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ATP and ADP Modulate a Cation Channel Formed by Hsc70 in Acidic Phospholipid Membranes

2000; Elsevier BV; Volume: 275; Issue: 40 Linguagem: Inglês

10.1074/jbc.m005226200

1083-351X

Nelson Arispe, Antonio De Maio,

Toxin Mechanisms and Immunotoxins

Heat shock proteins are molecular chaperones that participate in different cellular processes, particularly the folding and translocation of polypeptides across membranes. In this regard, members of the Hsp70 family of heat shock proteins have been observed in close proximity to cellular membranes. In this study, the direct interaction between Hsc70, which is constitutively expressed in cells, and lipid membranes was investigated. Recombinant Hsc70 was incorporated into artificial lipid bilayers, and a transmembrane ion flow was detected, suggesting the incorporation of an ion pathway. This ion flow was very stable and occurred in well defined, multilevel discrete electrical current events, indicating the formation of a multiconductance ion channel. The Hsc70 channel activity is ATP-dependent and is reversibly blocked by ADP. This channel has cationic selectivity. Thus, Hsc70 can directly interact with lipid membranes to create functionally stable ATP-dependent cationic pathways. Heat shock proteins are molecular chaperones that participate in different cellular processes, particularly the folding and translocation of polypeptides across membranes. In this regard, members of the Hsp70 family of heat shock proteins have been observed in close proximity to cellular membranes. In this study, the direct interaction between Hsc70, which is constitutively expressed in cells, and lipid membranes was investigated. Recombinant Hsc70 was incorporated into artificial lipid bilayers, and a transmembrane ion flow was detected, suggesting the incorporation of an ion pathway. This ion flow was very stable and occurred in well defined, multilevel discrete electrical current events, indicating the formation of a multiconductance ion channel. The Hsc70 channel activity is ATP-dependent and is reversibly blocked by ADP. This channel has cationic selectivity. Thus, Hsc70 can directly interact with lipid membranes to create functionally stable ATP-dependent cationic pathways. heat shock protein picosiemens Heat shock proteins (hsps)1 are molecular chaperones that participate in different normal cellular processes, including the folding of newly synthesized proteins and their import into subcellular compartments such as the endoplasmic reticulum and mitochondria, the oligomeric assembly of proteins, and protein degradation (1Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 316-377Crossref Scopus (4362) Google Scholar, 2Morimoto R.I. Cancer Cells. 1991; 3: 295-301PubMed Google Scholar, 3De Maio A. Shock. 1999; 11: 1-12Crossref PubMed Scopus (497) Google Scholar). hsps also play an important role in the recovery of cells from a variety of stresses. In this process, they participate in the resolubilization of proteins that become denatured as a consequence of the stress, as well as the stabilization of cellular pathways such as transcription and translation (3De Maio A. Shock. 1999; 11: 1-12Crossref PubMed Scopus (497) Google Scholar). hsps are grouped into families according to size and sequence homology. This most studied of the hsps is the Hsp70 family, which is composed of four members: Hsc70, Hsp70, BIP, and Mtp70. All members of the Hsp70 family contain three structural and functional domains. The domain at the N terminus of the molecule (44 kDa) binds and hydrolyzes ATP. The subsequent region (18 kDa) participates in the interaction with target proteins (peptide binding domain). The C terminus of the molecule (10 kDa) seems to be involved in the association with co-chaperone molecules such as DnaJ (2Morimoto R.I. Cancer Cells. 1991; 3: 295-301PubMed Google Scholar, 4Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (398) Google Scholar). The interaction of Hsp70s with peptides is modulated by the presence and hydrolysis of ATP. Thus, ATP is necessary for the recognition of the peptide, whereas hydrolysis of ATP to ADP increases the affinity for the peptide (5Hightower L.E. Sadis S.E. Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 179-207Google Scholar). Proteins in the Hsp70 family are located in different subcellular compartments. Hsc70 and Hsp70 are found in the cytosol, whereas BIP and Mtp70 are located in the endoplasmic reticulum and mitochondrial matrix, respectively. This subcellular localization is necessary for the coordinated action of these proteins in the translocation of polypeptides across membranes. Consequently, it is not surprising to observe these proteins in close proximity to cellular membranes (6Welch W.J. Suhan J.P. J. Cell Biol. 1985; 101: 1198-1211Crossref PubMed Scopus (321) Google Scholar, 7Kurucz I. Tombor B. Prechl J. Erdo F. Hegedus E. Nagy Z. Vitai M. Koranyi L. Laszlo L. Cell Stress Chaperones. 1999; 4: 139-152Crossref PubMed Scopus (42) Google Scholar). The interaction of Hsp70s with membranes may be necessary for the translocation of polypeptides across these lipid barriers. In the present study, the direct interaction of Hsc70 with membrane lipid moieties and the effects of ATP and ADP were analyzed using artificial lipids in a planar lipid bilayer system. This technique allows the study of direct protein-lipid interaction using purified components. When insertion across the lipid membrane occurs, the protein may create a transmembrane pathway that permits the flow of ions.DISCUSSIONThe finding that Hsc70 can form a well defined and stable cationic selective channel in artificial lipid bilayers is intriguing. Although Hsc70 is predominately a cytosolic protein, the presence of Hsp70s in association with or in close proximity to cellular membranes has been previously reported (6Welch W.J. Suhan J.P. J. Cell Biol. 1985; 101: 1198-1211Crossref PubMed Scopus (321) Google Scholar, 7Kurucz I. Tombor B. Prechl J. Erdo F. Hegedus E. Nagy Z. Vitai M. Koranyi L. Laszlo L. Cell Stress Chaperones. 1999; 4: 139-152Crossref PubMed Scopus (42) Google Scholar, 10Multhoff G. Botzler C. Wiesnet M. Muller E. Meier T. Wilmanns W. Int. J. Cancer. 1995; 61: 272-279Crossref PubMed Scopus (374) Google Scholar). The capacity of Hsc70 to form channels is a function that cannot be predicted a priorifrom the structure and chaperone role of this protein. Perhaps the interaction of Hsc70 with lipids is important in the processes of translocation and folding of membrane proteins. The activity of Hsc70 ion pathways was also found to be regulated by the presence of ATP and ADP, which is consistent with the role these nucleotides play in the other functions of Hsc70. Binding of ATP produces a change of conformation in Hsc70 that modulates its interaction with target polypeptides (5Hightower L.E. Sadis S.E. Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 179-207Google Scholar). The effect of ATP and ADP on the Hsc70 ion pathway may be similar. A conformational change of Hsc70 may be necessary for the opening of the channel. Previous observations have also suggested a possible interaction between Hsp70s and lipids. Alder et al.(11Alder G.M. Austen B.M. Bashford C.L. Mehlert A. Pasternak C.A. Biosci. Rep. 1990; 10: 509-518Crossref PubMed Scopus (49) Google Scholar) showed that Hsp70s were capable of opening pores into liposomes, producing a leakage of the liposome content. In addition, they observed Hsp70-induced currents in artificial lipid bilayers. In this early work, the Hsp70-induced currents were not characterized. The addition of exogenous Hsp70 to patch-clamped membranes was reported to activate potassium channels (12Negulyaev Y.A. Vedernikova E.A. Kinev A.V. Voronin A.P. Biochem. Biophys. Acta. 1996; 1282: 156-162Crossref PubMed Scopus (28) Google Scholar). Recently, we reported that Hsc70 is also capable of inducing liposome aggregation in a nucleotide regulated process (13Arispe N. Doh M. De Maio A. Biophys. J. 2000; 78 (abstr.): 36Google Scholar).All members of the Hsp70 family contain two major structural and functional domains. The N terminus of the molecule binds and hydrolyzes ATP, and the C terminus participates in the interaction with target proteins (peptide binding domain) (2Morimoto R.I. Cancer Cells. 