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1. Asymmetric protonation of glutamate residues drives a preferred transport pathway in EmrE

   https://doi.org/10.1073/pnas.2110790118  

   Li, Jianping ; Sae Her, Ampon ; Traaseth, Nathaniel J. ( October 2021 , Proceedings of the National Academy of Sciences)

   EmrE is anEscherichia colimultidrug efflux pump and member of the small multidrug resistance (SMR) family that transports drugs as a homodimer by harnessing energy from the proton motive force. SMR family transporters contain a conserved glutamate residue in transmembrane 1 (Glu14 in EmrE) that is required for binding protons and drugs. Yet the mechanism underlying proton-coupled transport by the two glutamate residues in the dimer remains unresolved. Here, we used NMR spectroscopy to determine acid dissociation constants (pK_a) for wild-type EmrE and heterodimers containing one or two Glu14 residues in the dimer. For wild-type EmrE, we measured chemical shifts of the carboxyl side chain of Glu14 using solid-state NMR in lipid bilayers and obtained unambiguous evidence on the existence of asymmetric protonation states. Subsequent measurements of pK_avalues for heterodimers with a single Glu14 residue showed no significant differences from heterodimers with two Glu14 residues, supporting a model where the two Glu14 residues have independent pK_avalues and are not electrostatically coupled. These insights support a transport pathway with well-defined protonation states in each monomer of the dimer, including a preferred cytoplasmic-facing state where Glu14 is deprotonated in monomer A and protonated in monomer B under pH conditions in the cytoplasm ofE. coli. Our findings also lead to a model, hop-free exchange, which proposes how exchangers with conformation-dependent pK_avalues reduce proton leakage. This model is relevant to the SMR family and transporters comprised of inverted repeat domains.

    

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2. Golgi‐localized cation/proton exchangers regulate ionic homeostasis and skotomorphogenesis in Arabidopsis

   https://doi.org/10.1111/pce.13452  

   Wang, Yuan ; Tang, Ren‐Jie ; Yang, Xiyan ; Zheng, Xiaojiang ; Shao, Qiaolin ; Tang, Qing‐Lin ; Fu, Aigen ; Luan, Sheng ( November 2018 , Plant, Cell & Environment)

   Multiple transporters and channels mediate cation transport across the plasma membrane and tonoplast to regulate ionic homeostasis in plant cells. However, much less is known about the molecular function of transporters that facilitate cation transport in other organelles such as Golgi. We report here thatArabidopsisKEA4, KEA5, and KEA6, members of cation/proton antiporters‐2 (CPA2) superfamily were colocalized with the known Golgi marker, SYP32‐mCherry. Although singlekea4,5,6mutants showed similar phenotype as the wild type under various conditions,kea4/5/6triple mutants showed hypersensitivity to low pH, high K⁺, and high Na⁺and displayed growth defects in darkness, suggesting that these three KEA‐type transporters function redundantly in controlling etiolated seedling growth and ion homeostasis. Detailed analysis indicated that thekea4/5/6triple mutant exhibited cell wall biosynthesis defect during the rapid etiolated seedling growth and under high K⁺/Na⁺condition. The cell wall‐derived pectin homogalacturonan (GalA)₃partially suppressed the growth defects and ionic toxicity in thekea4/5/6triple mutants when grown in the dark but not in the light conditions. Together, these data support the hypothesis that the Golgi‐localized KEAs play key roles in the maintenance of ionic and pH homeostasis, thereby facilitating Golgi function in cell wall biosynthesis during rapid etiolated seedling growth and in coping with high K⁺/Na⁺stress.

    

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3. Structure and function of LCI1: a plasma membrane CO 2 channel in the Chlamydomonas CO 2 concentrating mechanism

   https://doi.org/10.1111/tpj.14745  

   Kono, Alfredo ; Chou, Tsung‐Han ; Radhakrishnan, Abhijith ; Bolla, Jani Reddy ; Sankar, Kannan ; Shome, Sayane ; Su, Chih‐Chia ; Jernigan, Robert L. ; Robinson, Carol V. ; Yu, Edward W. ; et al ( April 2020 , The Plant Journal)

   Microalgae and cyanobacteria contribute roughly half of the global photosynthetic carbon assimilation. Faced with limited access to CO₂in aquatic environments, which can vary daily or hourly, these microorganisms have evolved use of an efficient CO₂concentrating mechanism (CCM) to accumulate high internal concentrations of inorganic carbon (C_i) to maintain photosynthetic performance. For eukaryotic algae, a combination of molecular, genetic and physiological studies using the model organismChlamydomonas reinhardtii, have revealed the function and molecular characteristics of many CCM components, including active C_iuptake systems. Fundamental to eukaryotic C_iuptake systems are C_itransporters/channels located in membranes of various cell compartments, which together facilitate the movement of C_ifrom the environment into the chloroplast, where primary CO₂assimilation occurs. Two putative plasma membrane C_itransporters, HLA3 and LCI1, are reportedly involved in active C_iuptake. Based on previous studies, HLA3 clearly plays a meaningful role in HCO₃⁻transport, but the function of LCI1 has not yet been thoroughly investigated so remains somewhat obscure. Here we report a crystal structure of the full‐length LCI1 membrane protein to reveal LCI1 structural characteristics, as well asin vivophysiological studies in an LCI1 loss‐of‐function mutant to reveal the C_ispecies preference for LCI1. Together, these new studies demonstrate LCI1 plays an important role in active CO₂uptake and that LCI1 likely functions as a plasma membrane CO₂channel, possibly a gated channel.

