Reduced intracellular ionic strength as the initial trigger for activation of endothelial volume-regulated anion channels

Abstract

Most mammalian cell types, including endothelial cells, respond to cell swelling by activating a Cl ؊ current termed I Cl,swell , but it is not known how the physical stimulus of cell swelling is transferred to the channels underlying I Cl,swell . We have investigated the precise relation between cell volume and I Cl,swell in endothelial cells by performing whole-cell current recordings while continuously monitoring cell thickness (T c ) as a measure for cell volume. The time course of T c was accurately predicted by a theoretical model that describes volume changes of patch-clamped cells in response to changes in the extracellular osmolality (OSM o ). This model also predicts significant changes in intracellular ionic strength (⌫ i ) when OSM o is altered. Under all experimental conditions I Cl,swell closely followed the changes in ⌫ i , whereas I Cl,swell and cell volume were often found to change independently. These results do not support the hypothesis that ⌫ i regulates the volume set point for activation of I Cl,swell . Instead, they are in complete agreement with a model in which a decrease of ⌫ i rather than an increase in cell volume is the initial trigger for activation of I Cl,swell .All living cells are programmed to activate a series of cellular processes to counter the harmful effects of cell swelling. One of the first detectable effects of cell swelling in most vertebrate cells is an increase in the plasma membrane permeability to anions, through the opening of anion channels (1). Although different types of swelling-activated anion currents have been functionally described, one phenotype seems to be predominant. This outwardly rectifying current, which under normal conditions is mainly carried by Cl Ϫ , has been termed I Cl,swell for swelling-activated Cl Ϫ current, and the underlying channel has been termed VRAC for volume-regulated anion channel. The biophysical and pharmacological properties of I Cl,swell ͞VRAC have been extensively studied (for recent reviews, see refs. 2-4), but the precise activation mechanism of I Cl,swell is still not resolved. Particularly, it remains unclear how the cell ''senses'' changes in its volume and translates this physical stimulus into the opening of VRAC.Hypotonicity-induced cell swelling is accompanied by a dilution of the intracellular medium, resulting in a decrease of ⌫ i , the intracellular ionic strength. Interestingly, it was recently shown that ⌫ i modulates I Cl,swell , independent of the molecular nature of the intracellular ions. A model was proposed in which ⌫ i regulates the volume set point for activation of VRAC, by modulating a putative ''volume sensor '' (5-7).In the present study we tested this model by investigating the relation between cell volume, ⌫ i , and I Cl,swell in endothelial cells. To this end, we set up a combined system that enables the simultaneous measurement of whole-cell currents and cell thickness (T c ). A theoretical model was developed that accurately predicts the osmotically induced volume changes and the conco...

Bestrophin 1 is indispensable for volume regulation in human retinal pigment epithelium cells

“…As suggested by Voets et al (44), this finding may be explained by VRAC activation in Best1 −/− spermatozoa before ejaculation and could be triggered by reduced intracellular ionic strength. As spermatozoa pass from the testis to the cauda of the epididymis, before ejaculation they are stored in a concentrated environment of high osmolality and low intracellular ionic strength compared with the testical environment (45).…”

Extracellular acidification elicits a chloride current that shares characteristics with ICl(swell)

“…I Cl(swell) is known to be activated by a number of maneuvers in nonswollen cells, e.g., activation by guanosine 5Ј-O-(3-thiotriphosphate) (GTP␥S) and low ionic strength (7,11,28,45). Because these maneuvers shift the set point of I Cl(swell) , osmotic shrinkage is known to be effective in blocking Cl Ϫ currents activated by the nonswelling maneuvers.…”

Role of volume-regulated and calcium-activated anion channels in cell volume homeostasis, cancer and drug resistance

“…7,[18][19][20] Activation and modulation of VRAC activity A decrease in the intracellular ionic strength due to cellular hydration is the main signal for VRAC activation. 21,22 Furthermore, VRAC is also activated under isotonic conditions by an increase in the intracellular concentration of GTP-g-S, presumably by a mechanism that involves the small G-protein RhoA and F-actin polymerization. 23,24 In agreement with this, VRAC is in several cell types demonstrated to be Rho and Rho-kinase regulated.…”

“…45 They show outward-rectification and a similar permeability sequence as described for VRAC (SCN ->I ->NO 3 ->Br ->Cl ->gluconate), they are activated by osmotic cell swelling in the presence of extracellular Ca 2C40 and requires cellular ATP. 22,43,44,46 However, VRAC and ANO1/6 activities can be distinguished by (i) their sensitivity to strongly buffered intracellular Ca 2C , i.e. VRAC can be activated whereas ANO1 cannot, (ii) activation/deactivation at positive potentials, i.e., VRAC deactivates 9,41,47 whereas ANO1/6 activates ( Fig.…”

“…[39][40][41][42] Finally, intracellular ATP seems to be required for VRAC activation. 22,43,44 For further discussion of VRAC activation and modulation see. 7,9,19 VRAC versus anoctamins The Ca 2C-activated channels ANO1 and ANO6 have been discussed as VRAC candidates.…”