1991; 3: 295-301PubMed Google Scholar). Members of the Hsp70 family self-associate to form oligomers of low order in the absence of peptide targets or denatured proteins, also in an ATP-dependent manner (14Schlossman D.M. Schmid S.L. Brael W.A. Rothman J.E. J. Cell Biol. 1984; 99: 723-733Crossref PubMed Scopus (286) Google Scholar, 15Freiden P.J. Gaut J.R. Hendershot L.M. EMBO J. 1992; 11: 63-70Crossref PubMed Scopus (150) Google Scholar, 16Blond-Elguindi S. Fourie A.M. Sambrook J.F. Gething M.J. J. Biol. Chem. 1993; 268: 12730-12735Abstract Full Text PDF PubMed Google Scholar, 17Gao B. Eisenberg E. Greene L. J. Biol. Chem. 1996; 271: 16792-16797Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The C terminus of Hsc70 has been shown to be essential for self-association (18Benaroudj N. Fouchaq B. Ladjimi M.M. J. Biol. Chem. 1997; 272: 8744-8751Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). It is quite likely that the actual Hsc70 channel may be formed by oligomers of this protein. The characteristic multiconductance nature and the various patterns of channel activity observed after incorporation of Hsc70 into the lipid bilayer may be the result of the incorporation of different order oligomers. However, we cannot discard the possibility that multiconductance is due to conformational changes of Hsc70. These oligomers may be inserted into the lipid bilayer by the C terminus of the molecule. This assumption is based on the observation that channel activity is reversibly regulated by ATP and ADP. Thus, the ATP binding domain (N-terminal) should be exposed to the aqueous phase. We have recently observed that Hsp70-mediated liposome aggregation is also modulated by ATP and ADP. This observation is consistent with the assumption that the nucleotide binding site is accessible to the nucleotides in the aqueous solution after the insertion of Hsc70 into the liposome membrane (13Arispe N. Doh M. De Maio A. Biophys. J. 2000; 78 (abstr.): 36Google Scholar). The three-dimensional structure of fragments containing the N-terminal (ATP binding site) and C-terminal (peptide binding site) of Hsc70 have been resolved by x-ray crystallography (19Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar, 20Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar) and NMR spectroscopy (21Morshauser R.C. Hu W. Wang H. Pang Y. Flynn G.C. Zuiderweg E.R.P. J. Mol. Biol. 1999; 289: 1387-1403Crossref PubMed Scopus (134) Google Scholar). This region contains a cluster of β-sheets and a long α-helix. These regions may associate directly with other Hsc70 molecules to create a complex that has the proper configuration to spontaneously insert into the lipid bilayer. The peptide binding domain of Hsc70 is over 50 Å in length, large enough to span a biological membrane. There are several examples of molecules with clusters of β-sheet structures that interact with membranes to form ion channels, such as some members of the annexin family (22Rojas E. Arispe N. Haigler H.T. Burns A.L. Pollard H.B. Bone Miner. 1992; 17: 214-218Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 23Arispe N. Rojas E. Genge B.R. Wu L.N.Y. Wuthier R.E. Biophys. J. 1996; 71: 1764-1775Abstract Full Text PDF PubMed Scopus (80) Google Scholar), Alzheimer's β-amyloid (8Arispe N. Rojas E. Pollard H.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 567-571Crossref PubMed Scopus (780) Google Scholar), and human amylin (24Mirzabekov T.A. Lin M.