Correlation between Steady-state ATP Hydrolysis and Vanadate-induced ADP Trapping in Human P-glycoprotein

P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resistance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis. The objective of this study was to understand how substrates affect the catalytic cycle of ATP hydrolysis by Pgp. The ATPase activity of purified and reconstituted recombinant human Pgp was measured using a continuous cycling assay. Pgp hydrolyzes ATP in the absence of drug at a basal rate of 0.5 μmol·min·mg−1 with aKm for ATP of 0.33 mm. This basal rate can be either increased or decreased depending on the Pgp substrate used, without an effect on the Km for ATP or 8-azidoATP and Ki for ADP, suggesting that substrates do not affect nucleotide binding to Pgp. Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal rate, they do not completely inhibit the activity. Therefore, these drugs can be classified as substrates. Vanadate (Vi)-induced trapping of [α-32P]8-azidoADP was used to probe the effect of substrates on the transition state of the ATP hydrolysis reaction. TheKm for [α-32P]8-azidoATP (20 μm) is decreased in the presence of Vi; however, it is not changed by drugs such as verapamil or cyclosporin A. Strikingly, the extent of Vi-induced [α-32P]8-azidoADP trapping correlates directly with the fold stimulation of ATPase activity at steady state. Furthermore, Pi exhibits very low affinity for Pgp (Ki ∼30 mm for Vi-induced 8-azidoADP trapping). In aggregate, these data demonstrate that the release of Vi trapped [α-32P]8-azidoADP from Pgp is the rate-limiting step in the steady-state reaction. We suggest that substrates modulate the rate of ATPase activity of Pgp by controlling the rate of dissociation of ADP following ATP hydrolysis and that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp. P-glycoprotein (Pgp) is a transmembrane protein conferring multidrug resistance to cells by extruding a variety of amphipathic cytotoxic agents using energy from ATP hydrolysis. The objective of this study was to understand how substrates affect the catalytic cycle of ATP hydrolysis by Pgp. The ATPase activity of purified and reconstituted recombinant human Pgp was measured using a continuous cycling assay. Pgp hydrolyzes ATP in the absence of drug at a basal rate of 0.5 μmol·min·mg−1 with aKm for ATP of 0.33 mm. This basal rate can be either increased or decreased depending on the Pgp substrate used, without an effect on the Km for ATP or 8-azidoATP and Ki for ADP, suggesting that substrates do not affect nucleotide binding to Pgp. Although inhibitors of Pgp activity, cyclosporin A, its analog PSC833, and rapamycin decrease the rate of ATP hydrolysis with respect to the basal rate, they do not completely inhibit the activity. Therefore, these drugs can be classified as substrates. Vanadate (Vi)-induced trapping of [α-32P]8-azidoADP was used to probe the effect of substrates on the transition state of the ATP hydrolysis reaction. TheKm for [α-32P]8-azidoATP (20 μm) is decreased in the presence of Vi; however, it is not changed by drugs such as verapamil or cyclosporin A. Strikingly, the extent of Vi-induced [α-32P]8-azidoADP trapping correlates directly with the fold stimulation of ATPase activity at steady state. Furthermore, Pi exhibits very low affinity for Pgp (Ki ∼30 mm for Vi-induced 8-azidoADP trapping). In aggregate, these data demonstrate that the release of Vi trapped [α-32P]8-azidoADP from Pgp is the rate-limiting step in the steady-state reaction. We suggest that substrates modulate the rate of ATPase activity of Pgp by controlling the rate of dissociation of ADP following ATP hydrolysis and that ADP release is the rate-limiting step in the normal catalytic cycle of Pgp. P-glycoprotein polyacrylamide gel electrophoresis vanadate 2[N-morpholino]ethane sulfonic acid Multidrug resistance, the reduced sensitivity to a variety of structurally unrelated, hydrophobic, chemotherapeutic agents, is a major problem in cancer treatment. This phenomenon is often associated with the overexpression of the human multidrug resistance gene (MDR1) (1Ambudkar S.V. Dey S. Hrycyna C.A. Ramachandra M. Pastan I. Gottesman M.M. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 361-398Crossref PubMed Scopus (1924) Google Scholar, 2Gottesman M.M. Hrycyna C.A. Schoenlein P.V. Germann U.A. Pastan I. Annu. Rev. Genet. 