William R. Harvey

Animal plasma membrane energization by proton-motive V-ATPases.

Proton‐translocating, vacuolar‐type ATPases, well known energizers of eukaryotic, vacuolar membranes, now emerge as energizers of many plasma membranes. Just as Na+ gradients, imposed by Na+/K+ ATPases, energize basolateral plasma membranes of epithelia, so voltage gradients, imposed by H+ V‐ATPases, energize apical plasma membranes. The energized membranes acidify or alkalinize compartments, absorb or secrete ions and fluids, and underwrite cellular homeostasis. V‐ATPases acidify extracellular spaces of single cells such as phagocytes and osteoclasts and of polarized epithelia, such as vertebrate kidney and epididymis. They alkalinize extracellular spaces of lepidopteran midgut. V‐ATPases energize fluid secretion by insect Malpighian tubules and fluid absorption by insect oocytes. They hyperpolarize external plasma membranes for Na+ uptake by amphibian skin and fish gills. Indeed, it is likely that ion uptake by osmotically active membranes of all fresh water organisms is energized by V‐ATPases. Awareness of plasma membrane energization by V‐ATPases provides new perspectives for basic science and presents new opportunities for medicine and agriculture. BioEssays 21:637–648, 1999.

Stoichiometry of K^+/H^+ antiport helps to explain extracellular pH 11 in a model epithelium

The stoichiometry of K+/H+ antiport was measured fluorometrically by the static head method in highly purified vesicles from goblet cell apical membranes of larval lepidopteran midgut. The measured stoichiometry of 1 K+/2 H+ explains how the antiport results in electrophoretic exchange of extracellular H+ for intracellular K+, driven by the voltage component of the proton‐motive force of an H+ translocating V‐ATPase that is located in the same membrane. In turn, the exchange of K+ for H+ helps to explain how the midgut contents are alkalinized to a pH of 11.

Alkalinization by chloride/bicarbonate pathway in larval mosquito midgut.

The midgut of mosquito larvae maintains a specific lumen alkalinization profile with large longitudinal gradients (pH ≈ 3 units⋅mm−1) in which an extremely alkaline (pH ≈ 11) anterior midgut lies between near-neutral posterior midgut and gastric cecum (pH 7–8). A plasma membrane H+ V-ATPase energizes this alkalinization but the ion carriers involved are unknown. Capillary zone electrophoresis of body samples with outlet conductivity detection showed a specific transepithelial distribution of chloride and bicarbonate/carbonate ions, with high concentrations of both anions in the midgut tissue: 68.3 ± 5.64 and 50.8 ± 4.21 mM, respectively. Chloride was higher in the hemolymph, 57.6 ± 7.84, than in the lumen, 3.51 ± 2.58, whereas bicarbonate was higher in the lumen, 58.1 ± 7.34, than the hemolymph, 3.96 ± 2.89. Time-lapse video assays of pH profiles in vivo revealed that ingestion of the carbonic anhydrase inhibitor acetazolamide and the ion exchange inhibitor DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid), at 10−4 M eliminates lumen alkalinization. Basal application of these inhibitors in situ also reduced gradients recorded with self-referencing pH-sensitive microelectrodes near the basal membrane by ≈65% and 85% respectively. Self-referencing chloride-selective microelectrodes revealed a specific spatial profile of transepithelial chloride transport with an efflux maximum in anterior midgut. Both acetazolamide and DIDS reduced chloride effluxes. These data suggest that an H+ V-ATPase-energized anion exchange occurs across the apical membrane of the epithelial cells and implicate an electrophoretic Cl−/HCO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} exchanger and carbonic anhydrase as crucial components of the steady-state alkalinization in anterior midgut of mosquito larvae.

ISOLATION OF GOBLET CELL APICAL MEMBRANE FROM TOBACCO HORNWORM MIDGUT AND PURIFICATION OF ITS VACUOLAR-TYPE ATPASE

Publisher Summary This chapter focuses on the isolation of goblet cell apical membrane from tobacco hornworm midgut and purification of its vacuolar-type ATPase. The only known method for isolation of the goblet cell apical membrane from larval lepidopteran midgut is described in this chapter. This chapter also described is the purification of the ATPase associated with this membrane as well as procedures for measuring both membrane-bound and purified solubilized ATPase activity. The chapter also discusses a detailed description of reliable and sensitive colorimetric methods for measurement of inorganic phosphate and protein is included. This chapter presents a discussion on the isolation of the goblet cell apical membrane. The isolated posterior midgut segment is placed lumen side down and the Malpighian tubules are removed.this chapter concludes with the discussion on Purification of the goblet cell apical membrane ATPase.

Molecular cloning, phylogeny and localization of AgNHA1: the first Na+/H+ antiporter (NHA) from a metazoan, Anopheles gambiae.