-C. Kagan B.L. J. Biol. Chem. 1996; 271: 1988-1992Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). In addition, hydrophobic patches, which are thought to be likely regions for interaction with membrane lipids, have been observed in the C-terminal of the ATP binding domain (19Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar) and in the N-terminal of the peptide binding site (21Morshauser R.C. Hu W. Wang H. Pang Y. Flynn G.C. Zuiderweg E.R.P. J. Mol. Biol. 1999; 289: 1387-1403Crossref PubMed Scopus (134) Google Scholar), which may be involved in the interaction with the lipid bilayer.The preceding observations demonstrate that Hsc70 can stably interact with lipids. The biological significance of this observation remains to be elucidated. A possibility is that ion pathways generated by Hsc70 may create the proper ionic environment necessary for the process of polypeptide translocation across membranes. Echoing this assumption, O'Brien and McKay (25O'Brien M.C. McKay D.B. J. Biol. Chem. 1995; 270: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) have shown that the ATPase activity of Hsc70 is modulated by potassium ions. In addition, a potassium ion requirement is necessary for the dissociation of denatured proteins from bacterial Hsp70 (26Palleros D.R. Reid K.L. Shi L. Welch W.J. Fink A.L. Nature. 1993; 365: 664-666Crossref PubMed Scopus (347) Google Scholar). Given these observations, the fact that Hsc70 can form channels with selectivity for cations opens new and exciting possibilities for the biological role of this chaperone protein. Heat shock proteins (hsps)1 are molecular chaperones that participate in different normal cellular processes, including the folding of newly synthesized proteins and their import into subcellular compartments such as the endoplasmic reticulum and mitochondria, the oligomeric assembly of proteins, and protein degradation (1Lindquist S. Craig E.A. Annu. Rev. Genet. 1988; 22: 316-377Crossref Scopus (4362) Google Scholar, 2Morimoto R.I. Cancer Cells. 1991; 3: 295-301PubMed Google Scholar, 3De Maio A. Shock. 1999; 11: 1-12Crossref PubMed Scopus (497) Google Scholar). hsps also play an important role in the recovery of cells from a variety of stresses. In this process, they participate in the resolubilization of proteins that become denatured as a consequence of the stress, as well as the stabilization of cellular pathways such as transcription and translation (3De Maio A. Shock. 1999; 11: 1-12Crossref PubMed Scopus (497) Google Scholar). hsps are grouped into families according to size and sequence homology. This most studied of the hsps is the Hsp70 family, which is composed of four members: Hsc70, Hsp70, BIP, and Mtp70. All members of the Hsp70 family contain three structural and functional domains. The domain at the N terminus of the molecule (44 kDa) binds and hydrolyzes ATP. The subsequent region (18 kDa) participates in the interaction with target proteins (peptide binding domain). The C terminus of the molecule (10 kDa) seems to be involved in the association with co-chaperone molecules such as DnaJ (2Morimoto R.I. Cancer Cells. 1991; 3: 295-301PubMed Google Scholar, 4Cyr D.M. Langer T. Douglas M.G. Trends Biochem. Sci. 1994; 19: 176-181Abstract Full Text PDF PubMed Scopus (398) Google Scholar). The interaction of Hsp70s with peptides is modulated by the presence and hydrolysis of ATP. Thus, ATP is necessary for the recognition of the peptide, whereas hydrolysis of ATP to ADP increases the affinity for the peptide (5Hightower L.E. Sadis S.E. Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 179-207Google Scholar). Proteins in the Hsp70 family are located in different subcellular compartments. Hsc70 and Hsp70 are found in the cytosol, whereas BIP and Mtp70 are located in the endoplasmic reticulum and mitochondrial matrix, respectively. This subcellular localization is necessary for the coordinated action of these proteins in the translocation of polypeptides across membranes. Consequently, it is not surprising to observe these proteins in close proximity to cellular membranes (6Welch W.J. Suhan J.P. J. Cell Biol. 1985; 101: 1198-1211Crossref PubMed Scopus (321) Google Scholar, 7Kurucz I. Tombor B. Prechl J. Erdo F. Hegedus E. Nagy Z. Vitai M. Koranyi L. Laszlo L. Cell Stress Chaperones. 