The Lepidopteran KAAT1 and CAATCH1: Orthologs to Understand Structure–Function Relationships in Mammalian SLC6 Transporters

To the SLC6 family belong 20 human transporters that utilize the sodium electrochemical gradient to move biogenic amines, osmolytes, amino acids and related compounds into cells. They are classified into two functional groups, the Neurotransmitter transporters (NTT) and Nutrient amino acid transporters (NAT). Here we summarize how since their first cloning in 1998, the insect (Lepidopteran) Orthologs of the SLC6 family transporters have represented very important tools for investigating functional–structural relationships, mechanism of transport, ion and pH dependence and substate interaction of the mammalian (and human) counterparts.

Rational design of electron/proton transfer mechanisms in the exoelectrogenic bacteria Geobacter sulfurreducens

The redox potential values of cytochromes can be modulated by the protonation/deprotonation of neighbor groups (redox-Bohr effect), a mechanism that permits the proteins to couple electron/proton transfer. In the respiratory chains, this effect is particularly relevant if observed in the physiological pH range, as it may contribute to the electrochemical gradient for ATP synthesis. A constitutively produced family of five triheme cytochromes (PpcA−E) from the bacterium Geobacter sulfurreducens plays a crucial role in extracellular electron transfer, a hallmark that permits this bacterium to be explored for several biotechnological applications. Two members of this family (PpcA and PpcD) couple electron/proton transfer in the physiological pH range, a feature not shared with PpcB and PpcE. That ability is crucial for G. sulfurreducens’ growth in Fe(III)-reducing habitats since extra contributors to the electrochemical gradient are needed. It was postulated that the redox-Bohr effect is determined by the nature of residue 6, a leucine in PpcA/PpcD and a phenylalanine in PpcB/PpcE. To confirm this hypothesis, Phe6 was replaced by leucine in PpcB and PpcE. The functional properties of these mutants were investigated by NMR and UV-visible spectroscopy to assess their capability to couple electron/proton transfer in the physiological pH range. The results obtained showed that the mutants have an increased redox-Bohr effect and are now capable of coupling electron/proton transfer. This confirms the determinant role of the nature of residue 6 in the modulation of the redox-Bohr effect in this family of cytochromes, opening routes to engineer Geobacter cells with improved biomass production.

Diversity in Starvation Survival Strategies and Outcomes among Heterotrophic Proteobacteria

Heterotrophic Proteobacteria are versatile opportunists that have been extensively studied as model organisms in the laboratory, as both pathogens and beneficial symbionts of plants and animals, and as ubiquitous organisms found free-living in many environments. Succeeding in these niches requires an ability to persist for potentially long periods of time in growth-arrested states when essential nutrients become limiting. The tendency of these bacteria to grow in dense biofilm communities frequently leads to the development of steep nutrient gradients and deprivation of interior cells even when the environment is nutrient rich. Surviving within host environments also likely requires tolerating growth arrest due to the host limiting access to nutrients and transitioning between hosts may require a period of survival in a nutrient-poor environment. Interventions to maximise plant-beneficial activities and minimise infections by bacteria will require a better understanding of metabolic and regulatory networks that contribute to starvation survival, and how these networks function in diverse organisms. Here we focus on carbon starvation as a growth-arresting condition that limits availability not only of substrates for biosynthesis but also of energy for ongoing maintenance of the electrochemical gradient across the cell envelope and cellular integrity. We first review models for studying bacterial starvation and known strategies that contribute to starvation survival<i>.</i> We then present the results of a survey of carbon starvation survival strategies and outcomes in ten bacterial strains, including representatives from the orders Enterobacterales and Pseudomonadales (both Gammaproteobacteria) and Burkholderiales (Betaproteobacteria). Finally, we examine differences in gene content between the highest and lowest survivors to identify metabolic and regulatory adaptations that may contribute to differences in starvation survival.

Computational Model for Membrane Transporters. Potential Implications for Cancer

To explain the increased transport of nutrients and metabolites and to control the movement of drug molecules through the transporters to the cancer cells, it is important to understand the exact mechanism of their structure and activity, as well as their biological and physical characteristics. We propose a computational model that reproduces the functionality of membrane transporters by quantifying the flow of substrates through the cell membrane. The model identifies the force induced by conformational changes of the transporter due to hydrolysis of ATP, in ABC transporters, or by an electrochemical gradient of ions, in secondary transporters. The transport rate is computed by averaging the velocity generated by the force along the paths followed by the substrates. The results obtained are in accordance with the experiments. The model provides an overall framework for analyzing the membrane transport proteins that regulate the flows of ions, nutrients and other molecules across the cell membranes, and their activities.

Application of a Statistical and Linear Response Theory to Multi-Ion Na+ Conduction in NaChBac

Biological ion channels are fundamental to maintaining life. In this manuscript we apply our recently developed statistical and linear response theory to investigate Na+ conduction through the prokaryotic Na+ channel NaChBac. This work is extended theoretically by the derivation of ionic conductivity and current in an electrochemical gradient, thus enabling us to compare to a range of whole-cell data sets performed on this channel. Furthermore, we also compare the magnitudes of the currents and populations at each binding site to previously published single-channel recordings and molecular dynamics simulations respectively. In doing so, we find excellent agreement between theory and data, with predicted energy barriers at each of the four binding sites of ∼4,2.9,3.6, and 4kT.

