobic molecules near the extracellular membrane surface, and/or association with additional protein partners.

Tweety homologs (TTYHs) are abundant in the brain. The three human paralogs were suggested to function as anion channels that are activated either by Ca2+ or cell swelling. To uncover their unknown architecture and its relationship to function, Sukalskaia et al. 2021 determined the structures of human TTYH1-3 by cryo-EM. All structures display equivalent features of a dimeric membrane protein that contains five TMSs and an extended extracellular domain. As none of the proteins shows attributes reminiscent of an anion channel, the authors revisited functional experiments and did not find an indication of ion conduction. Instead, they found density in an extended hydrophobic pocket contained in the extracellular domain that emerges from the lipid bilayer, which suggested to them a role of TTYH proteins in interactions with lipid-like compounds residing in the membrane (Sukalskaia et al. 2021).

Homologues of Tweety are found in vertebrates, insects, worms and probably plants. They are prevalent in animals but scarce in plants. A search is in progress to identify homologues elsewhere.

References associated with 1.A.48 family:

He, Y., A.J. Ramsay, M.L. Hunt, A.K. Whitbread, S.A. Myers, and J.D. Hooper. (2008). N-glycosylation analysis of the human Tweety family of putative chloride ion channels supports a penta-spanning membrane arrangement: impact of N-glycosylation on cellular processing of Tweety homologue 2 (TTYH2). Biochem. J. 412: 45-55. 18260827
Li, B., C.M. Hoel, and S.G. Brohawn. (2021). Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca-dependent switch from intra- to intermembrane dimerization. Nat Commun 12: 6913. 34824283
Sukalskaia, A., M.S. Straub, D. Deneka, M. Sawicka, and R. Dutzler. (2021). Cryo-EM structures of the TTYH family reveal a novel architecture for lipid interactions. Nat Commun 12: 4893. 34385445
Suzuki, M. (2006). The Drosophila tweety family: molecular candidates for large-conductance Ca2+-activated Cl- channels. Exp Physiol 91: 141-147. 16219661
Suzuki, M. and A. Mizuno. (2004). A novel human Cl- channel family related to Drosophila flightless locus. J. Biol. Chem. 279: 22461-22468. 15010458