1999; 4: 139-152Crossref PubMed Scopus (42) Google Scholar). The interaction of Hsp70s with membranes may be necessary for the translocation of polypeptides across these lipid barriers. In the present study, the direct interaction of Hsc70 with membrane lipid moieties and the effects of ATP and ADP were analyzed using artificial lipids in a planar lipid bilayer system. This technique allows the study of direct protein-lipid interaction using purified components. When insertion across the lipid membrane occurs, the protein may create a transmembrane pathway that permits the flow of ions. DISCUSSIONThe finding that Hsc70 can form a well defined and stable cationic selective channel in artificial lipid bilayers is intriguing. Although Hsc70 is predominately a cytosolic protein, the presence of Hsp70s in association with or in close proximity to cellular membranes has been previously reported (6Welch W.J. Suhan J.P. J. Cell Biol. 1985; 101: 1198-1211Crossref PubMed Scopus (321) Google Scholar, 7Kurucz I. Tombor B. Prechl J. Erdo F. Hegedus E. Nagy Z. Vitai M. Koranyi L. Laszlo L. Cell Stress Chaperones. 1999; 4: 139-152Crossref PubMed Scopus (42) Google Scholar, 10Multhoff G. Botzler C. Wiesnet M. Muller E. Meier T. Wilmanns W. Int. J. Cancer. 1995; 61: 272-279Crossref PubMed Scopus (374) Google Scholar). The capacity of Hsc70 to form channels is a function that cannot be predicted a priorifrom the structure and chaperone role of this protein. Perhaps the interaction of Hsc70 with lipids is important in the processes of translocation and folding of membrane proteins. The activity of Hsc70 ion pathways was also found to be regulated by the presence of ATP and ADP, which is consistent with the role these nucleotides play in the other functions of Hsc70. Binding of ATP produces a change of conformation in Hsc70 that modulates its interaction with target polypeptides (5Hightower L.E. Sadis S.E. Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 179-207Google Scholar). The effect of ATP and ADP on the Hsc70 ion pathway may be similar. A conformational change of Hsc70 may be necessary for the opening of the channel. Previous observations have also suggested a possible interaction between Hsp70s and lipids. Alder et al.(11Alder G.M. Austen B.M. Bashford C.L. Mehlert A. Pasternak C.A. Biosci. Rep. 1990; 10: 509-518Crossref PubMed Scopus (49) Google Scholar) showed that Hsp70s were capable of opening pores into liposomes, producing a leakage of the liposome content. In addition, they observed Hsp70-induced currents in artificial lipid bilayers. In this early work, the Hsp70-induced currents were not characterized. The addition of exogenous Hsp70 to patch-clamped membranes was reported to activate potassium channels (12Negulyaev Y.A. Vedernikova E.A. Kinev A.V. Voronin A.P. Biochem. Biophys. Acta. 1996; 1282: 156-162Crossref PubMed Scopus (28) Google Scholar). Recently, we reported that Hsc70 is also capable of inducing liposome aggregation in a nucleotide regulated process (13Arispe N. Doh M. De Maio A. Biophys. J. 2000; 78 (abstr.): 36Google Scholar).All members of the Hsp70 family contain two major structural and functional domains. The N terminus of the molecule binds and hydrolyzes ATP, and the C terminus participates in the interaction with target proteins (peptide binding domain) (2Morimoto R.I. Cancer Cells. 1991; 3: 295-301PubMed Google Scholar). Members of the Hsp70 family self-associate to form oligomers of low order in the absence of peptide targets or denatured proteins, also in an ATP-dependent manner (14Schlossman D.M. Schmid S.L. Brael W.A. Rothman J.E. J. Cell Biol. 1984; 99: 723-733Crossref PubMed Scopus (286) Google Scholar, 15Freiden P.J. Gaut J.R. Hendershot L.M. EMBO J. 1992; 11: 63-70Crossref PubMed Scopus (150) Google Scholar, 16Blond-Elguindi S. Fourie A.M. Sambrook J.F. Gething M.J. J. Biol. Chem. 1993; 268: 12730-12735Abstract Full Text PDF PubMed Google Scholar, 17Gao B. Eisenberg E. Greene L. J. Biol. Chem. 1996; 271: 16792-16797Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The C terminus of Hsc70 has been shown to be essential for self-association (18Benaroudj N. Fouchaq B. Ladjimi M.M. J. Biol. Chem. 1997; 