Plant Proton Pumps and Cytosolic pH-Homeostasis.
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ABSTRACT:
SUBMITTER: Cosse M
PROVIDER: S-EPMC8220075 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In plants, environmental stressors trigger plasma membrane depolarizations. Being electrically interconnected via plasmodesmata, proper functional dissection of electrical signaling by electrophysiology is basically impossible. The green alga Chlamydomonas reinhardtii evolved blue light-excited channelrhodopsins (ChR1, 2) to navigate. When expressed in excitable nerve and muscle cells, ChRs can be used to control the membrane potential via illumination. In Arabidopsis plants, we used the algal ChR2-light switches as tools to stimulate plasmodesmata-interconnected photosynthetic cell networks by blue light and monitor the subsequent plasma membrane electrical responses. Blue-dependent stimulations of ChR2 expressing mesophyll cells, resting around -160 to -180 mV, reproducibly depolarized the membrane potential by 95 mV on average. Following excitation, mesophyll cells recovered their prestimulus potential not without transiently passing a hyperpolarization state. By combining optogenetics with voltage-sensing microelectrodes, we demonstrate that plant plasma membrane AHA-type H+-ATPase governs the gross repolarization process. AHA2 protein biochemistry and functional expression analysis in Xenopus oocytes indicates that the capacity of this H+ pump to recharge the membrane potential is rooted in its voltage- and pH-dependent functional anatomy. Thus, ChR2 optogenetics appears well suited to noninvasively expose plant cells to signal specific depolarization signatures. From the responses we learn about the molecular processes, plants employ to channel stress-associated membrane excitations into physiological responses.
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Project description:Cancer cells reprogram their metabolism to meet elevated energy demands and favor glycolysis for energy production. This boost in glycolytic flux supports proliferation, but also generates acid in the form of hydrogen ions that must be eliminated from the cytoplasm to maintain the alkaline intracellular pH (pHi) associated with transformation. To cope with acid production, tumor cells employ ion transport systems, including the family of sodium-hydrogen exchangers (NHE). Here, we identify NHE7 as a novel regulator of pHi in pancreatic ductal adenocarcinoma (PDAC). We determine that NHE7 suppression causes alkalinization of the Golgi, leading to a buildup of cytosolic acid that diminishes tumor cell fitness mainly through the dysregulation of actin. Importantly, NHE7 knockdown in vivo leads to the abrogation of tumor growth. These results identify Golgi acidification as a mechanism to control pHi and point to the regulation of pHi as a possible therapeutic vulnerability in PDAC. SIGNIFICANCE: NHE7 regulates cytosolic pH through Golgi acidification, which points to the Golgi as a "proton sink" for metabolic acid. Disruption of cytosolic pH homeostasis via NHE7 suppression compromises PDAC cell viability and tumor growth.See related commentary by Ward and DeNicola, p. 768.This article is highlighted in the In This Issue feature, p. 747.
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Project description:Ion transporters are key players of cellular processes. The mechanistic properties of ion transporters have been well elucidated by biophysical methods. Meanwhile, the understanding of their exact functions in cellular homeostasis is limited by the difficulty of monitoring their activity in vivo. The development of biosensors to track subtle changes in intracellular parameters provides invaluable tools to tackle this challenging issue. AtCLCa (Arabidopsis thaliana Chloride Channel a) is a vacuolar NO3 -/H+ exchanger regulating stomata aperture in A thaliana Here, we used a genetically encoded biosensor, ClopHensor, reporting the dynamics of cytosolic anion concentration and pH to monitor the activity of AtCLCa in vivo in Arabidopsis guard cells. We first found that ClopHensor is not only a Cl- but also, an NO3 - sensor. We were then able to quantify the variations of NO3 - and pH in the cytosol. Our data showed that AtCLCa activity modifies cytosolic pH and NO3 - In an AtCLCa loss of function mutant, the cytosolic acidification triggered by extracellular NO3 - and the recovery of pH upon treatment with fusicoccin (a fungal toxin that activates the plasma membrane proton pump) are impaired, demonstrating that the transport activity of this vacuolar exchanger has a profound impact on cytosolic homeostasis. This opens a perspective on the function of intracellular transporters of the Chloride Channel (CLC) family in eukaryotes: not only controlling the intraorganelle lumen but also, actively modifying cytosolic conditions.
