Cellular Physiology and Pathophysiology of EAAT Anion Channels

Excitatory amino acid transporters (EAATs) optimize the temporal resolution and energy demand of mammalian excitatory synapses by quickly removing glutamate from the synaptic cleft into surrounding neuronal and glial cells and ensuring low resting glutamate concentrations. In addition to secondary active glutamate transport, EAATs also function as anion channels. The channel function of these transporters is conserved in all homologs ranging from archaebacteria to mammals; however, its physiological roles are insufficiently understood. There are five human EAATs, which differ in their glutamate transport rates. Until recently the high-capacity transporters EAAT1, EAAT2, and EAAT3 were believed to conduct only negligible anion currents, with no obvious function in cell physiology. In contrast, the low-capacity glutamate transporters EAAT4 and EAAT5 are thought to regulate neuronal signaling as glutamate-gated channels. In recent years, new experimental approaches and novel animal models, together with the discovery of a human genetic disease caused by gain-of-function mutations in EAAT anion channels have enabled identification of the first physiological and pathophysiological roles of EAAT anion channels.

Properties, Structures, and Physiological Roles of Three Types of Anion Channels Molecularly Identified in the 2010’s

Molecular identification was, at last, successfully accomplished for three types of anion channels that are all implicated in cell volume regulation/dysregulation. LRRC8A plus LRRC8C/D/E, SLCO2A1, and TMEM206 were shown to be the core or pore-forming molecules of the volume-sensitive outwardly rectifying anion channel (VSOR) also called the volume-regulated anion channel (VRAC), the large-conductance maxi-anion channel (Maxi-Cl), and the acid-sensitive outwardly rectifying anion channel (ASOR) also called the proton-activated anion channel (PAC) in 2014, 2017, and 2019, respectively. More recently in 2020 and 2021, we have identified the S100A10-annexin A2 complex and TRPM7 as the regulatory proteins for Maxi-Cl and VSOR/VRAC, respectively. In this review article, we summarize their biophysical and structural properties as well as their physiological roles by comparing with each other on the basis of their molecular insights. We also point out unsolved important issues to be elucidated soon in the future.

Consensus channelome of dinoflagellates revealed by transcriptomic analysis sheds light on their physiology

Ion channels are membrane protein complexes mediating passive ion flux across the cell membranes. Every organism has a certain set of ion channels that define its physiology. Dinoflagellates are ecologically important microorganisms characterized by effective physiological adaptability, which backs up their massive proliferations that often result in harmful blooms (red tides). In this study, we used a bioinformatics approach to identify homologs of known ion channels that belong to 36 ion channel families. We demonstrated that the versatility of the dinoflagellate physiology is underpinned by a high diversity of ion channels including homologs of animal and plant proteins, as well as channels unique to protists. The analysis of 27 transcriptomes allowed reconstructing a consensus ion channel repertoire (channelome) of dinoflagellates including the members of 31 ion channel families: inwardly-rectifying potassium channels, two-pore domain potassium channels, voltage-gated potassium channels (Kv), tandem Kv, cyclic nucleotide-binding domain-containing channels (CNBD), tandem CNBD, eukaryotic ionotropic glutamate receptors, large-conductance calcium-activated potassium channels, intermediate/small-conductance calcium-activated potassium channels, eukaryotic single-domain voltage-gated cation channels, transient receptor potential channels, two-pore domain calcium channels, four-domain voltage-gated cation channels, cation and anion Cys-loop receptors, small-conductivity mechanosensitive channels, large-conductivity mechanosensitive channels, voltage-gated proton channels, inositole-1,4,5- trisphosphate receptors, slow anion channels, aluminum-activated malate transporters and quick anion channels, mitochondrial calcium uniporters, voltage-dependent anion channels, vesicular chloride channels, ionotropic purinergic receptors, animal volage-insensitive cation channels, channelrhodopsins, bestrophins, voltage-gated chloride channels H+/Cl- exchangers, plant calcium-permeable mechanosensitive channels, and trimeric intracellular cation channels. Overall, dinoflagellates represent cells able to respond to physical and chemical stimuli utilizing a wide range of Gprotein coupled receptors- and Ca2+-dependent signaling pathways. The applied approach not only shed light on the ion channel set in dinoflagellates, but also provided the information on possible molecular mechanisms underlying vital cellular processes dependent on the ion transport.

