Short-Term Regulation of the Proximal Tubule Na+,K+-ATPase: Increased/Decreased Na+,K+-ATPase Activity Mediated by Protein Kinase C Isoforms

In different species and tissues, a great variety of hormones modulate Na⁺,K⁺-ATPase activity in a short-term fashion. Such regulation involves the activation of distinct intracellular signaling networks that are often hormone- and tissue-specific. This minireview focuses on our own experimental observations obtained by studying the regulation of the rodent proximal tubule Na⁺,K⁺-ATPase. We discuss evidence that hormones responsible for regulating kidney proximal tubule sodium reabsorption may not affect the intrinsic catalytic activity of the Na⁺,K⁺-ATPase, but rather the number of active units within the plasma membrane due to shuttling Na⁺,K⁺-ATPase molecules between intracellular compartments and the plasma membrane. These processes are mediated by different isoforms of protein kinase C and depend largely on variations in intracellular sodium concentrations.     

CO2 and intracellular pH

Abstract The experimental determination of cytoplasmic and vacuolar pH values is discussed. Despite variation in these values evidence indicates that intracellular pH values are normally regulated within narrow limits. The regulatory mechanisms proposed involve the metabolic consumption of OH^& and the active efflux of H ⁺. The evidence for intracellular pH modification in response to CO₂ hydration and the production of HCO^?₃ and H⁺ is examined. Theoretical calculations and experimental data indicate that CO₂ concentrations as high as 5% will lower intracellular pH. Conversely, variation in CO₂ levels around atmospheric concentrations is unlikely to perturb intracellular pH. High CO₂ levels are found in bulky tissues, and flooded root systems. Evidence is presented that the slow diffusion of dissolved CO₂ compared to gaseous CO₂ results in its accumulation. It is proposed that the accumulation of respiratory CO₂ may reduce intracellular pH values when plant tissues, cells or protoplasts are maintained in a liquid culture medium. Finally, the possible role of dark CO₂ fixation and organic acid synthesis in the regulation of intracellular pH is examined.     

Quantification of Ca2+ binding to melanin supports the hypothesis that melanosomes serve a functional role in regulating calcium homeostasis

Calcium regulation in melanocytes affects numerous biological pathways including protecting the redox balance in the cell and regulating the supply of substrate, l ‐tyrosine, for melanogenesis. The pigment contained in the melanocytes, melanin, has been implicated in maintaining calcium homeostasis in the cell and is known to be involved with calcium ion regulation in the inner ear. Herein, the association constant for Ca²⁺ binding to Sepia melanin is determined by isothermal titration calorimetry to be 3.3 (±0.2) × 10³/M. This value is comparable with other well‐established intracellular calcium‐binding proteins that serve to buffer calcium concentrations, lending further support to the hypothesis that melanosomes serve as intracellular mediators of calcium homeostasis in melanocytes. Using this binding constant and the data from a fluorescent Ca²⁺ displacement assay, the pK_a of the carboxyl group coordinated to Ca²⁺ is determined to be 3.1 ± 0.1.     

Sensitivity-dependent model of protein-protein interaction networks

The scale free structure p(k)-k(-gamma) of protein-protein interaction networks can be reproduced by a static physical model in simulation. We inspect the model theoretically, and find the key reason for the model generating apparent scale free degree distributions. This explanation provides a generic mechanism of 'scale free' networks. Moreover, we predict the dependence of gamma on experimental protein concentrations or other sensitivity factors in detecting interactions, and find experimental evidence to support the prediction.     

Dysregulation of Calcium Homeostasis in Alzheimer’s Disease

The accumulation of oligomeric species of β-amyloid protein in the brain is considered to be a key factor that causes Alzheimer’s disease (AD). However, despite many years of research, the mechanism of neurotoxicity in AD remains obscure. Recent evidence strongly supports the theory that Ca²⁺ dysregulation is involved in AD. Amyloid proteins have been found to induce Ca²⁺ influx into neurons, and studies on transgenic mice suggest that this Ca²⁺ influx may alter neuronal excitability. The identification of a risk factor gene for AD that may be involved in the regulation of Ca²⁺ homeostasis and recent findings which suggest that presenilins may be involved in the regulation of intracellular Ca²⁺ stores provide converging lines of evidence that support the idea that Ca²⁺ dysregulation is a key step in the pathogenesis of AD. Special issue article in Honor of Dr. Graham Johnston.     