1995; 29: 607-649Crossref PubMed Scopus (463) Google Scholar). MDR1 encodes a 170-kDa plasma membrane protein, P-glycoprotein (Pgp),1 that uses the energy from ATP hydrolysis to expel a variety of anticancer drugs from cells, thus making them ineffective during chemotherapy. The secondary structure of Pgp is predicted to consist of two homologous halves each containing six putative transmembrane helices and a nucleotide-binding domain. These structural elements are common to a large family of membrane transporters called the ATP-binding cassette superfamily (3Gottesman M.M. Pastan I. Annu. Rev. Biochem. 1993; 62: 385-427Crossref PubMed Scopus (3564) Google Scholar,4Croop J.M. Methods Enzymol. 1998; 292: 101-116Crossref PubMed Scopus (53) Google Scholar). The widely accepted hypothesis of Pgp function is that substrate transport is coupled to ATP hydrolysis. However, the mechanism for this reaction is not well understood. In the absence of added substrate, Pgp catalyzes basal ATP hydrolysis (ATPase activity). This basal activity has been suggested to occur because of an endogenous lipid substrate(s) or an uncoupling of ATP hydrolysis and drug extrusion (5Gottesman M.M. Pastan I. Ambudkar S.V. Curr. Opin. Genet. Dev. 1996; 6: 610-617Crossref PubMed Scopus (509) Google Scholar, 6Ramachandra M. Ambudkar S.V. Gottesman M.M. Pastan I. Hrycyna C.A. Mol. Biol. Cell. 1996; 7: 1485-1498Crossref PubMed Scopus (68) Google Scholar). The basal rate is stimulated by adding any one of a variety of hydrophobic drug substrates; these drugs bind with and affect the ATPase activity of Pgp to Germann U.A. Biol. PubMed Google Scholar, S.V. Gottesman M.M. Pastan I. S. A. PubMed Scopus Google Scholar, Mol. Pharmacol. Google Scholar). However, a of the of ATP hydrolysis in the presence of amphipathic drugs for the of the rate-limiting step in the catalytic cycle has not been and of endogenous and six human Pgp in a are well S.V. Methods Enzymol. 1998; 292: PubMed Scopus Google Scholar, M. Ambudkar S.V. Hrycyna C.A. Dey S. Gottesman M.M. Pastan I. 1998; PubMed Scopus Google Scholar, Methods Enzymol. 1998; 292: PubMed Scopus Google Scholar). on the ATPase activity of Pgp in membrane on the ATPase activity of Pgp (5Gottesman M.M. Pastan I. Ambudkar S.V. Curr. Opin. Genet. Dev. 1996; 6: 610-617Crossref PubMed Scopus (509) Google such with protein can be to the lipid of this membrane protein the ATPase activity and for of Biochem. 1995; PubMed Scopus Google Scholar, 1993; PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, 1999; PubMed Scopus Google Scholar). for the catalytic cycle of Pgp been on binding and Vi-induced ADP trapping 1996; PubMed Scopus Google Scholar, Biol. 1995; PubMed Scopus Google Scholar, S. Biol. 1995; PubMed Scopus Google Scholar, I. 1998; PubMed Scopus Google Scholar, C.A. Ramachandra M. Ambudkar S.V. Pastan I. Gottesman M.M. Biol. 1998; PubMed Scopus Google Scholar). Vi is a transition state analog that Pi its release ATP hydrolysis. of Pgp has been in the catalytic cycle of that in the Pgp reaction are Biol. 1995; PubMed Scopus Google Scholar). and Biol. 1995; PubMed Scopus Google Scholar, S. Biol. 1995; PubMed Scopus Google the Vi-induced ADP trapping reaction and that Pi release ADP release and ADP release is the rate-limiting step in 1995; PubMed Scopus Google Scholar). objective in this study is to understand how Pgp drug substrates affect the mechanism of Pgp. for drugs that affect the steady-state ATPase activity of Pgp are and the extent of Vi-induced ADP trapping is in the presence and absence of Pgp drug substrates. The of these has suggest that ADP release is a rate-limiting step in the catalytic cycle of and substrates and effect on ATPase activity by this and cyclosporin from ATP ADP, and from was from was from rapamycin was from the and was the of and from as from and [α-32P]8-azidoATP was from Pgp was and purified as M. Ambudkar S.V. Hrycyna C.A. Dey S. Gottesman M.M. Pastan I. 1998; PubMed Scopus Google with Pgp was using cells in and with a recombinant that a that encodes Pgp. by U.A. Methods Enzymol. 