Stability of Intracellular Protein Concentration under Extreme Osmotic Challenge

Cell volume (CV) regulation is typically studied in short-term experiments to avoid complications resulting from cell growth and division. By combining quantitative phase imaging (by transport-of-intensity equation) with CV measurements (by the exclusion of an external absorbing dye), we were able to monitor the intracellular protein concentration (PC) in HeLa and 3T3 cells for up to 48 h. Long-term PC remained stable in solutions with osmolarities ranging from one-third to almost twice the normal. When cells were subjected to extreme hypoosmolarity (one-quarter of normal), their PC did not decrease as one might expect, but increased; a similar dehydration response was observed at high concentrations of ionophore gramicidin. Highly dilute media, or even moderately dilute in the presence of cytochalasin, caused segregation of water into large protein-free vacuoles, while the surrounding cytoplasm remained at normal density. These results suggest that: (1) dehydration is a standard cellular response to severe stress; (2) the cytoplasm resists prolonged dilution. In an attempt to investigate the mechanism behind the homeostasis of PC, we tested the inhibitors of the protein kinase complex mTOR and the volume-regulated anion channels (VRAC). The initial results did not fully elucidate whether these elements are directly involved in PC maintenance.

Structures of tweety homolog proteins TTYH2 and TTYH3 reveal a Ca2+-dependent switch from intra- to intermembrane dimerization

Tweety homologs (TTYHs) comprise a conserved family of transmembrane proteins found in eukaryotes with three members (TTYH1-3) in vertebrates. They are widely expressed in mammals including at high levels in the nervous system and have been implicated in cancers and other diseases including epilepsy, chronic pain, and viral infections. TTYHs have been reported to form Ca2+- and cell volume-regulated anion channels structurally distinct from any characterized protein family with potential roles in cell adhesion, migration, and developmental signaling. To provide insight into TTYH family structure and function, we determined cryo-EM structures of Mus musculus TTYH2 and TTYH3 in lipid nanodiscs. TTYH2 and TTYH3 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+, TTYH2 and TTYH3 form homomeric cis-dimers bridged by extracellularly coordinated Ca2+. Strikingly, 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. All TTYH structures lack ion conducting pathways and we do not observe TTYH2-dependent channel activity in cells. We conclude TTYHs are not pore forming subunits of anion channels and their function may involve Ca2+-dependent changes in quaternary structure, interactions with hydrophobic molecules near the extracellular membrane surface, and/or association with additional protein partners.

In-cell structures of conserved supramolecular protein arrays at the mitochondria–cytoskeleton interface in mammalian sperm

Mitochondria–cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria–cytoskeleton interfaces in any cell type. Here, we use cryofocused ion beam milling-enabled cryoelectron tomography to image mammalian sperm, where mitochondria wrap around the flagellar cytoskeleton. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage-dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types in which mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.

VDAC1 Negatively Regulates Floral Transition in Arabidopsis thaliana

Voltage-dependent anion channels (VDACs) are the most important proteins in mitochondria. They localize to the outer mitochondrial membrane and contribute to the metabolite transport between the mitochondria and cytoplasm, which aids plant growth regulation. Here, we report that Arabidopsis thaliana VDAC1 is involved in the floral transition, with the loss of AtVDAC1 function, resulting in an early-flowering phenotype. AtVDAC1 is expressed ubiquitously in Arabidopsis. To identify the flowering pathway integrators that may be responsible for AtVDAC1′s function during the floral transition, an RNA-seq analysis was performed. In total, 106 differentially expressed genes (DEGs) were identified between wild-type and atvdac1-5 mutant seedlings. However, none were involved in flowering-related pathways. In contrast, AtVDAC1 physically associated with FLOWERING LOCUS T. Thus, in the floral transition, AtVDAC1 may function partly through the FLOWERING LOCUS T protein.

Emerging Roles of γ Aminobutyric Acid (GABA) Gated Channels in Plant Stress Tolerance

The signaling role for γ-Aminobutyric acid (GABA) has been documented in animals for over seven decades. However, a signaling role for GABA in plants is just beginning to emerge with the discovery of putative GABA binding site/s and GABA regulation of anion channels. In this review, we explore the role of GABA in plant growth and development under abiotic stress, its interactions with other signaling molecules and the probability that there are other anion channels with important roles in stress tolerance that are gated by GABA.