The inorganic ion content of native aquatic bacteria.

In this study we have quantified the ionic content and volume of native aquatic, and two cultured bacteria, by X-ray microanalysis (XRMA) in the transmission electron microscope (TEM). The cellular concentrations of magnesium (means of 630 and 710 mM) were more than an order of a magnitude higher than the outside concentrations. The internal concentrations of sodium were on average 50-180 mM, and the [K+]/[Na+] ratios were in the range of 0.1-0.5; lowest for apparently nonactive bacteria. Magnesium and chloride probably act as the major components of cell turgor, since no other inorganic ions were present in comparable amounts. Our carbon and nitrogen measurements indicated that organic solutes are not likely to be present at significant concentrations. The estimated charge of inorganic ions (Na, Mg, P, Cl, K, and Ca) gave a positive net internal charge for most cells. However, in cultures of Vibrio natriegens, the high internal chloride concentration made the net inorganic charge negative in these cells. Our results suggest that growing marine bacterioplankton have an internal environment in which magnesium is the dominating cation. These results suggest that actively growing marine bacteria are physiologically adapted to high internal concentrations of both magnesium and chloride.     

Acting antisense: plasmid- and chromosome-encoded sRNAs from Gram-positive bacteria

sRNAs that act by base pairing were first discovered in plasmids, phages and transposons, where they control replication, maintenance and transposition. Since 2001, however, computational searches were applied that led to the discovery of a plethora of sRNAs in bacterial chromosomes. Whereas the majority of these chromsome-encoded sRNAs have been investigated in Escherichia coli, Salmonella and other Gram-negative bacteria, only a few well-studied examples are known from Gram-positive bacteria. Here, the author summarizes our current knowledge on plasmid- and chromosome-encoded sRNAs from Gram-positive species, thereby focusing on regulatory mechanisms used by these RNAs and their biological role in complex networks. Furthermore, regulatory factors that control the expression of these RNAs will be discussed and differences between sRNAs from Gram-positive and Gram-negative bacteria highlighted. The main emphasis of this review is on sRNAs that act by base pairing (i.e., by an antisense mechanism). Thereby, both plasmid-encoded and chromosome-encoded sRNAs will be considered.     

The role of volume-sensitive ion transport systems in regulation of epithelial transport

This review focuses on using the knowledge on volume-sensitive transport systems in Ehrlich ascites tumour cells and NIH-3T3 cells to elucidate osmotic regulation of salt transport in epithelia. Using the intestine of the European eel (Anguilla anguilla) (an absorptive epithelium of the type described in the renal cortex thick ascending limb (cTAL)) we have focused on the role of swelling-activated K+- and anion-conductive pathways in response to hypotonicity, and on the role of the apical (luminal) Na+-K+-2Cl- cotransporter (NKCC2) in the response to hypertonicity. The shrinkage-induced activation of NKCC2 involves an interaction between the cytoskeleton and protein phosphorylation events via PKC and myosin light chain kinase (MLCK). Killifish (Fundulus heteroclitus) opercular epithelium is a Cl(-)-secreting epithelium of the type described in exocrine glands, having a CFTR channel on the apical side and the Na+/K+ ATPase, NKCC1 and a K+ channel on the basolateral side. Osmotic control of Cl- secretion across the operculum epithelium includes: (i) hyperosmotic shrinkage activation of NKCC1 via PKC, MLCK, p38, OSR1 and SPAK; (ii) deactivation of NKCC by hypotonic cell swelling and a protein phosphatase, and (iii) a protein tyrosine kinase acting on the focal adhesion kinase (FAK) to set levels of NKCC activity.     

第二信使分子c-di-AMP调控细菌中钾离子转运的机制

钾离子(K~+)是维持生命体存活的必需元素。原核生物进化出一系列K~+转运系统,如Kdp系统﹑Ktr系统和Trk系统等,来维持胞内相对恒定的K~+浓度。环二腺苷酸单磷酸(cyclic diadenosine monophosphate,c-di-AMP)是新发现的第二信使分子,可以与K~+转运系统中的KdpD、KtrA和TrkA结合。当胞内c-di-AMP浓度高时,c-di-AMP会与K~+转运蛋白结合,降低其转运活性。c-di-AMP的靶标除蛋白质外,还有RNA元件,即c-di-AMP的核糖开关。高浓度的c-di-AMP与其核糖开关结合后,可抑制下游K~+转运蛋白编码基因,如kdp、ktr和trk操纵子以及kup基因的转录,从而调控K~+的转运。总之,胞内高浓度的c-di-AMP抑制细菌对K~+的吸收。c-di-AMP调控K~+转运机制的研究,不仅丰富了K~+转运的调控方式,而且也扩大了c-di-AMP的调控范围,为细菌的利用与防治提供了新思路。     

Receptor regulation of ion transport in the intestinal epithelium

The active transport of ions by the intestinal epithelium is regulated by a number of enteric neurotransmitters, hormones and other substances. Our knowledge of the receptors mediating the actions of these substances is generally fragmentary. This review summarizes current knowledge on the location and functional characteristics of transmitter receptors regulating transport function in the small intestine, highlighting recent research on cholinergic and bradykinin receptors.     

