This study investigated the volume-regulated anion channel (VRAC) of human cervical cancer SiHa cells under various culture conditions, testing the hypothesis that the progression of the cell cycle is accompanied by differential expression of VRAC activity. Exponentially growing SiHa cells expressed VRACs, as indicated by the presence of large outwardly rectifying currents activated by hypotonic stress with the anion permeability sequence I- > Br- > Cl-. VRACs were potently inhibited by tamoxifen with an IC50 of 4.6 [mu]M. Fluorescence-activated cell sorting (FACS) experiments showed that 59 +/- 0.5, 5 +/- 0.5 and 36 +/- 1.1% of unsynchronized, exponentially growing cervical cancer SiHa cells were in G0/G1, S and G2/M stage, respectively. Treatment with aphidicolin (5 [mu]M) arrested 88 +/- 1.4% of cells at the G0/G1 stage. Arrest of cell growth in the G0/G1 phase was accompanied by a significant decrease of VRAC activity. The normalized hypotonicity-induced current decreased from 48 +/- 5.2 pA pF-1 at +100 mV in unsynchronized cells to 15 +/- 2.6 pA pF-1 at +100 mV in aphidicolin-treated cells. After removal of aphidicolin, culturing in medium containing 10% fetal calf serum triggered a rapid re-entry into the cell cycle and a concomitant recovery of VRAC density. Pharmacological blockade of VRACs by tamoxifen or NPPB caused proliferating cervical cancer cells to arrest in the G0/G1 stage, suggesting that activity of this channel is critical for G1/S checkpoint progression. This study provides new information on the functional significance of VRACs in the cell cycle clock of human cervical cancer cells.

In human erythrocytes infected with the mature form of the malaria parasite Plasmodium falciparum, the cytosolic concentration of Na(+) is increased and that of K(+) is decreased. In this study, the membrane transport changes underlying this perturbation were investigated using a combination of (86)Rb(+), (43)K(+), and (22)Na(+) flux measurements and a semiquantitative hemolysis technique. From >15 h postinvasion, there appeared in the infected erythrocyte membrane new permeation pathways (NPP) that caused a significant increase in the basal ion permeability of the erythrocyte membrane and that were inhibited by furosemide (0.1 mM). The NPP showed the selectivity sequence Cs(+) > Rb(+) > K(+) > Na(+), with the K(+)-to-Na(+) permeability ratio estimated as 2.3. From 18 to 36 h postinvasion, the activity of the erythrocyte Na(+)/K(+) pump increased in response to increased cytosolic Na(+) (a consequence of the increased leakage of Na(+) via the NPP) but underwent a progressive decrease in the latter 12 h of the parasite's occupancy of the erythrocyte (36-48 h postinvasion). Incorporation of the measured ion transport rates into a mathematical model of the human erythrocyte indicates that the induction of the NPP, together with the impairment of the Na(+)/K(+) pump, accounts for the altered Na(+) and K(+) levels in the host cell cytosol, as well as predicting an initial decrease, followed by a lytic increase in the volume of the host erythrocyte.

A recent study on malaria‐infected human red blood cells (RBCs) has shown induced ion channel activity in the host cell membrane, but the questions of whether they are host‐ or parasite‐derived and their molecular nature have not been resolved. Here we report a comparison of a malaria‐induced anion channel with an endogenous anion channel in Plasmodium falciparum‐infected human RBCs. Ion channel activity was measured using the whole‐cell, cell‐attached and excised inside‐out configurations of the patch‐clamp method. Parasitised RBCs were cultured in vitro, using co‐cultured uninfected RBCs as controls. Unstimulated uninfected RBCs possessed negligible numbers of active anion channels. However, anion channels could be activated in the presence of protein kinase A (PKA) and ATP in the pipette solution or by membrane deformation. These channels displayed linear conductance (∼15 pS), were blocked by known anion channel inhibitors and showed the permeability sequence I− > Br− > Cl−. In addition, in less than 5 % of excised patches, an outwardly rectifying anion channel (∼80 pS, outward conductance) was spontaneously active. The host membrane of malaria‐infected RBCs possessed spontaneously active anion channel activity, with identical conductances, pharmacology and selectivity to the linear conductance channel measured in stimulated uninfected RBCs. Furthermore, the channels measured in malaria‐infected RBCs were shown to have a low open‐state probability (Po) at positive potentials, which explains the inward rectification of membrane conductance observed when using the whole‐cell configuration. The data are consistent with the presence of two endogenous anion channels in human RBCs, of which one (the linear conductance channel) is up‐regulated by the malaria parasite P. falciparum.

The KCl cotransporter (KCC) plays a significant role in the ionic and osmotic homeostasis of many cell types. Four KCC isoforms have been cloned. KCC1 and KCC4 activity is osmolality-sensitive and involved in volume regulation. KCC2, a neuronal-specific isoform, can lower intracellular Cl ؊ and is critical for inhibitory GABA responses in the mature central nervous system. KCC3, initially cloned from vascular endothelial cells, is widely but not universally distributed and has an unknown physiological significance. Here we show a tight link between the expression and activity of KCC3 and cell growth by a NIH͞3T3 fibroblast expression system. KCC3 activity is sensitive to [(dihydroindenyl)oxy] alkanoic acid (DIOA) and N-ethylmaleimide and is regulated by tyrosine phosphorylation. Osmotic swelling does not activate KCC3, and the process of regulatory volume decrease is refractory to DIOA, indicating that KCC3 is not involved in volume regulation. KCC3 expression enhances cell proliferation, and this growth advantage can be abolished by the inhibition of KCC3 by DIOA. Fluorescence-activated cell sorting measurements and Western blot analysis show DIOA caused a significant reduction of the cell fraction in proliferative phase and a change in phosphorylation of retinoblastoma protein (Rb) and cdc2, suggesting that KCC3 activity is important for cell cycle progression. Insulin-like growth factor-1 up-regulates KCC3 expression and stimulates cell growth. Tumor necrotic factor-␣ down-regulates KCC3 expression and causes growth arrest. These data indicate that KCC3 is an important KCC isoform that may be involved in cell proliferation. The KCl cotransporter (KCC) has been implicated not only in regulatory volume decrease but also in transepithelial salt absorption, renal K ϩ secretion, myocardial K ϩ loss during ischemia, and regulation of neuronal Cl Ϫ concentrations (1-3). A major advance in the understanding of KCC has been the recent identification of genes that encode four KCC isoforms. KCC1 is a ''housekeeping'' isoform for cell volume regulation (4). KCC2, a neuronal-specific isoform, by lowering intracellular Cl Ϫ , is critical for inhibitory GABA responses in the mature central nervous system (5, 6). The KCC3 gene is located on chromosome 15q13, which colocalizes with the gene for myoclonal epilepsy, and the mRNA of KCC3 has been found in vascular endothelial cells, heart, kidney, brain, placenta, liver, and lung (7-9). Despite the wide distribution of KCC3, next to nothing is known about the functional and regulatory properties of this isoform. KCC4, predominantly found in heart and kidney, is volume-sensitive and also contributes to volume regulation (9, 10).The present study was aimed at characterizing the regulation and function of the recently cloned human KCC3 by using the NIH͞3T3 fibroblast expression system. NIH͞3T3 cells, which, unlike many other cell types [such as human embryonic kidney (HEK)-293 cells and Xenopus oocytes], do not have endogenous KCC activity and therefore provide a suitable system fo...