1998; 292: PubMed Scopus Google and membrane with in the presence of and lipid to affinity from in the presence of and purified Pgp was with mm M. Ambudkar S.V. Hrycyna C.A. Dey S. Gottesman M.M. Pastan I. 1998; PubMed Scopus Google Scholar). Pgp in mm was to and at Pgp was by M. Ambudkar S.V. Hrycyna C.A. Dey S. Gottesman M.M. Pastan I. 1998; PubMed Scopus Google and by protein as Biochem. PubMed Scopus Google Scholar). Pgp was with a lipid and S.V. Gottesman M.M. Pastan I. S. A. PubMed Scopus Google for in the of This was with at with mm mm and using from by at for at and in was by the as ATPase activity of reconstituted Pgp was measured by two the Pi and the continuous cycling assay. In activity was as the Vi ATPase activity. agents added from in that the was this of effect on the activity of Pgp or the cycling the Pi the of at was ATPase mm mm mm and mm was with either mm or of reconstituted and Pgp drug substrates for a at The reaction was by the of mm ATP and with the of Pi was using a as M. Ambudkar S.V. Hrycyna C.A. Dey S. Gottesman M.M. Pastan I. 1998; PubMed Scopus Google Scholar). the cycling cycling 0.33 mm and and added to the ATPase with to the hydrolysis of ATP directly with the of Methods Enzymol. 1998; 292: PubMed Scopus Google Scholar). reconstituted Pgp in of was with Pgp drug substrates at in a The reaction was by the of ATP and at using for The rate of in was to using an this is to ATP The ATPase in the presence of ATP and Pgp drug substrates to is the ATPase activity at of is with as the activity, is the drug substrate is the at activity, and is the basal rate in the absence of added The fold stimulation of ATPase activity by a substrate is for ATP and 8-azidoATP in the presence of Pgp drug verapamil μm) and cyclosporin for basal activity the absence of added an of was ATPase using nucleotide to is the of ATP or 8-azidoATP is the of at activity. for ADP using of ADP and ATP in the presence and absence of ATPase to Ki is the of ADP at in using for Vi-induced 8-azidoADP trapping ATPase mm either or purified Pgp Pgp drug substrate, and in low or in the absence of [α-32P]8-azidoATP at for by the of and by the of ATP to on for and to and The extent of was using the The of Pgp was using for and in as Ambudkar S.V. S. A. PubMed Scopus Google Scholar). Pgp was reconstituted and in the ATPase containing mm Vi and μm) of [α-32P]8-azidoATP in the at for The reaction was by of mm ATP and the on of Pgp the was basal and in the presence of either or cyclosporin A. on an the in the Pgp was on a TheKm for 8-azidoATP basal and in the presence of of verapamil and cyclosporin by the data to using the as containing purified Pgp in the ATPase containing mm The with or cyclosporin either in the presence of mm or [α-32P]8-azidoATP was added in the and at for The reaction was by with mm ATP and on on an the extent of trapping of 8-azidoADP was as of in the ATPase in the presence of or cyclosporin for at and to on [α-32P]8-azidoATP was added to each in the and at for The with on as ATP was added to by at at for by using in a and the in on a gel at and to at for The the Pgp was using the and the The lipid was by and in the of by and to S.V. Methods Enzymol. 1998; 292: PubMed Scopus Google Scholar). This was at at in a mm was using as U.A. Ambudkar S.V. Pastan I. Gottesman M.M. Biol. 1996; PubMed Scopus Google using the from The was used for protein of was by a mm in for and was by using The was for the of large of Pgp M. Ambudkar S.V. Hrycyna C.A. Dey S. Gottesman M.M. Pastan I. 1998; PubMed Scopus Google Scholar). cells of cells in of membrane protein was of this membrane protein was with and of purified Pgp was following affinity The of purified Pgp an lipid is to a Pgp of the a common for Pgp Methods Enzymol. 1998; 292: PubMed Scopus Google Scholar, S.V. Methods Enzymol. 1998; 292: PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). The ATPase activity of reconstituted Pgp was measured using two an Pi release the of Pi by the hydrolysis of ATP in a of and a continuous cycling the hydrolysis of ATP to the of using and substrates. The cycling was to the Pi because of its to the ATPase reaction in in this the of ATPase activity as Although the cycling was used to the of ATP and Pgp drug in the the cycling decrease in the at from the of activity using a substrate, using the Pi assay. Furthermore, the ATPase activity not be in the presence of ADP because of its effect of cycling in the absence of ATP hydrolysis. ATPase cycling is in the rate of ATPase activity to Pgp is as the activity in the presence or absence of The rate of of is to using an This is directly with the ATPase activity of Pgp as ATP ATPase activity of purified and reconstituted Pgp was ATP is with the using the Pi assay. This activity is for the human Pgp reconstituted by using the M. Ambudkar S.V. Hrycyna C.A. Dey S. Gottesman M.M. 