272: 8744-8751Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). It is quite likely that the actual Hsc70 channel may be formed by oligomers of this protein. The characteristic multiconductance nature and the various patterns of channel activity observed after incorporation of Hsc70 into the lipid bilayer may be the result of the incorporation of different order oligomers. However, we cannot discard the possibility that multiconductance is due to conformational changes of Hsc70. These oligomers may be inserted into the lipid bilayer by the C terminus of the molecule. This assumption is based on the observation that channel activity is reversibly regulated by ATP and ADP. Thus, the ATP binding domain (N-terminal) should be exposed to the aqueous phase. We have recently observed that Hsp70-mediated liposome aggregation is also modulated by ATP and ADP. This observation is consistent with the assumption that the nucleotide binding site is accessible to the nucleotides in the aqueous solution after the insertion of Hsc70 into the liposome membrane (13Arispe N. Doh M. De Maio A. Biophys. J. 2000; 78 (abstr.): 36Google Scholar). The three-dimensional structure of fragments containing the N-terminal (ATP binding site) and C-terminal (peptide binding site) of Hsc70 have been resolved by x-ray crystallography (19Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar, 20Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar) and NMR spectroscopy (21Morshauser R.C. Hu W. Wang H. Pang Y. Flynn G.C. Zuiderweg E.R.P. J. Mol. Biol. 1999; 289: 1387-1403Crossref PubMed Scopus (134) Google Scholar). This region contains a cluster of β-sheets and a long α-helix. These regions may associate directly with other Hsc70 molecules to create a complex that has the proper configuration to spontaneously insert into the lipid bilayer. The peptide binding domain of Hsc70 is over 50 Å in length, large enough to span a biological membrane. There are several examples of molecules with clusters of β-sheet structures that interact with membranes to form ion channels, such as some members of the annexin family (22Rojas E. Arispe N. Haigler H.T. Burns A.L. Pollard H.B. Bone Miner. 1992; 17: 214-218Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 23Arispe N. Rojas E. Genge B.R. Wu L.N.Y. Wuthier R.E. Biophys. J. 1996; 71: 1764-1775Abstract Full Text PDF PubMed Scopus (80) Google Scholar), Alzheimer's β-amyloid (8Arispe N. Rojas E. Pollard H.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 567-571Crossref PubMed Scopus (780) Google Scholar), and human amylin (24Mirzabekov T.A. Lin M.-C. Kagan B.L. J. Biol. Chem. 1996; 271: 1988-1992Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). In addition, hydrophobic patches, which are thought to be likely regions for interaction with membrane lipids, have been observed in the C-terminal of the ATP binding domain (19Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar) and in the N-terminal of the peptide binding site (21Morshauser R.C. Hu W. Wang H. Pang Y. Flynn G.C. Zuiderweg E.R.P. J. Mol. Biol. 1999; 289: 1387-1403Crossref PubMed Scopus (134) Google Scholar), which may be involved in the interaction with the lipid bilayer.The preceding observations demonstrate that Hsc70 can stably interact with lipids. The biological significance of this observation remains to be elucidated. A possibility is that ion pathways generated by Hsc70 may create the proper ionic environment necessary for the process of polypeptide translocation across membranes. Echoing this assumption, O'Brien and McKay (25O'Brien M.C. McKay D.B. J. Biol. Chem. 1995; 270: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) have shown that the ATPase activity of Hsc70 is modulated by potassium ions. In addition, a potassium ion requirement is necessary for the dissociation of denatured proteins from bacterial Hsp70 (26Palleros D.R. Reid K.L. Shi L. Welch W.J. Fink A.L. Nature. 