Competition between protons and substrate for binding to the major facilitator superfamily multidrug/H+ antiporter MdtM

Proton electrochemical gradient-driven multidrug efflux activity of representatives of the major facilitator superfamily (MFS) of secondary active transporters contributes to antimicrobial resistance of pathogenic bacteria. Integral to the mechanism of these transporters is a proposed competition between substrate and protons for the binding site of the protein. The current work investigated the competition between protons and antimicrobial substrate for binding to the Escherichia coli MFS multidrug/H+ antiporter MdtM by measuring the quench of intrinsic protein fluorescence upon titration of substrate tetraphenylphosphonium into a solution of purified MdtM over a range of pH values between pH 8.8 and 5.9. The results, which revealed that protons inhibit binding of substrate to MdtM in a competitive manner, are consistent with those reported in a study on the related MFS multidrug/H+ antiporter MdfA and provide further evidence that competition for binding between substrate and protons is a general feature of secondary multidrug efflux.

The Janus face of Ouabain in Na+, K+-ATPase and calcium signalling in neurons

Na+,K+ATPase (NKA), a transmembrane protein essential for maintaining the electrochemical gradient across the plasma membrane, acts as a receptor for cardiotonic steroids (CTS) such as ouabain. CTS binding to NKA, triggers signalling pathways or inhibits NKA activity in a concentration-dependent manner, resulting in a modulation of Ca2+ levels, which are essential for homeostasis in neurons. However, most of the pharmacological strategies for avoiding neuronal death do not target NKA activity, due to its complexity and poor comprehension of the mechanisms involved in NKA modulation. The present review aims to discuss two points regarding the interplay between NKA and Ca2+ signalling in the brain: NKA impairment causing illness as well as neuronal death due to Ca2+ signalling and benefits to the brain by modulating NKA activity. These interactions play an essential role in neuronal cell fate determination and are relevant to finding new targets for the treatment of neurodegenerative diseases.

Mechanistic picture for chemo-mechanical couplings in a bacterial proton-coupled oligopeptide transporter from Streptococcus thermophilus

Proton-coupled oligopeptide transporters (POTs) use the proton electrochemical gradient to transport peptides across the cell membrane. Despite the significant biological and biomedical relevance of these proteins, a detailed mechanistic picture for chemo-mechanical couplings involved in substrate/proton transport and protein structural changes is missing. We therefore performed microsecond-level molecular dynamics (MD) simulations of bacterial POT transporter PepTSt, which shares ~80% sequence identity with the human POT, PepT1, in the substrate binding region. Three different conformational states of PepTSt were simulated including, (i) occluded, apo, (ii) inward-facing, apo, and (iii) inward-facingoccluded, Leu-Ala bound. We propose that the interaction of R33 with E299 and E300 acts as a conformational switch (i.e., to trigger the conformational change from an inward-to outward-facing state) in the substrate transport. Additionally, E299 and E400 should disengage from interacting with the substrate either through protonation or through co-ordination with a cation for the substrate to get transported.

Mitochondrial Calcium Signaling as a Therapeutic Target for Alzheimer’s Disease

Mitochondria absorb calcium (Ca2+) at the expense of the electrochemical gradient generated during respiration. The influx of Ca2+ into the mitochondrial matrix helps maintain metabolic function and results in increased cytosolic Ca2+ during intracellular Ca2+ signaling. Mitochondrial Ca2+ homeostasis is tightly regulated by proteins located in the inner and outer mitochondrial membranes and by the cross-talk with endoplasmic reticulum Ca2+ signals. Increasing evidence indicates that mitochondrial Ca2+ overload is a pathological phenotype associated with Alzheimer’s Disease (AD). As intracellular Ca2+ dysregulation can be observed before the appearance of typical pathological hallmarks of AD, it is believed that mitochondrial Ca2+ overload may also play an important role in AD etiology. The high mitochondrial Ca2+ uptake can easily compromise neuronal functions and exacerbate AD progression by impairing mitochondrial respiration, increasing reactive oxygen species formation and inducing apoptosis. Additionally, mitochondrial Ca2+ overload can damage mitochondrial recycling via mitophagy. This review will discuss the molecular players involved in mitochondrial Ca2+ dysregulation and the pharmacotherapies that target this dysregulation. As most of the current AD therapeutics are based on amyloidopathy, tauopathy, and the cholinergic hypothesis, they achieve only symptomatic relief. Thus, determining how to reestablish mitochondrial Ca2+ homeostasis may aid in the development of novel AD therapeutic interventions.

Neuronal AMPK coordinates mitochondrial energy sensing and hypoxia resistance

ABSTRACTOrganisms adapt to their environment through coordinated changes in mitochondrial function and metabolism. The mitochondrial protonmotive force (PMF) is an electrochemical gradient that powers ATP synthesis and adjusts metabolism to energetic demands via cellular signaling. It is unknown how or where transient PMF changes are sensed and signaled due to lack of precise spatiotemporal control in vivo. We addressed this by expressing a light-activated proton pump in mitochondria to spatiotemporally “turn off” mitochondrial function through PMF dissipation in tissues with light. We applied our construct – mitochondria-OFF (mtOFF) – to understand how metabolic status impacts hypoxia resistance, a response that relies on mitochondrial function. mtOFF activation induced starvation-like behavior mediated by AMP-activated protein kinase (AMPK). We found prophylactic mtOFF activation increased survival following hypoxia, and that protection relied on neuronal AMPK. Our study links spatiotemporal control of mitochondrial PMF to cellular metabolic changes that mediate behavior and stress resistance.