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Biochem. 1995; PubMed Scopus Google for nucleotide hydrolysis by P-glycoprotein in the presence of using the ATPase cycling drug activity in the absence of drug substrates as using the ATPase Pi release drug activity in the absence of drug substrates as using the ATPase Pi release drug activity in the absence of drug substrates as of Km for ATP and 8-azidoATP for ADP in the presence and absence of Pgp substrates ATPase and basal activity cyclosporin A, as not using the ATPase cycling activity in the absence of drug substrates as using the ATPase Pi release in a of Km for ATP and 8-azidoATP for ADP in the presence and absence of Pgp substrates ATPase and basal activity cyclosporin A, as not hydrolysis at Pgp is with or and Vi in the presence or absence of Pgp drug substrates. The reaction ATP hydrolysis to Vi for Pi and [α-32P]8-azidoADP on the Biol. 1995; PubMed Scopus Google Scholar). The reaction is by adding ATP at to inhibit any [α-32P]8-azidoATP binding and to the extent of trapped or and gel the [α-32P]8-azidoADP Pgp is by and in the extent of Vi-induced [α-32P]8-azidoADP trapping in the presence of Pgp substrates. This drug effect is to be the of or of Pgp because of the the extent of Vi-induced 8-azidoADP trapping and the fold stimulation of the ATPase activity in the presence of a substrate as In the in the extent of Vi-induced [α-32P]8-azidoADP trapping in the presence of drugs in is not to of protein in the gel the in in in used for the of Pgp with the Biochem. Pharmacol. 1998; PubMed Scopus Google Scholar, M. I. A. Biol. 1998; PubMed Scopus Google drug in the extent of ADP trapping Pgp. [α-32P]8-azidoADP is of the Pgp in the reaction depending on the Pgp drug substrates these are with for Pgp in the presence of verapamil 1998; Google Scholar). be that is using reconstituted the protein of the used in these is in been to Furthermore, [α-32P]8-azidoADP trapping in the absence of Vi or the reaction is at a at 8-azidoATP is not by Pgp not We that Pgp substrates trapping of 8-azidoADP in the transition state the effect of substrates on the ATPase activity and the extent of it is to the for 8-azidoADP trapping Pgp. the in the presence of of [α-32P]8-azidoATP and Vi and the in the Pgp one to and of trapped of [α-32P]8-azidoATP during We verapamil and cyclosporin to the effect of agents, either or decrease the ATPase activity of is from the data in that the [α-32P]8-azidoATP in the presence of verapamil and is reduced in the presence of cyclosporin A. TheKm and basal in the presence of verapamil and cyclosporin A, however, not by the presence of verapamil or cyclosporin A. the Km for [α-32P]8-azidoATP was in the presence of to the extent of trapping in the presence of drugs in the presence of is that the affinity of 8-azidoATP for Pgp is increased in the presence of Vi μm) with the 0.5 Vi the affinity of for Pgp is not at S. Biol. 1995; PubMed Scopus Google Scholar, Ambudkar S.V. S. A. PubMed Scopus Google the trapped using at However, with reaction the dissociation of the is to not Although steady-state Pgp ATPase activity at S. Biol. 1995; PubMed Scopus Google M. and S. for the of Vi trapping the to be The extent of Vi trapping with and in the presence of Pgp drug substrates correlates with the fold stimulation of ATPase activity by drugs in the steady-state reaction with the data used for this are in and This is using [α-32P]8-azidoATP to μm) for Vi-induced In was with a Vi-induced [α-32P]8-azidoADP and the fold stimulation of ATPase activity was not The demonstrate that are substrates of Pgp affect ATP hydrolysis and the Vi-induced trapping of This the as to the substrates nucleotide binding or a step during hydrolysis. We this directly by [α-32P]8-azidoATP to bind Pgp in the absence and presence of verapamil or cyclosporin A. We that of verapamil and cyclosporin do not affect nucleotide binding suggesting that the substrate at a step or that binding of nucleotide and S. Biol. in Scholar). of a membrane transport protein, such as large of purified protein that activity are The used in this study human Pgp at a in the absence of cytotoxic drug and the absence of such the of the of human Pgp. Pgp is an membrane protein, a in protein and activity Biochem. 