Lang, Florian, Gillian L. Busch, Markus Ritter, Harald Volkl, Siegfried Waldegger, Erich Gulbins, and Dieter Haussinger. Functional Significance of Cell Volume Regulatory Mechanisms. Physiol. Rev. ...

Functional Significance of Cell Volume Regulatory Mechanisms

The influence of macromolecular crowding and macromolecular confinement on biochemical reactions in physiological media.

Diversity and ubiquity of K channels.

Magnesium. An update on physiological, clinical and analytical aspects.

Microbial cells operate under governing constraints that limit their range of possible functions. With the availability of annotated genome sequences, it has become possible to reconstruct genome-scale biochemical reaction networks for microorganisms. The imposition of governing constraints on a reconstructed biochemical network leads to the definition of achievable cellular functions. In recent years, a substantial and growing toolbox of computational analysis methods has been developed to study the characteristics and capabilities of microorganisms using a constraint-based reconstruction and analysis (COBRA) approach. This approach provides a biochemically and genetically consistent framework for the generation of hypotheses and the testing of functions of microbial cells.

Genome-scale models of microbial cells: evaluating the consequences of constraints

Excess hemoglobin digestion and the osmotic stability ofPlasmodium falciparum–infected red blood cells

The physiological actions of Ca2+ as a trigger and second messenger depend on the maintenance of large inward resting Ca2+ gradients across the cell plasma membrane. An ATP-fuelled Ca-pump, originally discovered and still best characterized in human red cells1–4, is now believed to mediate resting Ca2+ extrusion in most animal cells. However, even in red cells, the truly physiological pump-leak turnover rate and cytoplasmic free Ca2+ level are unknown. Previous estimates5,6 were only very imprecise upper limits because normal intact red cells have a minute total pool of exchangeable Ca of less than 1 µmol l−1 cells7; Ca fluxes could not be measured without artificially increasing that pool with ionophores8–10 or disrupting the membrane to incorporate Ca buffers5. Both procedures leave the membrane considerably leakier than in intact cells. Here, we have increased the exchangeable Ca pool by non-disruptively loading a Ca-chelator into intact cells, using intracellular hydrolysis of a membrane-permeant ester11,12. The trapped chelator made the free cytoplasmic calcium concentration, [Ca2+]i, an easily defined function of directly measurable total cell Ca. We were then able to establish the physiological steady-state [Ca2+]i and pump-leak turnover rate of fresh cells suspended in their own plasma. If [Ca2+]i was lowered below the normal resting level, the Ca pump rate decreased according to the square of [Ca2+]i, and the inward Ca leak increased. The increase in leak did not develop if the cells were depleted of ATP and ADP.

Physiological [Ca2+]i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator.

Publisher Summary This chapter concentrates on the Ca-sensitive K permeability mechanism of red cells. Although the functional role of this process is completely unknown in these cells, the red cell membrane represents an extremely convenient experimental model for the study of such a mechanism. Ca-induced K flux is so easy to measure and so richly responsive to all sorts of treatments that it has stimulated more extensive than analytical research. The chapter explores that as an experimental model, the red cell has clear advantages in that Ca 2+ can be assessed and controlled better than in any other cell, and in that even minute selective changes in K permeability can be unambiguously determined by relatively simple methods. Furthermore, investigation of the molecular nature of the Ca-sensitive K channel is still at a very early stage. Moreover, a few attempts have been made to search for specific high affinity inhibitors with which the channels can be “labeled,” their surface density can be measured, but the results so far are not very promising.

Calcium Transport and the Properties of a Calcium-Activated Potassium Channel in Red Cell Membranes

DURING the formation of the mature mammalian erythrocyte, the reticulocyte sheds its nucleus, endoplasmic reticulum and mitochondria leaving a featureless cytoplasm with a high haemoglobin content surrounded by a plasma membrane. All Ca-accumulating organelles are lost and the mature red cell is maintained virtually Ca-free throughout its life1 by the low Ca permeability of its membrane2 perhaps aided by a powerful Ca-extrusion pump3. Most of our knowledge of Ca transport in red cells comes from studies with resealed ghosts, but in this preparation the results are often affected by alterations to the membrane and metabolism of the cell2–4. The main difficulty of using intact fresh cells in physiological conditions has been the impossibility of increasing their Ca content in a controlled way and of assessing the fraction of Ca which is ionised. Using a divalent-cation ionophore, A23187 (ref. 5), however, we have overcome these difficulties and found that cytoplasmic Ca buffering occurs as if the cell had a single large-capacity, low-affinity Ca buffer and that there are two Ca-translocating sites of equal affinity at the internal surface of the Ca pump.

Use of ionophore A23187 to measure cytoplasmic Ca buffering and activation of the Ca pump by internal Ca

A basic mathematical model of human red cells is presented which integrates the charge and nonideal osmotic behavior of hemoglobin and of other impermeant cell solutes with the ion transport properties of the red cell membrane. The computing strategy was designed to predict the behavior of all measurable variables in time in ways that optimize comparison with experimentally determined behavior. The need and applications of such a model are illustrated in three separate examples covering different areas of experimentation in the physiology and pathophysiology of red cells.

Virgilio L. Lew

Papers

Excess hemoglobin digestion and the osmotic stability ofPlasmodium falciparum–infected red blood cells

Physiological [Ca2+]i level and pump-leak turnover in intact red cells measured using an incorporated Ca chelator.

Calcium Transport and the Properties of a Calcium-Activated Potassium Channel in Red Cell Membranes

Use of ionophore A23187 to measure cytoplasmic Ca buffering and activation of the Ca pump by internal Ca

Volume, pH, and ion-content regulation in human red cells: Analysis of transient behavior with an integrated model