Cryo-EM structure and electrophysiological characterization of ALMT from Glycine max reveal a previously uncharacterized class of anion channels.

Aluminum-activated malate transporters (ALMTs) form an anion channel family that plays essential roles in diverse functions in plants. Arabidopsis ALMT12, also named QUAC1 (quick anion channel 1), regulates stomatal closure in response to environmental stimuli. However, the molecular basis of ALMT12/QUAC1 activity remains elusive. Here, we describe the cryo-EM structure of ALMT12/QUAC1 from Glycine max at 3.5-Å resolution. GmALMT12/QUAC1 is a symmetrical dimer, forming a single electropositive T-shaped pore across the membrane. The transmembrane and cytoplasmic domains are assembled into a twisted two-layer architecture, with their associated dimeric interfaces nearly perpendicular. GmALMT12/QUAC1-mediated currents display rapid kinetics of activation/deactivation and a bell-shaped voltage dependency, reminiscent of the rapid (R)-type anion currents. Our structural and functional analyses reveal a domain-twisting mechanism for malate-mediated activation. Together, our study uncovers the molecular basis for a previously uncharacterized class of anion channels and provides insights into the gating and modulation of the ALMT12/QUAC1 anion channel.

Ccp1-Ndc80 switch at the N terminus of CENP-T regulates kinetochore assembly.

Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain-deleted mutant phenocopies ccp1Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle.

[Effect and mechanism of moxibustion on oxidative stress injury in rats with Parkinson's disease by activating Nrf2/ARE pathway].

OBJECTIVE: To observe the effect of moxibustion on oxidative stress injury of nigrostriatal system in rats with Parkinson's disease (PD) based on nuclear factor erythroid 2-related factor (Nrf2)/antioxidant response element (ARE) pathway, and to explore its mechanism. METHODS: A total of 48 SD rats were randomized into a blank group, a sham-operation group, a model group and a moxibustion group, 12 rats in each group. Unilateral two-point injection with 6-hydroxydopamine (6-OHDA) was adopted in the model group and the moxibustion group to establish the PD model; the operation manipulation in the sham-operation group was the same as the model group and the moxibustion group, and the same volume of 0.9% sodium chloride solutions was given by unilateral two-point injection. Moxibustion was adopted at "Baihui" (GV 20) and "Sishencong" (EX-HN 1) in the moxibustion group for 20 min, once a day, 6 times a week for 6 weeks. No intervention was given in the other 3 groups. Morphology of right mesencephalon substantia nigra was observed by HE staining, the expression of tyrosine hydroxylase (TH) in right mesencephalon substantia nigra was detected by immunohistochemistry method, the expression of reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH), and glutathione peroxidase (GSH-Px) in corpus striatum was detected by colorimetry method, and the expression of Nrf2 and heme oxygenase-1 (HO-1) proteins was detected by Western blot in the 4 groups. RESULTS: Clear tissue structure and complete dopaminergic neurons of right mesencephalon substantia nigra were observed in the blank group and the sham-operation group; unclear tissue structure, decreased and swelling dopaminergic neurons were observed in the model group; compared with the model group, more neurons were observed and the swelling of cyton was reduced in the moxibustion group. Compared with the sham-operation group, the expression of TH in right mesencephalon substantia nigra was decreased in the model group (P<0.01); compared with the model group, the expression of TH in right mesencephalon substantia nigra was increased in the moxibustion group (P<0.05). Compared with the sham-operation group, the expression of ROS, MDA was increased (P<0.01), the expression of GSH, GSH-Px, Nrf2 and HO-1 was decreased in the model group (P<0.01, P<0.05); compared with the model group, the expression of ROS, MDA was decreased (P<0.05, P<0.01), the expression of GSH, GSH-Px, Nrf2 and HO-1 was increased in the moxibustion group (P<0.05, P<0.01). CONCLUSION: Moxibustion can alleviate oxidative stress injury of nigrostriatal system in rats with Parkinson's disease by activating the Nrf2/ARE pathway, and protect the dopamine neurons.

Structural basis for activity of TRIC counter-ion channels in calcium release.

Trimeric intracellular cation (TRIC) channels are thought to provide counter-ion currents that facilitate the active release of Ca2+ from intracellular stores. TRIC activity is controlled by voltage and Ca2+ modulation, but underlying mechanisms have remained unknown. Here we describe high-resolution crystal structures of vertebrate TRIC-A and TRIC-B channels, both in Ca2+-bound and Ca2+-free states, and we analyze conductance properties in structure-inspired mutagenesis experiments. The TRIC channels are symmetric trimers, wherein we find a pore in each protomer that is gated by a highly conserved lysine residue. In the resting state, Ca2+ binding at the luminal surface of TRIC-A, on its threefold axis, stabilizes lysine blockage of the pores. During active Ca2+ release, luminal Ca2+ depletion removes inhibition to permit the lysine-bearing and voltage-sensing helix to move in response to consequent membrane hyperpolarization. Diacylglycerol is found at interprotomer interfaces, suggesting a role in metabolic control.