functional-characterization-na-h-exchangers-intracellular

Functional Characterization of Na<sup>+</sup>/H<sup>+</sup> Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics

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.

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

"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.

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

Vesicular H(+)-ATPases and ClC-chloride transporters are described to acidify intracellular compartments, which also express the highly conserved Na(+)/H(+) exchangers NHE6, NHE7, and NHE9. Mutations of these exchangers cause autism-spectrum disorders and neurodegeneration. NHE6, NHE7, and NHE9 are hypothesized to exchange cytosolic K(+) for H(+) and alkalinize vesicles, but this notion has remained untested in K(+) because their intracellular localization prevents functional measurements. Using proton-killing techniques, we selected a cell line that expresses wild-type NHE7 at the plasma membrane, enabling measurement of the exchanger's transport parameters. We found that NHE7 transports Li(+) and Na(+), but not K(+), is nonreversible in physiological conditions and is constitutively activated by cytosolic H(+). Therefore, NHE7 acts as a proton-loading transporter rather than a proton leak. NHE7 mediates an acidification of intracellular vesicles that is additive to that of V-ATPases and that accelerates endocytosis. This study reveals an unexpected function for vesicular Na(+)/H(+) exchangers and provides clues for understanding NHE-linked neurological disorders.

Article	Year
Nongenomic effects of cisplatin: acute inhibition of mechanosensitive transporters and channels without actin remodeling. Cancer research\| PubMed ID: 20841472	2010
Teaching new dogs old tricks: membrane biophysical properties in drug delivery and resistance. Recent patents on anti-cancer drug discovery\| PubMed ID: 21756245	2011
The intracellular Na(+)/H(+) exchanger NHE7 effects a Na(+)-coupled, but not K(+)-coupled proton-loading mechanism in endocytosis. Cell reports\| PubMed ID: 24767989	2014

Nina Milosavljevic

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