SUMMARY We have cloned a cDNA encoding a new ion transporter from the alimentary canal of larval African malaria mosquito, Anopheles gambiae Giles sensu stricto. Phylogenetic analysis revealed that the corresponding gene is in a group that has been designated NHA, and which includes (Na+ or K+)/H+ antiporters; so the novel transporter is called AgNHA1. The annotation of current insect genomes shows that both AgNHA1 and a close relative, AgNHA2, belong to the cation proton antiporter 2 (CPA2) subfamily and cluster in an exclusive clade of genes with high identity from Aedes aegypti, Drosophila melanogaster, D. pseudoobscura, Apis mellifera and Tribolium castaneum. Although NHA genes have been identified in all phyla for which genomes are available, no NHA other than AgNHA1 has previously been cloned, nor have the encoded proteins been localized or characterized. The AgNHA1 transcript was localized in An. gambiae larvae by quantitative real-time PCR (qPCR) and in situ hybridization. AgNHA1 message was detected in gastric caeca and rectum, with much weaker transcription in other parts of the alimentary canal. Immunolabeling of whole mounts and longitudinal sections of isolated alimentary canal showed that AgNHA1 is expressed in the cardia, gastric caeca, anterior midgut, posterior midgut, proximal Malpighian tubules and rectum, as well as in the subesophageal and abdominal ganglia. A phylogenetic analysis of NHAs and KHAs indicates that they are ubiquitous. A comparative molecular analysis of these antiporters suggests that they catalyze electrophoretic alkali metal ion/hydrogen ion exchanges that are driven by the voltage from electrogenic H+ V-ATPases. The tissue localization of AgNHA1 suggests that it plays a key role in maintaining the characteristic longitudinal pH gradient in the lumen of the alimentary canal of An. gambiae larvae.

The Plasma Membrane H+-V-ATPase from Tobacco Hornworm Midgut

The midgut plasma membrane V-ATPase from larval Manduca sexta,the tobacco hornworm, is the sole energizer of any epithelial ion transportin this tissue and is responsible for the alkalinization of the gut lumen upto a pH of more than 11. This mini-review deals with those topics of researchon this enzyme which may have contributed or are expected to contribute noveland general aspects to the field of V-ATPases. Topics dealt with includenovel subunits or the quaternary structure of the V1 complex, aswell as the regulation of the enzymes function by reversible dissociation ofthe V1 from the V0 complexes and by genetic control onthe transcriptional and posttranscriptional level.

A novel electrogenic amino acid transporter is activated by K+ or Na+, is alkaline pH-dependent, and is Cl--independent.

A new eukaryotic nutrient amino acid transporter has been cloned from an epithelium that is exposed to high voltages and alkaline pH. The full-length cDNA encoding this novel CAATCH1 (cation-anion-activated Amino acidtransporter/channel) was isolated using a polymerase chain reaction-based strategy, and its expression product inXenopus oocytes displayed a combination of several unique, unanticipated functional properties. CAATCH1 electrophysiological properties resembled those of Na+,Cl−-coupled neurotransmitter amine transporters, although CAATCH1 was cloned from a gut absorptive epithelium rather than from an excitable tissue. Amino acids such as l-proline, l-threonine, andl-methionine elicited complex current-voltage relationships in alkaline pH-dependent CAATCH1 that were reminiscent of the behavior of the dopamine, serotonin, and norepinephrine transporters (DAT, SERT, NET) in the presence of their substrates and pharmacological inhibitors such as cocaine or antidepressants. These I-V relationships indicated a combination of substrate-associated carrier current plus an independent CAATCH1-associated leakage current that could be blocked by certain amino acids. However, unlike all structurally related proteins, CAATCH1 activity is absolutely independent of Cl−. Unlike related KAAT1, CAATCH1 possesses a methionine-inhibitable constitutive leakage current and is able to switch its narrow substrate selectivity, preferring threonine in the presence of K+ but preferring proline in the presence of Na+.

Cationic pathway of pH regulation in larvae of Anopheles gambiae.