1993; 365: 664-666Crossref PubMed Scopus (347) Google Scholar). Given these observations, the fact that Hsc70 can form channels with selectivity for cations opens new and exciting possibilities for the biological role of this chaperone protein. The finding that Hsc70 can form a well defined and stable cationic selective channel in artificial lipid bilayers is intriguing. Although Hsc70 is predominately a cytosolic protein, the presence of Hsp70s in association with or in close proximity to cellular membranes has been previously reported (6Welch W.J. Suhan J.P. J. Cell Biol. 1985; 101: 1198-1211Crossref PubMed Scopus (321) Google Scholar, 7Kurucz I. Tombor B. Prechl J. Erdo F. Hegedus E. Nagy Z. Vitai M. Koranyi L. Laszlo L. Cell Stress Chaperones. 1999; 4: 139-152Crossref PubMed Scopus (42) Google Scholar, 10Multhoff G. Botzler C. Wiesnet M. Muller E. Meier T. Wilmanns W. Int. J. Cancer. 1995; 61: 272-279Crossref PubMed Scopus (374) Google Scholar). The capacity of Hsc70 to form channels is a function that cannot be predicted a priorifrom the structure and chaperone role of this protein. Perhaps the interaction of Hsc70 with lipids is important in the processes of translocation and folding of membrane proteins. The activity of Hsc70 ion pathways was also found to be regulated by the presence of ATP and ADP, which is consistent with the role these nucleotides play in the other functions of Hsc70. Binding of ATP produces a change of conformation in Hsc70 that modulates its interaction with target polypeptides (5Hightower L.E. Sadis S.E. Morimoto R.I. Tissieres A. Georgopoulos C. The Biology of Heat Shock Proteins and Molecular Chaperones. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994: 179-207Google Scholar). The effect of ATP and ADP on the Hsc70 ion pathway may be similar. A conformational change of Hsc70 may be necessary for the opening of the channel. Previous observations have also suggested a possible interaction between Hsp70s and lipids. Alder et al.(11Alder G.M. Austen B.M. Bashford C.L. Mehlert A. Pasternak C.A. Biosci. Rep. 1990; 10: 509-518Crossref PubMed Scopus (49) Google Scholar) showed that Hsp70s were capable of opening pores into liposomes, producing a leakage of the liposome content. In addition, they observed Hsp70-induced currents in artificial lipid bilayers. In this early work, the Hsp70-induced currents were not characterized. The addition of exogenous Hsp70 to patch-clamped membranes was reported to activate potassium channels (12Negulyaev Y.A. Vedernikova E.A. Kinev A.V. Voronin A.P. Biochem. Biophys. Acta. 1996; 1282: 156-162Crossref PubMed Scopus (28) Google Scholar). Recently, we reported that Hsc70 is also capable of inducing liposome aggregation in a nucleotide regulated process (13Arispe N. Doh M. De Maio A. Biophys. J. 2000; 78 (abstr.): 36Google Scholar). All members of the Hsp70 family contain two major structural and functional domains. The N terminus of the molecule binds and hydrolyzes ATP, and the C terminus participates in the interaction with target proteins (peptide binding domain) (2Morimoto R.I. Cancer Cells. 1991; 3: 295-301PubMed Google Scholar). Members of the Hsp70 family self-associate to form oligomers of low order in the absence of peptide targets or denatured proteins, also in an ATP-dependent manner (14Schlossman D.M. Schmid S.L. Brael W.A. Rothman J.E. J. Cell Biol. 1984; 99: 723-733Crossref PubMed Scopus (286) Google Scholar, 15Freiden P.J. Gaut J.R. Hendershot L.M. EMBO J. 1992; 11: 63-70Crossref PubMed Scopus (150) Google Scholar, 16Blond-Elguindi S. Fourie A.M. Sambrook J.F. Gething M.J. J. Biol. Chem. 1993; 268: 12730-12735Abstract Full Text PDF PubMed Google Scholar, 17Gao B. Eisenberg E. Greene L. J. Biol. Chem. 1996; 271: 16792-16797Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The C terminus of Hsc70 has been shown to be essential for self-association (18Benaroudj N. Fouchaq B. Ladjimi M.M. J. Biol. Chem. 1997; 272: 8744-8751Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). It is quite likely that the actual Hsc70 channel may be formed by oligomers of this protein. The characteristic multiconductance nature and the various patterns of