Calcium-sensing receptor regulation of renal mineral ion transport

Extracellular calcium has long been known to affect the rate and magnitude of renal calcium and phosphate recovery. In this review, we consider some of these findings in light of our present understanding of the tubular localization of the calcium-sensing receptor (CaSR). Experiments directly implicating the CaSR in regulating calcium and phosphate transport are described. These results point to an important role of the CaSR in regulating PTH-dependent calcium absorption by cortical thick ascending limbs and on PTH-sensitive proximal tubule phosphate transport. Possible avenues for further investigation are suggested.     

Mechanisms and regulation of ion transport in the renal collecting duct

1. In the present paper the ion transport function of the renal mammalian collecting duct and its regulation is briefly reviewed. 2. The epithelium is characterized by different cell types: principal cells, intercalated cells, type A, and intercalated cells, type B. 3. Using microelectrodes and various microscopic techniques active Na+ absorption as well as K+ secretion has been localized to the principal cells, while Cl- absorption was found to proceed largely, though not exclusively, through the tight junctions between cells. 4. Intercalated cells of type A, which prevail in the outer medullary collecting duct, secrete H+ and intercalated cells of type B, which are most frequent in the late cortical collecting duct, secrete HCO3-. 5. This specialization of different cells in transporting individual ions provides the basis for the efficient adaptive regulation of urinary ion excretion.     

Calcium and ATP regulation of ion transport in larval frog skin

Ion transport measured as short circuit current (Isc) across the skin of larval frogs is activated by amiloride, acetylcholine, and ATP. In many epithelia, ATP stimulation of Isc involves an increase in intracellular calcium. To define the role of changes in intracellular calcium in ATP stimulation of Isc in larval frog skin, epithelial cells were loaded with calcium by adding 5 μM ionomycin to a 2 mM calcium apical Ringer's solution. Calcium loading had no observable effect on baseline Isc or on stimulation by ATP. Minimizing changes in intracellular calcium by loading the cell with the calcium chelator BAPTA also had no measurable effect on ATP stimulation of Isc. When the apical side was bathed with Ca²⁺-free Ringer's solution, ionomycin increased Isc up to 15 μA. This increase was partially blocked by 2 mM Ca²⁺, 2 mM Mg²⁺, and 10 μM W-7. Other experiments showed that baseline-stimulated and ATP-stimulated Isc were always larger in 2 mM Mg²⁺ Ringer's compared to 2 mM Ca²⁺. In dissociated cells bathed in 2 mM Ca²⁺ Ringer's, ATP had no effect on intracellular calcium as measured by Fluo-LR fluorescence changes. In conclusion, ATP apparently stimulates Isc without concomitant changes in intracellular calcium. This is consistent with a directly ligand-gated receptor at the apical membrane with P2X-like characteristics. Accepted: 21 April 1999     

Separate regulation of transport and biosynthesis of leucine, isoleucine, and valine in bacteria.

Since both transport activity and the leucine biosynthetic enzymes are repressed by growth on leucine, the regulation of leucine, isoleucine, and valine biosynthetic enzymes was examined in Escherichia coli K-12 strain EO312, a constitutively derepressed branched-chain amino acid transport mutant, to determine if the transport derepression affected the biosynthetic enzymes. Neither the iluB gene product, acetohydroxy acid synthetase (acetolactate synthetase, EC 4.1.3.18), NOR THE LEUB gene product, 3-isopropylmalate dehydrogenase (2-hydroxy-4-methyl-3-carboxyvalerate-nicotinamide adenine dinucleotide oxido-reductase, EC 1.1.1.85), were significantly affected in their level of derepression or repression compared to the parental strain. A number of strains with alterations in the regulation of the branched-chain amino acid biosynthetic enzymes were examined for the regulation of the shock-sensitive transport system for these amino acids (LIV-I). When transport activity was examined in strains with mutations leading to derepression of the iluB, iluADE, and leuABCD gene clusters, the regulation of the LIV-I transport system was found to be normal. The regulation of transport in an E. coli strain B/r with a deletion of the entire leucine biosynthetic operon was normal, indicating none of the gene products of this operon are required for regulation of transport. Salmonella typhimurium LT2 strain leu-500, a single-site mutation affecting both promotor-like and operator-like function of the leuABCD gene cluster, also had normal regulation of the LIV-I transport system. All of the strains contained leucine-specific transport activity, which was also repressed by growth in media containing leucine, isoleucine and valine. The concentrated shock fluids from these strains grown in minimal medium or with excess leucine, isoleucine, and valine were examined for proteins with leucine-binding activity, and the levels of these proteins were found to be regulated normally. It appears that the branched-chain amino acid transport systems and biosynthetic enzymes in E. coli strains K-12 and B/r and in S. typhimurium strain LT2 are not regulated together by a cis-dominate type of mechanism, although both systems may have components in common.     