MD simulations of the central pore of ryanodine receptors and sequence comparison with 2B protein from coxsackie virus

The regulation of intracellular Ca^2 + triggers a multitude of vital processes in biological cells. Ca^2 + permeable ryanodine receptors (RyRs) are the biggest known ion channels and play a key role in the regulation of intracellular calcium concentrations, particularly in muscle cells. In this study, we construct a computational model of the pore region of the skeletal RyR and perform molecular dynamics (MD) simulations. The dynamics and distribution of Ca^2 + around the luminal pore entry of the RyR suggest that Ca^2 + ions are channeled to the pore entry due to the arrangement of (acidic) amino acids at the extramembrane surface of the protein. This efficient mechanism of Ca^2 + supply is thought to be part of the mechanism of Ca^2 + conductance of RyRs. Viral myocarditis is predominantly caused by coxsackie viruses that induce the expression of the protein 2B which is known to affect intracellular Ca^2 + homeostasis in infected cells. From our sequence comparison, it is hypothesized, that modulation of RyR could be due to replacement of its transmembrane domains (TMDs) by those domains of the viral channel forming protein 2B of coxsackie virus. This article is part of a Special Issue entitled: Viral Membrane Proteins — Channels for Cellular Networking.     

Calcium-dependent regulation of exocytosis

A rapid increase in intracellular calcium directly triggers regulated exocytosis. In addition, changes in intracellular calcium concentration can adjust the extent of exocytosis (quantal content) or the magnitude of individual release events (quantal size) in both the short- and long-term. It is generally agreed that calcium achieves this regulation via an interaction with a number of different molecular targets located at or near to the site of membrane fusion. We review here the synaptic proteins with defined calcium-binding domains and protein kinases activated by calcium, summarize what is known about their function in membrane fusion and the experimental evidence in support of their involvement in synaptic plasticity.     

Pig sperm membrane microdomains contain a highly glycosylated 15-25-kDa wheat germ agglutinin-binding protein

A highly glycosylated protein, which has unique, novel features in localization, structure, and potential function, is found in pig sperm, and named WGA-gp due to its high binding property with wheat germ agglutinin (WGA). WGA-gp is localized mainly in flagella and enriched in membrane microdomains or lipid rafts. It is not detected by ordinary protein staining methods due to a high content of both N- and O-glycans consisting of neutral monosaccharides. Interestingly, WGA-gp may be involved in intracellular Ca²⁺ regulation. Treatment of sperm with anti-WGA-gp antibody enhances the amplitude of Ca²⁺ oscillation without changing the basal intracellular Ca²⁺ concentrations. All these features of WGA-gp, except for different carbohydrate structures occupying most part of the molecules, are similar to those of flagellasialin in sea urchin sperm, which regulates the intracellular Ca²⁺ concentration. Presence of carbohydrate-enriched flagellar proteins involved in intracellular Ca²⁺ regulation may be a common feature among animal sperm.     

Membrane mechanisms of excretion of physiologically active substances by bacteria (review)

The review covers the evidence on the mechanisms of regulation of intracellular concentrations of low-molecular weight physiologically active substances, as well as of other intermediate or end metabolites by means of their excretion from bacterial cells. These processes can be considered as a particular level of metabolic regulation that can be called "membrane regulation".     

Alkali Metal Cation Transport and Homeostasis in Yeasts

Summary: The maintenance of appropriate intracellular concentrations of alkali metal cations, principally K⁺ and Na⁺, is of utmost importance for living cells, since they determine cell volume, intracellular pH, and potential across the plasma membrane, among other important cellular parameters. Yeasts have developed a number of strategies to adapt to large variations in the concentrations of these cations in the environment, basically by controlling transport processes. Plasma membrane high-affinity K⁺ transporters allow intracellular accumulation of this cation even when it is scarce in the environment. Exposure to high concentrations of Na⁺ can be tolerated due to the existence of an Na⁺, K⁺-ATPase and an Na⁺, K⁺/H⁺-antiporter, which contribute to the potassium balance as well. Cations can also be sequestered through various antiporters into intracellular organelles, such as the vacuole. Although some uncertainties still persist, the nature of the major structural components responsible for alkali metal cation fluxes across yeast membranes has been defined within the last 20 years. In contrast, the regulatory components and their interactions are, in many cases, still unclear. Conserved signaling pathways (e.g., calcineurin and HOG) are known to participate in the regulation of influx and efflux processes at the plasma membrane level, even though the molecular details are obscure. Similarly, very little is known about the regulation of organellar transport and homeostasis of alkali metal cations. The aim of this review is to provide a comprehensive and up-to-date vision of the mechanisms responsible for alkali metal cation transport and their regulation in the model yeast Saccharomyces cerevisiae and to establish, when possible, comparisons with other yeasts and higher plants.     

The possible role of intracellular polyamines in mitochondrial metabolic regulation.

