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 transport of amino acids in kidney and intestine is critical for the supply of amino acids to all tissues and the homeostasis of plasma amino acid levels. This is illustrated by a number of inherited disorders affecting amino acid transport in epithelial cells, such as cystinuria, lysinuric protein intolerance, Hartnup disorder, iminoglycinuria, dicarboxylic aminoaciduria, and some other less well-described disturbances of amino acid transport. The identification of most epithelial amino acid transporters over the past 15 years allows the definition of these disorders at the molecular level and provides a clear picture of the functional cooperation between transporters in the apical and basolateral membranes of mammalian epithelial cells. Transport of amino acids across the apical membrane not only makes use of sodium-dependent symporters, but also uses the proton-motive force and the gradient of other amino acids to efficiently absorb amino acids from the lumen. In the basolateral membrane, antiporters cooperate with facilitators to release amino acids without depleting cells of valuable nutrients. With very few exceptions, individual amino acids are transported by more than one transporter, providing backup capacity for absorption in the case of mutational inactivation of a transport system.

Amino acid transport across mammalian intestinal and renal epithelia.

The NLRP3 (nucleotide-binding domain, leucine-rich-repeat-containing family, pyrin domain-containing 3) inflammasome mediates production of inflammatory mediators, such as IL-1β and IL-18, and as such is implicated in a variety of inflammatory processes, including infection, sepsis, autoinflammatory diseases, and metabolic diseases. The proximal steps in NLRP3 inflammasome activation are not well understood. Here we elucidate a critical role for Ca2+ mobilization in activation of the NLRP3 inflammasome by multiple stimuli. We demonstrate that blocking Ca2+ mobilization inhibits assembly and activation of the NLRP3 inflammasome complex, and that during ATP stimulation Ca2+ signaling is pivotal in promoting mitochondrial damage. C/EPB homologous protein, a transcription factor that can modulate Ca2+ release from the endoplasmic reticulum, amplifies NLRP3 inflammasome activation, thus linking endoplasmic reticulum stress to activation of the NLRP3 inflammasome. Our findings support a model for NLRP3 inflammasome activation by Ca2+-mediated mitochondrial damage.

https://www.pnas.org/content/pnas/109/28/11282.full.pdf

Critical role for calcium mobilization in activation of the NLRP3 inflammasome

Increased prevalence of oxidant stress and inflammation in patients with moderate to severe chronic kidney disease.

The maintenance of a constant volume in the face of extracellular and intracellular osmotic perturbation is essential for the normal function and survival of animal cells. Osmotically swollen cells restore their volume, exhibiting a regulatory volume decrease by releasing intracellular K+, Cl-, organic solutes, and obligated water. In many cell types, the volume regulatory effluxes of Cl- and some organic osmolytes are known to be induced by swelling-induced activation of anion channels that are characterized by their moderate outward rectification, cytosolic ATP dependency, and intermediate unitary conductance (10-100 pS). Recently, simultaneous measurements of cell size by light microscopy and whole cell Cl- current have shown that the Cl- current density is proportionally increased with an increase in the outer surface area, which is mainly achieved through unfolding of membrane invaginations by volume expansion. Thus this anion channel can somehow sense volume expansion and can be called the volume expansion-sensing outwardly rectifying (VSOR) anion channel. Its molecular identity and activation mechanism are yet to be elucidated. Three cloned proteins, ClC-2, P-glycoprotein, and pIcln, have been proposed as candidates for the VSOR anion channel. The unitary conductance, voltage dependency, anion selectivity, pH dependency, and pharmacology of the VSOR anion channel are distinct from the ClC-2 Cl- channel, which is also known to be sensitive to volume changes. Recent patch-clamp studies in combination with molecular biological techniques have shown that P-glycoprotein is not itself the channel protein but is a regulator of its volume sensitivity. Although there is still debate about another candidate protein, pIcln, the most recent study has suggested that this is likely to be a regulator of some other distinct Cl- channel. Identification of the VSOR anion channel protein per se, its volume-sensing mechanism, and its accessory/regulatory proteins at the molecular level is currently a subject of utmost physiological importance.

Volume expansion-sensing outward-rectifier Cl- channel: fresh start to the molecular identity and volume sensor.

[Hemodynamics of kidney medullary substance. Part I. Capillary passage time, blood volume, circulation, tissue hematocrit and oxygen consumption of kidney medullary substance in situ].

