[The Role of Membrane-Bound Heat Shock Proteins Hsp90 in Migration of Tumor Cells in vitro and Involvement of Cell Surface Heparan Sulfate Proteoglycans in Protein Binding to Plasma Membrane].

Heat shock protein Hsp90, detected in the extracellular space and on the membrane of cells, plays an important role in cell motility, migration, invasion and metastasis of tumor cells. At present, the functional role and molecular mechanisms of Hsp90 binding to plasma membrane are not elucidated. Using isoform-specific antibodies against Hsp90, Hsp9α and Hsp90ß, we showed that membrane-bound Hsp90α and Hsp90ß play a significant role in migration of human fibrosarcoma (HT1080) and glioblastoma (A-172) cells in vitro. Disorders of sulfonation of cell heparan sulfates, cleavage of cell heparan. sulfates by heparinase I/III as well as treatment of cells with heparin lead to an abrupt reduction in the expression level of Hsp90 isoforms. Furthermore, heparin significantly inhibits tumor cell migration. The results obtained demonstrate that two isoforms of membrane-bound Hsp90 are involved in migration of tumor cells in vitro and that cell surface heparan sulfate proteoglycans play a pivotal role in the "anchoring" of Hsp90α and Hsp90ß to the plasma membrane.

Regulation of mitochondrial KATP channel by redox agents.

The ATP-dependent K+ channel (KATP) was purified from the inner mitochondrial membrane and reconstituted into lipid bilayer membranes. KATP activity was inhibited by high concentrations of ATP and ADP, but activated by low concentrations (up to 200 microM) of ADP. p-Diethylaminoethylbenzoate (DEB) acted as a KATP opener: at micromolar concentrations, it reversed inhibition by ATP and ADP and it also prevented KATP rundown. Pelargonidine, extracted from flowers of Pelargonium, reduced spontaneous activity of KATP channels and diminished their potentiation by DEB. Their opposite action on KATP corresponded with their opposite redox properties in reactions with free radicals: DEB behaved as an electron donor, whereas pelargonidine acted as an electron acceptor. We hypothesize that thiol groups on mitoKATP are targets for redox-active ligans.

Inhibition of 2,4-dinitrophenol-induced potassium efflux by adenine nucleotides in mitochondria.

The influence of nucleotides on 2,4-dinitrophenol (DNP)-induced K+ efflux from intact rat liver mitochondria has been studied. ATP and ADP at micromolar concentrations were found to inhibit mitochondrial potassium transport, whereas GTP, GDP, CTP, and UTP did not show tha same effect. The values of half-maximal inhibition (IC50) were approximately 20 microM for ATP and approximately 60 microM for ADP. It is suggested that adenine nucleotides exert their inhibitory action at the matrix side of the inner mitochondrial membrane since the inhibitor of adenine nucleotide translocase atractyloside at concentration of 1 microM completely removed the inhibitory effect of ATP and ADP. The mitochondrial ATPase inhibitor oligomycin (2 microg/ml) was found to reduce slightly the rate of DNP-induced K+ efflux and had no effect on inhibition by adenine nucleotides; the latter was insensitive to Mg2+ and the changes in pH. It seems likely that the regulation of potassium transport is not due to phosphorylation of the channel-forming protein but to binding of the nucleotides in specific regulatory sites. The possibility of potassium efflux from mitochondria in the presence of uncoupler via the ATP-dependent potassium channel is discussed.

Reconstitution of the mitochondrial ATP-dependent potassium channel into bilayer lipid membrane.

Electrical properties and regulation of the mitochondrial ATP-dependent potassium channel were studied. The channel protein was solubilized from the mitochondrial membrane using an ethanol/water mixture. Reconstituted into a bilayer lipid membrane BLM), the protein formed a slightly voltage-dependent channel with a conductance of 10 pS in 100 mM KCl. Often, several channels worked simultaneously (clusters) when many channels were incorporated into the BLM. The elementary channel and the clusters were both highly potassium selective. At concentrations of 1 to 10 microM, ATP favors channel opening, while channels become closed at 1-3 mM ATP. GDP (0.5 mM) reactivated the ATP-closed channels without affecting the untreated channels. The sulfhydryl-reducing agent ditiothreitol increased the open probability at concentrations of 1 to 3 mM, but damaged the selectivity of the channel.