Phosphorylation by inorganic phosphate of sodium plus potassium ion transport adenosine triphosphatase. Four reactive states.

Native solium and potassium adenosine triphosphatase from guinea pig kidney accepted a phosphate group from radioactive inorganic phosphate to form an acyl phosphate bond at the active site in the presence or absence of sodium ion. Magnesium ion was always required. In the presence of sodium ion and absence of adenosine triphosphate, there was no phosphorylation by inorganic phosphate. Addition of unlabeled adenosine triphosphate produced a potassium-sensitive phosphoenzyme which exchanged its phosphate-group with radioactive inorganic phosphate. The dephosphoenzyme was an intermediate in this exchange. The rate constant for dephosphorylation was about 0.05 per second. Addition of rubidium ion, a congener of potassium ion, to the potassium-sensitive phosphoenzyme produced a phosphoenzyme labeled from inorganic phosphate with a corresponding rate constant of 0.26 per s. This was a rubidium-complexed phosphoenzyme. Addition of magnesium ion to potassium-sensitive phosphoenzyme converted it into insensitive phosphoenzyme, the splitting of which was not accelerated by potassium ion or by adenosine diphosphate. Its rate constant was 0.07 per s. In the absence of sodium ion and adenosine triphosphate, inorganic phosphate was incorporated directly into a similar insensitive phosphoenzyme. In the presence of potassium ion or rubidium ion, inorganic phosphate was incorporated into a potassium-complexed or rubidium-complexed phosphoenzyme which exchanged 32-P with inorganic phosphate completely in less than 3 s. Incorporation of inorganic phosphate into a complex of the enzyme with the inhibitor, ouabain, is already described in the literature. Its rate constant was about 0.02 per s. Thus there appear to be at least four reactive states of the phosphoenzyme which equilibrate measurably with inorganic phosphate, namely, potassium-sensitive phosphoenzyme, potassium-complexed phosphoenzyme, insensitive phosphoenzyme, and ouabain phosphoenzyme. Two of these reactive states are functional intermediates in native sodium and potassium ion transport adenosine triphosphatase. The results are compatible with control of the reactivity of the active site by conformational changes in the surrounding active center and with regulation of the energy level of the phosphate group according to the kind of monovalent cation bound to the enzyme.     

Basic and editing mechanisms underlying ion transport and regulation in NCX variants

Structure-dynamic analysis of archaeal NCX (NCX_Mj) provided new insights into the underlying mechanisms of ion selectivity, ion-coupled alternating access, ion occlusion, and transport catalysis. This knowledge is relevant, not only for prokaryotic and eukaryotic NCXs, but also for other families belonging to the superfamily of Ca²⁺/CA antiporters. In parallel with the ion transport mechanisms, the structure-dynamic determinants of regulatory CBD1 and CBD2 domains have been resolved according to which the Ca²⁺-induced allosteric signal is decoded at the two-domain interface and "secondarily" modified by a splicing segment at CBD2. The exon-dependent combinations within the splicing segment control the number of Ca²⁺ binding sites (from zero to three) at CBD2, as well as the Ca²⁺ binding affinity and Ca²⁺ off-rates at both CBDs. The exon-dependent combinations specifically rigidify the local segments at CBDs, yielding the Ca²⁺-dependent activation (through Ca²⁺ binding to CBD1) and Ca²⁺-dependent alleviation of Na⁺-induced inactivation (through Ca²⁺ binding with CBD2). The exon-dependent synergistic interactions between CBDs characteristically differ in NCX1 and NCX3, thereby underscoring the physiological relevance of structure-controlled shaping of ion-dependent regulation in tissue-specific NCX variants. How the ion-dependent regulatory modules operate in conjunction with other regulators (PIP₂, palmitoylation, XIP, among the others) of NCX is an open question that remains to be determined.