CD95-Mediated Proton Regulation.

The Na+/H+ exchanger NHE1 is at the crossroads of a large diversity of signaling pathways, whose activation modifies the cooperative response of the transporter to intracellular H+ ions. Here we show how the activation of the Na+/H+ exchanger NHE1 by the cleaved ligand of CD95 can be measured. We demonstrate two different methods designed to set intracellular pH at precise values. Then we show how these can be coupled to fast kinetics of lithium transport, which will enable to measure the NHE1 activity like for an enzyme, because they will yield rates of transport.

The cleaved FAS ligand activates the Na(+)/H(+) exchanger NHE1 through Akt/ROCK1 to stimulate cell motility.

Transmembrane CD95L (Fas ligand) can be cleaved to release a promigratory soluble ligand, cl-CD95L, which can contribute to chronic inflammation and cancer cell dissemination. The motility signaling pathway elicited by cl-CD95L remains poorly defined. Here, we show that in the presence of cl-CD95L, CD95 activates the Akt and RhoA signaling pathways, which together orchestrate an allosteric activation of the Na(+)/H(+) exchanger NHE1. Pharmacologic inhibition of Akt or ROCK1 independently blocks the cl-CD95L-induced migration. Confirming these pharmacologic data, disruption of the Akt and ROCK1 phosphorylation sites on NHE1 decreases cell migration in cells exposed to cl-CD95L. Together, these findings demonstrate that NHE1 is a novel molecular actor in the CD95 signaling pathway that drives the cl-CD95L-induced cell migration through both the Akt and RhoA signaling pathways.

NaV1.5 Na⁺ channels allosterically regulate the NHE-1 exchanger and promote the activity of breast cancer cell invadopodia.

The degradation of the extracellular matrix by cancer cells represents an essential step in metastatic progression and this is performed by cancer cell structures called invadopodia. NaV1.5 (also known as SCN5A) Na(+) channels are overexpressed in breast cancer tumours and are associated with metastatic occurrence. It has been previously shown that NaV1.5 activity enhances breast cancer cell invasiveness through perimembrane acidification and subsequent degradation of the extracellular matrix by cysteine cathepsins. Here, we show that NaV1.5 colocalises with Na(+)/H(+) exchanger type 1 (NHE-1) and caveolin-1 at the sites of matrix remodelling in invadopodia of MDA-MB-231 breast cancer cells. NHE-1, NaV1.5 and caveolin-1 co-immunoprecipitated, which indicates a close association between these proteins. We found that the expression of NaV1.5 was responsible for the allosteric modulation of NHE-1, rendering it more active at the intracellular pH range of 6.4-7; thus, it potentially extrudes more protons into the extracellular space. Furthermore, NaV1.5 expression increased Src kinase activity and the phosphorylation (Y421) of the actin-nucleation-promoting factor cortactin, modified F-actin polymerisation and promoted the acquisition of an invasive morphology in these cells. Taken together, our study suggests that NaV1.5 is a central regulator of invadopodia formation and activity in breast cancer cells.

Teaching new dogs old tricks: membrane biophysical properties in drug delivery and resistance.

"How do drugs cross the plasma membrane?" this may seem like a trivial question. This question is often overlooked to focus primarily on the different complex macro-molecular aspects involved in drug delivery or drug resistance. However, recent studies have highlighted the theme that to be fully understood, more knowledge of the underlying biology of the most complex biological processes involved in the delivery and resistance to drugs is needed. After all, why would a drug interact with a transporter then subsequently be excluded from P-glycoprotein (P-gp) expressing drug resistant cells? What are the determinants of this transition in behavior? Full consideration of the physical biology of drug delivery has allowed a better understanding of the reasons why specific membrane proteins are upregulated or overexpressed in drug resistant cells. This, in turn, allows us to identify new targets for drug chemicals. Better yet, it increases the significance of recents patents and underlines their importance in multi drug resistance.

Nongenomic effects of cisplatin: acute inhibition of mechanosensitive transporters and channels without actin remodeling.

Cisplatin is an antineoplastic drug, mostly documented to cause cell death through the formation of DNA adducts. In patients, it exhibits a range of short-term side effects that are unlikely to be related to its genomic action. As cisplatin has been shown to modify membrane properties in different cell systems, we investigated its effects on mechanosensitive ion transporters and channels. We show here that cisplatin is a noncompetitive inhibitor of the mechanosensitive Na(+)/H(+) exchanger NHE-1, with a half-inhibition concentration of 30 µg/mL associated with a decrease in V(max) and Hill coefficient. We also showed that it blocks the Cl(-) and K(+) mechanosensitive channels VSORC and TREK-1 at similar concentrations. In contrast, the nonmechanosensitive Cl(-) and K(+) channels CFTR and TASK-1 and the Na(+)-coupled glucose transport, which share functional features with VSORC, TREK-1, and NHE-1, respectively, were insensitive to cisplatin. We next investigated whether cisplatin action was due to a direct effect on membrane or to cortical actin remodeling that would affect mechanosensors. Using scanning electron microscopy, in vivo actin labeling, and atomic force microscopy, we did not observe any modification of the Young's modulus and actin cytoskeleton for up to 60 and 120 µg/mL cisplatin, whereas these concentrations modified membrane morphology. Our results reveal a novel mechanism for cisplatin, which affects mechanosensitive channels and transporters involved in cell fate programs and/or expressed in mechanosensitive organs in which cisplatin elicits strong secondary effects, such as the inner ear or the peripheral nervous system. These results might constitute a common denominator to previously unrelated effects of this drug.

Role of TASK2 in the control of apoptotic volume decrease in proximal kidney cells.

Apoptotic volume decrease (AVD) is prerequisite to apoptotic events that lead to cell death. In a previous study, we demonstrated in kidney proximal cells that the TASK2 channel was involved in the K+ efflux that occurred during regulatory volume decrease. The aim of the present study was to determine the role of the TASK2 channel in the regulation of AVD and apoptosis phenomenon. For this purpose renal cells were immortalized from primary cultures of proximal convoluted tubules (PCT) from wild type and TASK2 knock-out mice (task2-/-). Apoptosis was induced by staurosporine, cyclosporin A, or tumor necrosis factor alpha. Cell volume, K+ conductance, caspase-3, and intracellular reactive oxygen species (ROS) levels were monitored during AVD. In wild type PCT cells the K+ conductance activated during AVD exhibited characteristics of TASK2 currents. In task2-/- PCT cells, AVD and caspase activation were reduced by 59%. Whole cell recordings indicated that large conductance calcium-activated K+ currents inhibited by iberiotoxin (BK channels) partially compensated for the deletion of TASK2 K+ currents in the task2-/- PCT cells. This result explained the residual AVD measured in these cells. In both cell lines, apoptosis was mediated via intracellular ROS increase. Moreover AVD, K+ conductances, and caspase-3 were strongly impaired by ROS scavenger N-acetylcysteine. In conclusion, the main K+ channels involved in staurosporine, cyclosporin A, and tumor necrosis factor-alpha-induced AVD are TASK2 K+ channels in proximal wild type cells and iberiotoxin-sensitive BK channels in proximal task2-/- cells. Both K+ channels could be activated by ROS production.