channel activity observed after incorporation of Hsc70 into the lipid bilayer may be the result of the incorporation of different order oligomers. However, we cannot discard the possibility that multiconductance is due to conformational changes of Hsc70. These oligomers may be inserted into the lipid bilayer by the C terminus of the molecule. This assumption is based on the observation that channel activity is reversibly regulated by ATP and ADP. Thus, the ATP binding domain (N-terminal) should be exposed to the aqueous phase. We have recently observed that Hsp70-mediated liposome aggregation is also modulated by ATP and ADP. This observation is consistent with the assumption that the nucleotide binding site is accessible to the nucleotides in the aqueous solution after the insertion of Hsc70 into the liposome membrane (13Arispe N. Doh M. De Maio A. Biophys. J. 2000; 78 (abstr.): 36Google Scholar). The three-dimensional structure of fragments containing the N-terminal (ATP binding site) and C-terminal (peptide binding site) of Hsc70 have been resolved by x-ray crystallography (19Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar, 20Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar) and NMR spectroscopy (21Morshauser R.C. Hu W. Wang H. Pang Y. Flynn G.C. Zuiderweg E.R.P. J. Mol. Biol. 1999; 289: 1387-1403Crossref PubMed Scopus (134) Google Scholar). This region contains a cluster of β-sheets and a long α-helix. These regions may associate directly with other Hsc70 molecules to create a complex that has the proper configuration to spontaneously insert into the lipid bilayer. The peptide binding domain of Hsc70 is over 50 Å in length, large enough to span a biological membrane. There are several examples of molecules with clusters of β-sheet structures that interact with membranes to form ion channels, such as some members of the annexin family (22Rojas E. Arispe N. Haigler H.T. Burns A.L. Pollard H.B. Bone Miner. 1992; 17: 214-218Abstract Full Text PDF PubMed Scopus (42) Google Scholar, 23Arispe N. Rojas E. Genge B.R. Wu L.N.Y. Wuthier R.E. Biophys. J. 1996; 71: 1764-1775Abstract Full Text PDF PubMed Scopus (80) Google Scholar), Alzheimer's β-amyloid (8Arispe N. Rojas E. Pollard H.B. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 567-571Crossref PubMed Scopus (780) Google Scholar), and human amylin (24Mirzabekov T.A. Lin M.-C. Kagan B.L. J. Biol. Chem. 1996; 271: 1988-1992Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). In addition, hydrophobic patches, which are thought to be likely regions for interaction with membrane lipids, have been observed in the C-terminal of the ATP binding domain (19Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar) and in the N-terminal of the peptide binding site (21Morshauser R.C. Hu W. Wang H. Pang Y. Flynn G.C. Zuiderweg E.R.P. J. Mol. Biol. 1999; 289: 1387-1403Crossref PubMed Scopus (134) Google Scholar), which may be involved in the interaction with the lipid bilayer. The preceding observations demonstrate that Hsc70 can stably interact with lipids. The biological significance of this observation remains to be elucidated. A possibility is that ion pathways generated by Hsc70 may create the proper ionic environment necessary for the process of polypeptide translocation across membranes. Echoing this assumption, O'Brien and McKay (25O'Brien M.C. McKay D.B. J. Biol. Chem. 1995; 270: 2247-2250Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar) have shown that the ATPase activity of Hsc70 is modulated by potassium ions. In addition, a potassium ion requirement is necessary for the dissociation of denatured proteins from bacterial Hsp70 (26Palleros D.R. Reid K.L. Shi L. Welch W.J. Fink A.L. Nature. 1993; 365: 664-666Crossref PubMed Scopus (347) Google Scholar). Given these observations, the fact that Hsc70 can form channels with selectivity for cations opens new and exciting possibilities for the biological role of this chaperone protein. We thank Dylan Stewart for critically reading the manuscript and Michael Doh for technical assistance.