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Project description:Pancreas secretes fluid rich in digestive enzymes and bicarbonate. The alkaline secretion is important in buffering of acid chyme entering duodenum and for activation of enzymes. This secretion is formed in pancreatic ducts, and studies to date show that plasma membranes of duct epithelium express H(+)/HCO(3)(-) transporters, which depend on gradients created by the Na(+)/K(+)-ATPase. However, the model cannot fully account for high-bicarbonate concentrations, and other active transporters, i.e. pumps, have not been explored. Here we show that pancreatic ducts express functional gastric and non-gastric H(+)-K(+)-ATPases. We measured intracellular pH and secretion in small ducts isolated from rat pancreas and showed their sensitivity to H(+)-K(+) pump inhibitors and ion substitutions. Gastric and non-gastric H(+)-K(+) pumps were demonstrated on RNA and protein levels, and pumps were localized to the plasma membranes of pancreatic ducts. Quantitative analysis of H(+)/HCO(3)(-) and fluid transport shows that the H(+)-K(+) pumps can contribute to pancreatic secretion in several species. Our results call for revision of the bicarbonate transport physiology in pancreas, and most likely other epithelia. Furthermore, because pancreatic ducts play a central role in several pancreatic diseases, it is of high relevance to understand the role of H(+)-K(+) pumps in pathophysiology.
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Project description:The Na/K pump hydrolyzes ATP to export three intracellular Na (Nai) as it imports two extracellular K (Ko) across animal plasma membranes. Within the protein, two ion-binding sites (sites I and II) can reciprocally bind Na or K, but a third site (site III) exclusively binds Na in a voltage-dependent fashion. In the absence of Nao and Ko, the pump passively imports protons, generating an inward current (IH). To elucidate the mechanisms of IH, we used voltage-clamp techniques to investigate the [H]o, [Na]o, and voltage dependence of IH in Na/K pumps from ventricular myocytes and in ouabain-resistant pumps expressed in Xenopus oocytes. Lowering pHo revealed that Ho both activates IH (in a voltage-dependent manner) and inhibits it (in a voltage-independent manner) by binding to different sites. Nao effects depend on pHo; at pHo where no Ho inhibition is observed, Nao inhibits IH at all concentrations, but when applied at pHo that inhibits pump-mediated current, low [Na]o activates IH and high [Na]o inhibits it. Our results demonstrate that IH is a property inherent to Na/K pumps, not linked to the oocyte expression environment, explains differences in the characteristics of IH previously reported in the literature, and supports a model in which 1), protons leak through site III; 2), binding of two Na or two protons to sites I and II inhibits proton transport; and 3), pumps with mixed Na/proton occupancy of sites I and II remain permeable to protons.
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Project description:Proton pumping of the vacuolar-type H(+)-ATPase into the lumen of the central plant organelle generates a proton gradient of often 1-2 pH units or more. Although structural aspects of the V-type ATPase have been studied in great detail, the question of whether and how the proton pump action is controlled by the proton concentration on both sides of the membrane is not understood. Applying the patch clamp technique to isolated vacuoles from Arabidopsis mesophyll cells in the whole-vacuole mode, we studied the response of the V-ATPase to protons, voltage, and ATP. Current-voltage relationships at different luminal pH values indicated decreasing coupling ratios with acidification. A detailed study of ATP-dependent H(+)-pump currents at a variety of different pH conditions showed a complex regulation of V-ATPase activity by both cytosolic and vacuolar pH. At cytosolic pH 7.5, vacuolar pH changes had relative little effects. Yet, at cytosolic pH 5.5, a 100-fold increase in vacuolar proton concentration resulted in a 70-fold increase of the affinity for ATP binding on the cytosolic side. Changes in pH on either side of the membrane seem to be transferred by the V-ATPase to the other side. A mathematical model was developed that indicates a feedback of proton concentration on peak H(+) current amplitude (v(max)) and ATP consumption (K(m)) of the V-ATPase. It proposes that for efficient V-ATPase function dissociation of transported protons from the pump protein might become higher with increasing pH. This feature results in an optimization of H(+) pumping by the V-ATPase according to existing H(+) concentrations.