    

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4. A chloride channel blocker prevents the suppression by inorganic phosphate of the cytosolic calcium signals that control muscle contraction

   https://doi.org/10.1113/JP279917  

   Ferreira, Juan J. ; Pequera, Germán ; Launikonis, Bradley S. ; Ríos, Eduardo ; Brum, Gustavo ( October 2020 , The Journal of Physiology)

    Accumulation of inorganic phosphate (P_i) may contribute to muscle fatigue by precipitating calcium salts inside the sarcoplasmic reticulum (SR). Neither direct demonstration of this process nor definition of the entry pathway of P_iinto SR are fully established.

    We showed that P_ipromoted Ca²⁺release at concentrations below 10 mmand decreased it at higher concentrations. This decrease correlated well with that of [Ca²⁺]_SR.

    Pre‐treatment of permeabilized myofibres with 2 mmCl⁻channel blocker 9‐anthracenecarboxylic acid (9AC) inhibited both effects of P_i.

    The biphasic dependence of Ca²⁺release on [P_i] is explained by a direct effect of P_iacting on the SR Ca²⁺release channel, combined with the intra‐SR precipitation of Ca²⁺salts. The effects of 9AC demonstrate that P_ienters the SR via a Cl⁻pathway of an as‐yet‐undefined molecular nature.

   Fatiguing exercise causes hydrolysis of phosphocreatine, increasing the intracellular concentration of inorganic phosphate (P_i). P_idiffuses into the sarcoplasmic reticulum (SR) where it is believed to form insoluble Ca²⁺salts, thus contributing to the impairment of Ca²⁺release. Information on the P_ientrance pathway is still lacking. In amphibian muscles endowed with isoform 3 of the RyR channel, Ca²⁺spark frequency is correlated with the Ca²⁺load of the SR and can be used to monitor this variable. We studied the effects of P_ion Ca²⁺sparks in permeabilized fibres of the frog. Relative event frequency (f/f_ref) rose with increasing [P_i], reaching 2.54 ± 1.6 at 5 mm,and then decreased monotonically, reaching 0.09 ± 0.03 at [P_i] = 80 mm. Measurement of [Ca²⁺]_SRconfirmed a decrease correlated with spark frequency at high [P_i]. A large [Ca²⁺]_SRsurge was observed upon P_iremoval. Anion channels are a putative path for P_iinto the SR. We tested the effect of the chloride channel blocker 9‐anthracenecarboxylic acid (9AC) on P_ientrance. 9AC (400 µm)applied to the cytoplasm produced a non‐significant increase in spark frequency and reduced the P_ieffects on this parameter. Fibre treatment with 2 mm9AC in the presence of high cytoplasmic Mg²⁺suppressed the effects of P_ion [Ca²⁺]_SRand spark frequency up to 55 mm[P_i]. These results suggest that chloride channels (or transporters) provide the main pathway of inorganic phosphate into the SR and confirm that P_iimpairs Ca²⁺release by accumulating and precipitating with Ca²⁺inside the SR, thus contributing to myogenic fatigue.

    

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5. The intimate and controversial relationship between voltage‐gated proton channels and the phagocyte NADPH oxidase

   https://doi.org/10.1111/imr.12437  

   DeCoursey, Thomas E. ( August 2016 , Immunological Reviews)

   One of the most fascinating and exciting periods in my scientific career entailed dissecting the symbiotic relationship between two membrane transporters, the Nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase complex and voltage‐gated proton channels (H_V1). By the time I entered this field, there had already been substantial progress toward understandingNADPHoxidase, but H_V1 were known only to a tiny handful of cognoscenti around the world. Having identified the first proton currents in mammalian cells in 1991, I needed to find a clear function for these molecules if the work was to become fundable. The then‐recent discoveries of Henderson, Chappell, and colleagues in 1987–1988 that led them to hypothesize interactions of both molecules during the respiratory burst of phagocytes provided an excellent opportunity. In a nutshell, both transporters function by moving electrical charge across the membrane:NADPHoxidase moves electrons and H_V1 moves protons. The consequences of electrogenicNADPHoxidase activity on both membrane potential and pH strongly self‐limit this enzyme. Fortunately, both consequences specifically activate H_V1, and H_V1 activity counteracts both consequences, a kind of yin–yang relationship. Notwithstanding a decade starting in 1995 when many believed the opposite, these are two separate molecules that function independently despite their being functionally interdependent in phagocytes. The relationship betweenNADPHoxidase and H_V1 has become a paradigm that somewhat surprisingly has now extended well beyond the phagocyteNADPHoxidase – an industrial strength producer of reactive oxygen species (ROS) – to myriad other cells that produce orders of magnitude lessROSfor signaling purposes. These cells with their sevenNADPHoxidase (NOX) isoforms provide a vast realm of mechanistic obscurity that will occupy future studies for years to come.

    

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