Studies were made on the effects of very low concentrations of the polyamine, spermine, on rat liver mitochondrial metabolism associated with β-hydroxybutyrate. The respiratory control ratio and the rate of respiration during ADP-ATP conversion are significantly altered with shifts in spermine concentrations of as little as 15.7 nMoles/ml within the physiological Mg⁺⁺ concentration range. These spermine concentration changes are small compared to the estimated hepatic intracellular levels of spermine which have been reported to be between 200 and 1200 nMoles/gm wet weight under normal conditions. There is now evidence that exposure of an animal to certain environmental conditions induces changes of 164 nMoles/gm wet weight in intracellular levels of liver spermine in a few hours. Also there is evidence that the concentration of intracellular polyamines is influenced by endocrines since the levels of the enzymes responsible for their synthesis are markedly affected by hormonal changes. Therefore, alterations of polyamine levels may play a role in mitochondrial metabolic regulation $\text{in vivo}$.     

Experimental and Theoretical Investigation of Arachidonic Acid Uptake in Macrophages

The kinetics of ³H-labeled arachidonic acid (AA, 10^—10-10^—5 M) incorporation into murine peritoneal macrophages was investigated. During the incorporation of AA into the cells, the steady state was reached at 10 h. The level of incorporation consisted of 48-50% for nanomolar concentrations and 28-30% for micromolar concentrations of AA. Exogenous AA in micromolar but not nanomolar concentrations stimulated [³H]AA release from intracellular stores of pre-labeled cells. A mathematical model fitting the behavior of the experimental system is proposed. The difference in the level of uptake of AA in nanomolar and micromolar concentrations is explained by the activation of AA release from intracellular stores at high concentrations of exogenous AA.     

Homology modeling, molecular dynamics and QM/MM study of the regulatory protein PhoP from Corynebacterium pseudotuberculosis

Corynebacterium pseudotuberculosis is a facultatively intracellular Gram-positive bacterium that causes caseous lymphadenitis, principally in sheep and goats, though sometimes in other species of animals, leading to considerable economic losses. This pathogen has a TCS known as PhoPR, which consists of a sensory histidine kinase protein (PhoR) and an intracellular response regulator protein (PhoP). This system is involved in the regulation of proteins present in various processes, including virulence. The regulation is activated by PhoP protein phosphorylation, an event that requires a magnesium (Mg²⁺) ion. Here we describe the 3D structure of the regulatory response protein (PhoP) of C. pseudotuberculosis through molecular modeling by homology. The model generated provides the first structural information on a full-length member of the OmpR/PhoP subfamily. Classical molecular dynamics was used to investigate the stability of the model. In addition, we used quantum mechanical/molecular mechanical techniques to perform (internal, potential) energy optimizations to determine the interaction energy between the Mg²⁺ ion and the structure of the PhoP protein. Analysis of the interaction energy residue by residue shows that Asp-16 and Asp-59 play an important role in the protein–Mg²⁺ ion interactions. These results may be useful for the future development of a new vaccine against tuberculosis based on genetic attenuation via a point mutation that results in the polar residue Asp-16 and/or Asp-59 being replaced with a nonpolar residue in the DNA-binding domain of PhoP of C. pseudotuberculosis.     

THE NATURE OF THE AMINO ACID POOL USED FOR PROTEIN SYNTHESIS IN RAT BRAIN SLICES

The incorporation into brain slice protein of externally provided [1-¹⁴C]valine was measured at varying levels of valine in the medium, under conditions of constant protein synthesis and equilibration of intracellular valine specific activity. The results indicate that the valine pool used for protein synthesis is not identical to the pool of total free valine. Neither does the incorporation solely occur from an extracellular pool which is in equilibrium with the incubation medium. The data are compatible with a two-site activation model in which aminoacylation of tRNA occurs at both an internal site utilizing amino acid from the intracellular pool and an external (possibly membranous) site converting extracellular valine directly to valyl-tRNA. A good fit to the experimental observations is also provided by a compartmented intracellular valine pool model.     

Mechanisms of Beat-to-Beat Regulation of Cardiac Pacemaker Cell Function by Ca Cycling Dynamics

Whether intracellular Ca²⁺ cycling dynamics regulate cardiac pacemaker cell function on a beat-to-beat basis remains unknown. Here we show that under physiological conditions, application of low concentrations of caffeine (2–4 mM) to isolated single rabbit sinoatrial node cells acutely reduces their spontaneous action potential cycle length (CL) and increases Ca²⁺ transient amplitude for several cycles. Numerical simulations, using a modified Maltsev-Lakatta coupled-clock model, faithfully reproduced these effects, and also the effects of CL prolongation and dysrhythmic spontaneous beating (produced by cytosolic Ca²⁺ buffering) and an acute CL reduction (produced by flash-induced Ca²⁺ release from a caged Ca²⁺ buffer), which we had reported previously. Three contemporary numerical models (including the original Maltsev-Lakatta model) failed to reproduce the experimental results. In our proposed new model, Ca²⁺ releases acutely change the CL via activation of the Na⁺/Ca²⁺ exchanger current. Time-dependent CL reductions after flash-induced Ca²⁺ releases (the memory effect) are linked to changes in Ca²⁺ available for pumping into sarcoplasmic reticulum which, in turn, changes the sarcoplasmic reticulum Ca²⁺ load, diastolic Ca²⁺ releases, and Na⁺/Ca²⁺ exchanger current. These results support the idea that Ca²⁺ regulates CL in cardiac pacemaker cells on a beat-to-beat basis, and suggest a more realistic numerical mechanism of this regulation.     

