Hyaluronan Export through Plasma Membranes Depends on Concurrent K+ Efflux by Kir Channels

Hyaluronan is synthesized within the cytoplasm and exported into the extracellular matrix through the cell membrane of fibroblasts by the MRP5 transporter. In order to meet the law of electroneutrality, a cation is required to neutralize the emerging negative hyaluronan charges. As we previously observed an inhibiting of hyaluronan export by inhibitors of K+ channels, hyaluronan export was now analysed by simultaneously measuring membrane potential in the presence of drugs. This was done by both hyaluronan import into inside-out vesicles and by inhibition with antisense siRNA. Hyaluronan export from fibroblast was particularly inhibited by glibenclamide, ropivacain and BaCl2 which all belong to ATP-sensitive inwardly-rectifying Kir channel inhibitors. Import of hyaluronan into vesicles was activated by 150 mM KCl and this activation was abolished by ATP. siRNA for the K+ channels Kir3.4 and Kir6.2 inhibited hyaluronan export. Collectively, these results indicated that hyaluronan export depends on concurrent K+ efflux

Recovery of ΔF508-CFTR Function by Citrate

Treatment of cystic fibrosis relies so far on expensive and sophisticated drugs. A logical approach to rescuing the defective ΔF508-CFTR protein has not yet been published. Therefore, virtual docking of ATP and CFTR activators to the open conformation of the CFTR protein was performed. A new ATP binding site outside of the two known locations was identified. It was located in the cleft between the nucleotide binding domains NBD1 and NBD2 and comprised six basic amino acids in close proximity. Citrate and isocitrate were also bound to this site. Citrate was evaluated for its action on epithelial cells with intact CFTR and defective ΔF508-CFTR. It activated hyaluronan export from human breast carcinoma cells and iodide efflux, and recovered ΔF508-CFTR from premature intracellular degradation. In conclusion, citrate is an activator for ΔF508-CFTR and increases export by defective ΔF508-CFTR into the extracellular matrix of epithelial cells

Inhibition by exogenous hyaluronan.

<p>Human skin fibroblast were labelled with [³H] GlcN and incubated in the presence of the indicated exogenous hyaluronan concentrations. After 24 hours the radioactivity incorporated into [³H] hyaluronan was determined. The error bars indicate the mean of duplicate samples.</p

Inhibition of K_ir channels by siRNA.

<p>Human fibrosarcoma cells HT1080 were reverse transfected with 30 nM siRNA for K_ir3.4, three different K_ir6.2 siRNAs, a mixture of the three K_ir6.2 siRNAs or non-sense siRNA and incubated for 24 hours in serum free Quantum medium. <b>A.</b> Specific inhibition of mRNA. The downregulation of K_ir3.4 mRNA and K_ir6.2 mRNA was detected by rtPCR. <b>B.</b> The hyaluronan concentrations were determined in the culture media. The error bars indicate the standard error of 6 determinations. *p<0.01 (ANOVA test).</p

Effect of the K⁺ channel opener pinacidil.

<p>Human skin fibroblasts were grown in 96 well microtiter plates with increasing concentrations of pinacidil for 24 hours. The hyaluronan concentrations were determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039096#s4" target="_blank">methods</a> section. The error bars indicate the sd of 3 determinations.</p

Action profile of ion channel inhibitors, percentage of hyaluronan export and alteration of membrane potential at maximal inhibitory concentrations.

<p>Action profile of ion channel inhibitors, percentage of hyaluronan export and alteration of membrane potential at maximal inhibitory concentrations.</p

Effect of K⁺ export inhibitors on hyaluronan export and membrane potential.

<p>Human skin fibroblasts were grown in 96 well microtiter plates with increasing concentrations of valinomycin (A), glibenclamid (B), ropivacaine (C), amiloride (D), barium chloride (E) and verapamil (F) for 24 hours. The membrane potentials (- -) and hyaluronan concentrations (__) were determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039096#s4" target="_blank">methods</a> section. The error bars indicate the sd of 3 determinations.</p

Model of hyaluronan synthesis and export.

<p>The hyaluronan synthase assembles hyaluronan at the inner side of the plasma membrane, the chains are exported by the ABC transporter MRP5 from fibroblasts and retained on the cell surface by CD44. Concurrent K⁺ efflux is required for maintaining electroneutrality.</p