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Project description:Recent evidence indicates that H+ extrusion in macrophages is in part accomplished by a H(+)-ATPase of vacuolar type. The presence and plasma-membrane localization of such a mechanism in adherent resident macrophages was verified by inhibition of H+ extrusion, monitored by changes in both cytosolic pH (pHi) and extracellular pH, with low concentrations of the H(+)-ATPase inhibitors N-ethylmaleimide and 7-chloro-4-nitrobenz-2-oxa-1,3-diazole. The H(+)-ATPase was operative at physiological pHi levels, thus contributing to maintenance of steady-state pHi. It was further shown to be sensitive to the plasma-membrane potential, with hyperpolarization being strongly inhibitory. In addition, H+ extrusion mediated by the H(+)-ATPase and the generation and release of lactic acid caused acidification of the pericellular space and could enable secreted lysosomal hydrolases to act extracellularly.
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Project description:Ehrlichia chaffeensis causes human monocytic ehrlichiosis. Little is known about how this and other related tick-borne rickettsia pathogens maintain pH homeostasis in acidified phagosomes and the extracellular milieu. The membrane-bound sodium (cation)/proton antiporters are found in a wide range of organisms aiding pH homeostasis. We recently reported a mutation in an antiporter gene of E. chaffeensis (ECH_0379) which causes bacterial in vivo attenuation. The E. chaffeensis genome contains 10 protein coding sequences encoding for predicted antiporters. We report here that nine of these genes are transcribed during the bacterial growth in macrophages and tick cells. All E. chaffeensis antiporter genes functionally complemented antiporter deficient Escherichia coli. Antiporter activity for all predicted E. chaffeensis genes was observed at pH 5.5, while gene products of ECH_0179 and ECH_0379 were also active at pH 8.0, and ECH_0179 protein was complemented at pH 7.0. The antiporter activity was independently verified for the ECH_0379 protein by proteoliposome diffusion analysis. This is the first description of antiporters in E. chaffeensis and demonstrates that the pathogen contains multiple antiporters with varying biological functions, which are likely important for the pH homeostasis of the pathogen's replicating and infectious forms.
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Project description:The rate of phosphorylation of ADP by isolated mitochondria respiring on succinate was set by addition of ATP, ADP or ADP plus malonate. We measured the rates of phosphorylation and respiration and the protonmotive force under each of these conditions. We measured the oxygen consumption required to drive the proton leak at the protonmotive force reached under each condition and subtracted it from the respiration rate during phosphorylation to determine the oxygen consumption driving phosphorylation. By dividing the rate of phosphorylation by the rate of respiration driving phosphorylation we calculated the mechanistic P/O ratio (number of molecules of ADP phosphorylated per oxygen atom reduced). This ratio was the same at high, intermediate and low values of protonmotive force, indicating that the relative stoichiometries of the mitochondrial protonmotive-force-producing and protonmotive-force-consuming pumps (i.e. H+/O:H+/ATP) are independent of the protonmotive force. This greatly weakens the case for a decrease in stoichiometry, or 'slip', in the mitochondrial proton pumps at high protonmotive force.
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Project description:The ability to silence the activity of genetically specified neurons in a temporally precise fashion would provide the opportunity to investigate the causal role of specific cell classes in neural computations, behaviours and pathologies. Here we show that members of the class of light-driven outward proton pumps can mediate powerful, safe, multiple-colour silencing of neural activity. The gene archaerhodopsin-3 (Arch) from Halorubrum sodomense enables near-100% silencing of neurons in the awake brain when virally expressed in the mouse cortex and illuminated with yellow light. Arch mediates currents of several hundred picoamps at low light powers, and supports neural silencing currents approaching 900 pA at light powers easily achievable in vivo. Furthermore, Arch spontaneously recovers from light-dependent inactivation, unlike light-driven chloride pumps that enter long-lasting inactive states in response to light. These properties of Arch are appropriate to mediate the optical silencing of significant brain volumes over behaviourally relevant timescales. Arch function in neurons is well tolerated because pH excursions created by Arch illumination are minimized by self-limiting mechanisms to levels comparable to those mediated by channelrhodopsins or natural spike firing. To highlight how proton pump ecological and genomic diversity may support new innovation, we show that the blue-green light-drivable proton pump from the fungus Leptosphaeria maculans (Mac) can, when expressed in neurons, enable neural silencing by blue light, thus enabling alongside other developed reagents the potential for independent silencing of two neural populations by blue versus red light. Light-driven proton pumps thus represent a high-performance and extremely versatile class of 'optogenetic' voltage and ion modulator, which will broadly enable new neuroscientific, biological, neurological and psychiatric investigations.
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