Ca microdomains and the control of insulin secretion

Nutrient-induced increases in intracellular free Ca²⁺ concentrations are the key trigger for insulin release from pancreatic islet β-cells. These Ca²⁺ changes are tightly regulated temporally, occurring as Ca²⁺ influx-dependent baseline oscillations. We explore here the concept that locally high [Ca²⁺] concentrations (i.e. Ca²⁺ microdomains) may control exocytosis via the recruitment of key effector proteins to sites of exocytosis. Importantly, recent advances in the development of organelle- and membrane-targeted green fluorescent protein (GFP-) or aequorin-based Ca²⁺ indicators, as well as in rapid imaging techniques, are providing new insights into the potential role of these Ca²⁺ microdomains in β-cells. We summarise here some of the evidence indicating that Ca²⁺ microdomains beneath the plasma membrane and at the surface of large dense core vesicles may be important in the normal regulation of insulin secretion, and may conceivably contribute to “ATP-sensitive K⁺-channel independent” effects of glucose. We also discuss evidence that, in contrast to certain non-excitable cells, direct transfer of Ca²⁺ from the ER to mitochondria via localised physical contacts between these organelles is relatively less important for efficient mitochondrial Ca²⁺ uptake in β-cells. Finally, we discuss evidence from single cell imaging that increases in cytosolic Ca²⁺ are not required for the upstroke of oscillations in mitochondrial redox state, but may underlie the reoxidation process.     

Multicellular model for intercellular synchronization in circadian neural networks

We developed a multicellular model characterized by a high degree of heterogeneity to investigate possible mechanisms that underlie circadian network synchronization and rhythmicity in the suprachiasmatic nucleus (SCN). We populated a two-dimensional grid with 400 model neurons coupled via γ-aminobutyric acid (GABA) and vasoactive intestinal polypeptide (VIP) neurotransmitters through a putative Ca²⁺ mediated signaling cascade to investigate their roles in gene expression and electrical firing activity of cell populations. As observed experimentally, our model predicted that GABA would affect the amplitude of circadian oscillations but not synchrony among individual oscillators. Our model recapitulated experimental findings of decreased synchrony and average periods, loss of rhythmicity, and reduced circadian amplitudes as VIP signaling was eliminated. In addition, simulated increases of VIP reduced periodicity and synchrony. We therefore postulated a physiological range of VIP within which the system is able to produce sustained and synchronized oscillations. Our model recapitulated experimental findings of diminished amplitudes and periodicity with decreasing intracellular Ca²⁺ concentrations, suggesting that such behavior could be due to simultaneous decrease of individual oscillation amplitudes and population synchrony. Simulated increases in Cl⁻ levels resulted in increased Cl⁻ influx into the cytosol, a decrease of inhibitory postsynaptic currents, and ultimately a shift of GABA-elicited responses from inhibitory to excitatory. The simultaneous reduction of IPSCs and increase in membrane resting potential produced GABA dose-dependent increases in firing rates across the population, as has been observed experimentally. By integrating circadian gene regulation and electrophysiology with intracellular and intercellular signaling, we were able to develop the first (to our knowledge) multicellular model that allows the effects of clock genes, electrical firing, Ca²⁺, GABA, and VIP on circadian system behavior to be predicted.     

Effects of the calcium ionophore A-23187 on the regulation of sugar transport in muscle

The distribution of (¹⁴C)-3-0-methyl-D-glucose and of (⁴⁵Ca) was followed in perifused left atria and intact hemidiaphragms of the rat. The carboxylic calcium ionophore A-23187 affected sugar and Ca²⁺ influx in parallel, with low concentrations inhibiting and higher ones stimulating influx under basal conditions. The stimulation of sugar transport by insulin, high concentrations of adrenaline or ouabain, or by K⁺-free medium was antagonized by the calcium ionophore. Likewise, A-23187 counteracted the depression of sugar transport caused by low concentrations of ouabain or adrenaline. These results support a role of Ca²⁺ in the regulation of sugar transport in muscle. However, increased influx of Ca²⁺ cannot explain all the effects of A-23187. It is suggested that the ionophore may also act by releasing Ca²⁺ from intracellular storage and binding sites.