The ATP-sensitive potassium channel from the inner mitochondrial membrane (mitoK ATP ) is a highly selective conductor of K ؉ ions. When isolated in the presence of nonionic detergent and reconstituted in liposomes, mitoK ATP is inhibited with high affinity by ATP (K 1/2 ‫؍‬ 20 -30 M). We have suggested that holo-mitoK ATP is a heteromultimer consisting of an inwardly rectifying K The importance of the mitochondrial K ϩ cycle for volume regulation, reviewed by Garlid and Paucek (1) was recognized by Mitchell (2) long before any of the components of this cycle were discovered. K ϩ is driven into the matrix by the high membrane potential (⌬⌿) 1 generated by the proton-pumping electron transport system, and excess K ϩ is removed by the regulated K ϩ /H ϩ antiporter. Electrophoretic K ϩ influx occurs by diffusion and by means of ATP-sensitive potassium channel from the inner mitochondrial membrane (mitoK ATP ). At the high values of ⌬⌿ maintained by mitochondria, both of these processes increase exponentially with ⌬⌿ (3, 4) and are consequently very sensitive to fluctuations in ⌬⌿. These fluctuations, in turn, are high in tissues such as heart, which undergo large variations in energy demand and ATP synthesis rates (5). Thus, regulation of K ϩ influx and efflux pathways can be seen as a means of regulating volume in the face of the changing energy requirements of the cell. MitoK ATP plays more than a housekeeping role in cell physiology. There is now general agreement that mitoK ATP plays a key role in cardioprotection against ischemia-reperfusion injury (6, 7). The proposed mechanisms of this protective effect of mitoK ATP opening (5, 8, 9) are plausible; however, it is evident that more needs to be known about the functional properties of mitoK ATP before its role in vivo can be ascertained.By using a novel ethanol extraction technique, Mironova et al. (10) were the first to report reconstitution in lipid bilayer membranes of a 55-kDa K ϩ channel from mitochondria. Paucek et al. (3) used a detergent extraction technique and were the first to report reconstitution of mitoK ATP in liposomes. The latter channel was associated with two proteins of molecular mass 55 and 63 kDa, and we hypothesized that mitoK ATP is a heteromultimeric complex consisting of a 55-kDa inwardly rectifying K ϩ channel (mitoKIR) and a 63-kDa sulfonylurea receptor (mitoSUR), analogous to the plasma membrane ATPdependent K ϩ channel (cellK ATP ) (11,12). In this report, we focus on three interactions that address the key question of whether the 55-kDa K ϩ channel protein observed in the ethanol purification is the same as the 55-kDa protein purified with detergents. First, we show that UDP reverses ATP-inhibition of K ϩ flux mediated by both mitoKIR and mitoK ATP reconstituted in liposomes. Moreover, UDP exerts the same action in isolated mitochondria, and the affinities for the opening effect of UDP are about the same in each preparation. Second, we show that the mitoKIR opener p-diethylaminoethylbenzoate (DEB) also activates K ϩ flux via ...

The HSPs (heat-shock proteins) of the 70-kDa family, the constitutively expressed HSC70 (cognate 70-kDa heat-shock protein) and the stress-inducible HSP70 (stress-inducible 70-kDa heat-shock protein), have been reported to be actively secreted by various cell types. The mechanisms of the release of these HSPs are obscure, since they possess no consensus secretory signal sequence. We showed that baby hamster kidney (BHK-21) cells released HSP70 and HSC70 in a serum-free medium and that this process was the result of an active secretion of HSPs rather than the non-specific release of the proteins due to cell death. It was found that the secretion of HSP70 and HSC70 is independent of de novo protein synthesis. BFA (Brefeldin A) did not inhibit the basal secretion of HSPs, indicating that the secretion of HSP70 and HSC70 from cells occurs by a non-classical pathway. Exosomes did not contribute to the secretion of HSP70 and HSC70 by cells. MBC (methyl-beta-cyclodextrin), a substance that disrupts the lipid raft organization, considerably reduced the secretion of both HSPs, indicating that lipid rafts are involved in the secretion of HSP70 and HSC70 by BHK-21 cells. The results suggest that HSP70 and HSC70 are actively secreted by BHK-21 cells in a serum-free medium through a non-classical pathway in which lipid rafts play an important role.

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.

The extracellular heat shock protein 90 (Hsp90) is known to participate in cell migration and invasion. Recently, we have shown that cell surface heparan sulfate proteoglycans (HSPGs) are involved in the binding and anchoring of extracellular Hsp90 to the plasma membrane, but the biological relevance of this finding was unclear. Here, we demonstrated that the digestion of heparan sulfate (HS) moieties of HSPGs with a heparinase I/III blend and the metabolic inhibition of the sulfation of HS chains by sodium chlorate considerably impair the migration and invasion of human glioblastoma A-172 and fibrosarcoma HT1080 cells stimulated by extracellular native Hsp90. Heparin, a polysaccharide closely related to HS, also reduced the Hsp90-stimulated migration and invasion of cells. Phorbol 12-myristate 13-acetate, an intracellular inducer of cell motility bypassing the ligand activation of receptors, restored the basal migration of heparinase-and chlorate-treated cells almost to the control level, suggesting that the cell motility machinery was insignificantly affected in cells with degraded and undersulfated HS chains. On the other hand, the downstream phosphorylation of AKT in response to extracellular Hsp90 was substantially impaired in heparinase-and chloratetreated cells as compared to untreated cells. Taken together, our results demonstrated for the first time that cell surface HSPGs play an important role in the migration and invasion of cancer cells stimulated by extracellular Hsp90 and that plasma membraneassociated HSPGs are required for the efficient transmission of signal from extracellular Hsp90 into the cell.

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.

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Heat shock proteins (HSPs) hsp70/hsc70, hsp90 and hsp96 were separated from mammalian cells and tissues on a gel obtained by the reaction of beta-mercaptoethanol with divinyl sulfone-activated Sepharose CL-6B (thiophilic gel or T-gel). Hsp90 revealed a much higher affinity towards the T-gel than the other HSPs. One-step thiophilic interaction chromatography of proteins resulted in a more than 80% purity and 85% yield of hsp90. Based on this observation, a simple and efficient method for the purification of hsp90 and a procedure for the simultaneous purification of several HSPs (hsp70/hsc70, hsp90 and hsp96) using thiophilic interaction chromatography was developed. All the HSPs were recovered with a high yield and purity (90-99%). The results indicated that the thiophilic gel is a highly efficient affinity matrix for the purification of hsp90 and can be used in the protocols of purification of different HSPs from cells and tissues of various animal species.

Glucose-regulated protein 94 (grp94) is a major component of the endoplasmic reticulum (ER) lumen of eukaryotic cells. We showed that grp94 is released from baby hamster kidney (BHK-21) cells into a serum-free medium. The exit of grp94 into the medium was not related to the protein discharge due to cell death and was independent of de novo protein synthesis. The treatment of cells with brefeldin A and monensin, the inhibitors of the classical pathway of protein secretion, did not decrease the extracellular level of grp94, indicating that the discharge of grp94 from cells does not occur through the ER/Golgi-dependent pathway. Exosomes, membrane vesicles secreted by several cell types, were not involved in the release of grp94 from cells. Methyl-β-cyclodextrin, a substance that disrupts the lipid raft organization, considerably reduced the extracellular level of grp94, indicating that lipid rafts are involved in the liberation of grp94 from BHK-21 cells. The results suggest that BHK-21 cells release grp94 into the serum-free medium via the nonclassical secretory pathway in which lipid rafts play an important role.