The lysosomal compartment as intracellular calcium store in MDCK cells: a possible involvement in InsP3-mediated Ca2+ release

The Na(+)/H(+) exchangers (NHEs) are among the major ion transporters involved in cell volume regulation. NHE activation leads to a cellular influx of Na(+) ions and extrusion of H(+) ions, which are readily replenished from intracellular buffers. This will result in a net import of Na(+). In many systems NHE operates in parallel to Cl(-)/ HCO3(-) exchange, resulting in cellular uptake of NaCl. The influx of osmotically obliged water will consequently lead to cell swelling. This makes NHEs suitable to serve as powerful mechanisms for increasing cell volume (CV). The low volume threshold for NHE activation enables the cells to respond to very minute reductions of the CV. By the coupling to the export of H(+) ions cell volume regulatory NHE activation may lead to changes in intracellular pH. On the other hand NHEs are activated by a broad variety of ligands and by intracellular acidosis, which, in turn, may consequently lead to cell swelling. In addition, NHEs are linked to other intracellular proteins and structures, like e.g. the cytoskeleton, which themelves are involved in the regulation of numerous cellular processes. Therefore NHEs link CV regulation to a diversity of cellular functions, both in physiological and pathophysiological conditions. Six isoforms of the Na(+)/H(+) exchanger, termed NHE1--6, have been cloned so far. NHE 1--5 are located in the plasma membrane, whereas NHE6 is sorted to the mitochondrial membrane. NHE1 and NHE6 are the ubiquitously expressed isoforms. The expression of the isoforms NHE2 to NHE5 is restricted to specific tissues and the pattern of their expression, as well as their subcellular localization indicate that they fulfill specialized functions. Cell shrinkage induced activation has been shown for NHE1,2 and 4. In contrast, NHE3 is inhibited by cell shrinkage. In many cells several isoforms are present and assigned to specific membrane domains where they may serve a functional crosstalk between the different ion transporters.

Na(+)/H(+)exchangers: linking osmotic dysequilibrium to modified cell function.

Reconstitution of purified ICln in lipid bilayer leads to functional ion channels showing varying rectification. The reconstituted single channels have a conductance of ≅3 pS and their open probability is sensitive to nucleoside analogues. Mutation of a putative nucleotide binding site identified at the predicted extracellular mouth of the ICln channel protein leads to the reduction of the nucleoside-analogue sensitivity. Reconstituted ICln channels can be permeated both by cations and anions. The relative permeability of cations over anions depends on the presence of calcium. In the presence of calcium reconstituted ICln channels are more permeable to bromide than chloride, and more permeable to potassium than sodium. Similarly in NIH3T3 fibroblasts, the relative permeability of cations over anions of swelling-dependent chloride channels depends on extracellular calcium. Site-directed mutagenesis revealed the calcium-binding site responsible for the shift of the selectivity from cations towards anions of reconstituted ICln channels. Additional indirect structural information has been obtained by mutating a histidine in the predicted pore region of ICln. This histidine seems to have access to the ion-conducting tunnel of the pore. Our experiments show that ICln can act as an ionic channel, which does not exclude additional functions of the protein in regulatory mechanisms of the cell. Since knocking down the ICln protein in fibroblasts and epithelial cells leads to an impaired regulatory volume decrease (RVD) after cytoplasmic swelling and reconstituted ICln channels show several biophysical features of ion channels activated after swelling, ICln is a molecular candidate for these channels.

Functional reconstitution of ICln in lipid bilayers.

Fluorescence-optical measurements of the intracellular chloride concentration facilitate identification of chloride movements across the cell membrane of living cells. The two main dyes used for this purpose are 6-methoxy-N-(3-sulfopropyl)quinolinium (SPQ) and 6-methoxy-quinolyl acetoethyl ester (MQAE). The use of both substances is impaired by their poor membrane permeability and therefore limited loading of the cells to be studied. Here we report the use of 6-methoxy-N-ethylquinolinium iodide (MEQ), a chloride-sensitive dye for which a membrane-permeable form is easily prepared. This makes the loading procedure as easy as with the acetoxymethyl (AM) forms of other dyes for sensing intracellular ions. In addition, the original method, which described absolute concentration measurements of chloride in the cytosol, was modified in so far as only relative measurements were made. This avoids the known limitations of single wavelength excitation and emission dyes with respect to exact concentration measurements. More-over, to enhance the signal-to-noise ratio the driving force for chloride was considerably increased by changing the original direction of the anion flux in the cells under investigation. We verified the method by using fibroblasts and activating ICln, a putative chloride channel cloned from epithelial cells and of paramount importance in the regulatory volume decrease in these cells. In the presence of SCN− the MEQ quench measured in NIH 3T3 fibroblasts is dramatically enhanced in hypotonically challenged cells compared with cells under isotonic conditions. Antisense oligodeoxynucleotides sensing I Cln considerably impeded the swelling-induced chloride current (I Cl) in NIH 3T3 fibroblasts. Accordingly, the chloride movement measured by the SCN− quench of the MEQ signal was significantly reduced. Similar results can be obtained in the presence of 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB) or 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), two known blockers of chloride transport in the plasma membrane of a variety of cells. In conclusion, fluroscence-optical measurements using MEQ as the chloride-sensitive dye provide a reliable and easy-to-use method for measuring changes of the chloride flux across the cell membrane of living cells.

Fluorescence-optical measurements of chloride movements in cells using the membrane-permeable dye diH-MEQ

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