SUMMARY Anopheles gambiae larvae (Diptera: Culicidae) live in freshwater with low Na+ concentrations yet they use Na+ for alkalinization of the alimentary canal, for electrophoretic amino acid uptake and for nerve function. The metabolic pathway by which larvae accomplish these functions has anionic and cationic components that interact and allow the larva to conserve Na+ while excreting H+ and HCO3–. The anionic pathway consists of a metabolic CO2 diffusion process, carbonic anhydrase and Cl–/HCO3– exchangers; it provides weak HCO3– and weaker CO32– anions to the lumen. The cationic pathway consists of H+ V-ATPases and Na+/H+ antiporters (NHAs), Na+/K+ P-ATPases and Na+/H+ exchangers (NHEs) along with several (Na+ or K+):amino acid+/– symporters, a.k.a. nutrient amino acid transporters (NATs). This paper considers the cationic pathway, which provides the strong Na+ or K+ cations that alkalinize the lumen in anterior midgut then removes them and restores a lower pH in posterior midgut. A key member of the cationic pathway is a Na+/H+ antiporter, which was cloned recently from Anopheles gambiae larvae, localized strategically in plasma membranes of the alimentary canal and named AgNHA1 based upon its phylogeny. A phylogenetic comparison of all cloned NHAs and NHEs revealed that AgNHA1 is the first metazoan NHA to be cloned and localized and that it is in the same clade as electrophoretic prokaryotic NHAs that are driven by the electrogenic H+ F-ATPase. Like prokaryotic NHAs, AgNHA1 is thought to be electrophoretic and to be driven by the electrogenic H+ V-ATPase. Both AgNHA1 and alkalophilic bacterial NHAs face highly alkaline environments; to alkalinize the larva mosquito midgut lumen, AgNHA1, like the bacterial NHAs, would have to move nH+ inwardly and Na+ outwardly. Perhaps the alkaline environment that led to the evolution of electrophoretic prokaryotic NHAs also led to the evolution of an electrophoretic AgNHA1 in mosquito larvae. In support of this hypothesis, antibodies to both AgNHA1 and H+ V-ATPase label the same membranes in An. gambiae larvae. The localization of H+ V-ATPase together with (Na+ or K+):amino acid+/– symporter, AgNAT8, on the same apical membrane in posterior midgut cells constitutes the functional equivalent of an NHE that lowers the pH in the posterior midgut lumen. All NATs characterized to date are Na+ or K+ symporters so the deduction is likely to have wide application. The deduced colocalization of H+ V-ATPase, AgNHA1 and AgNAT8, on this membrane forms a pathway for local cycling of H+ and Na+ in posterior midgut. The local H+ cycle would prevent unchecked acidification of the lumen while the local Na+ cycle would regulate pH and support Na+:amino acid+/– symport. Meanwhile, a long-range Na+ cycle first transfers Na+ from the blood to gastric caeca and anterior midgut lumen where it initiates alkalinization and then returns Na+ from the rectal lumen to the blood, where it prevents loss of Na+ during H+ and HCO3– excretion. The localization of H+ V-ATPase and Na+/K+-ATPase in An. gambiae larvae parallels that reported for Aedes aegypti larvae. The deduced colocalization of the two ATPases along with NHA and NAT in the alimentary canal constitutes a cationic pathway for Na+-conserving midgut alkalinization and de-alkalinization which has never been reported before.

Cloning and sequencing of cDNA encoding the putative insect lasma membrane V-ATPase subunit A

For the first time a cDNA encoding subunit A of an invertebrate V‐ATPase has been sequenced. The cDNA library was prepared from larval midgut of the tobacco hornworm. Manduca sexta, and screened with monoclonal antibodies to the midgut plasma membrane subunit A. From the cDNA sequence the insect subunit A is predicted to consist of 617 amino acids with a relative molecular mass of 68.162. The predicted primary structure is similar to that of the published eukaryotic subunit A proteins (Bos, Daucus, Saccharomyces and Neurospora); it most closely resembles the bovine amino acid sequences with which it has an 83% sequence identity

Molecular characterization of the first aromatic nutrient transporter from the sodium neurotransmitter symporter family

SUMMARY Nutrient amino acid transporters (NATs, subfamily of sodium neurotransmitter symporter family SNF, a.k.a. SLC6) represent a set of phylogenetically and functionally related transport proteins, which perform intracellular absorption of neutral, predominantly essential amino acids. Functions of NATs appear to be critical for the development and survival in organisms. However, mechanisms of specific and synergetic action of various NAT members in the amino acid transport network are virtually unexplored. A new transporter, agNAT8, was cloned from the malaria vector mosquito Anopheles gambiae (SS). Upon heterologous expression in Xenopus oocytes it performs high-capacity, sodium-coupled (2:1) uptake of nutrients with a strong preference for aromatic catechol-branched substrates, especially phenylalanine and its derivatives tyrosine and L-DOPA, but not catecholamines. It represents a previously unknown SNF phenotype, and also appears to be the first sodium-dependent B0 type transporter with a narrow selectivity for essential precursors of catecholamine synthesis pathways. It is strongly and specifically transcribed in absorptive and secretory parts of the larval alimentary canal and specific populations of central and peripheral neurons of visual-, chemo- and mechano-sensory afferents. We have identified a new SNF transporter with previously unknown phenotype and showed its important role in the accumulation and redistribution of aromatic substrates. Our results strongly suggest that agNAT8 is an important, if not the major, provider of an essential catechol group in the synthesis of catecholamines for neurochemical signaling as well as ecdysozoan melanization and sclerotization pathways, which may include cuticle hardening/coloring, wound curing, oogenesis, immune responses and melanization of pathogens.