1995; PubMed Scopus Google Scholar, 1993; PubMed Scopus Google Scholar, Biochem. PubMed Scopus Google Scholar, 1999; PubMed Scopus Google Scholar). and in the presence of and with lipid containing and a for the of the ATPase activity of purified Pgp S.V. Gottesman M.M. Pastan I. S. A. PubMed Scopus Google Scholar, M. Ambudkar S.V. Hrycyna C.A. Dey S. Gottesman M.M. Pastan I. 1998; PubMed Scopus Google Scholar, S.V. Methods Enzymol. 1998; 292: PubMed Scopus Google Scholar). The ATPase activity of Pgp in the presence of Pgp drug substrates can be measured using reconstituted Pgp in the cycling assay. In membrane in the absence of ATP because of and in the presence of ATP and Vi because of the presence of the cycling on the of that in the such as Vi or Pgp drug do not affect the activity of these Furthermore, the of Pgp with respect to cycling and is to the from ATP hydrolysis to ATPase activity of Pgp is measured as the activity in the presence and absence of Pgp substrates. The in the activity of Pgp is to of because of these the ATPase activity of Pgp in the presence of drug substrates is using fold stimulation that The at Pgp drug substrates ATP activity are because Pgp drug substrates are hydrophobic and the membrane lipid Pgp at However, in this reconstituted is for The of for Pgp drug substrates is of Pgp are to the activity in the presence of the and this of Pgp a on the of drug to be used for for as in are however, the ATPase in the presence of these drugs are well Pgp drug substrates are hydrophobic with low making at drug these and ATPase are to is the of cyclosporin and Pgp to ATPase activity. is from the data that cyclosporin A, its analog PSC833, and rapamycin as Pgp at a ATPase rate basal and The that these drugs as substrates ATPase activity is by cyclosporin transport Biol. 1993; PubMed Google Scholar, 1995; Google Scholar, C.A. 1995; PubMed Scopus Google Scholar, A. 1995; 6: PubMed Scopus Google Scholar). human suggest with a common Pgp substrate, as well as cyclosporin S. A. PubMed Scopus Google Scholar). These demonstrate that the cycling a to a is a or a substrate of Pgp. is that the ATPase activity of Pgp is stimulated by a of drug substrates. The and of the drug binding of Pgp has been in the S. Ramachandra M. Pastan I. Gottesman M.M. Ambudkar S.V. S. A. PubMed Scopus Google 1998; PubMed Scopus Google Scholar, M. S. Biochem. PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, M. S. Biochem. 1998; PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar). Although these drugs on the stimulation of the ATPase activity of they effect on the of Km for nucleotide ATP and 8-azidoATP and Furthermore, the for ADP, a of in the presence of verapamil these data suggest that drug verapamil and cyclosporin A, effect on ATP binding to these are with suggested using Pgp I. 1998; PubMed Scopus Google Scholar). 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Biol. 1998; PubMed Scopus Google suggested a the extent of Vi-induced nucleotide trapping and the drug ATPase activity of Pgp. on the Vi-induced ADP trapped and the steady-state reaction in the presence of Pgp drug substrates that these are the step in the Pgp catalytic The Vi-induced ADP trapping the of ADP of the of ADP from the transition state by its that the rate-limiting step measured in the steady-state reaction is the release of ADP in These that the release of not to be the rate-limiting step of the catalytic The low affinity of Pi for Pgp this step an to as the rate-limiting step of the Pgp reaction cycle (Ki ∼30 mm for Vi-induced 8-azidoADP trapping in mm for ATP hydrolysis S. Biol. 1995; PubMed Scopus Google the the steady-state reaction and the of ADP trapping in the presence of drug substrates verapamil and cyclosporin that the rate-limiting step is a step Vi and and Pi release is a for Vi binding and The ADP release the rate-limiting step in the catalytic cycle is by that is an ADP release from the and the of the substrate binding to the following the transition state step Ambudkar S.V. S. A. PubMed Scopus Google Scholar). In the rate of the release of 8-azidoADP from the Pgp is not by the of such as ADP, or and S. Biol. in Scholar). is that ATP-binding cassette transporters ATP is stimulated by substrates. Vi-induced 8-azidoADP trapping has been in transporters such as and ATP-binding cassette M. M. Biol. PubMed Scopus Google Scholar, Biol. 1999; PubMed Scopus Google Scholar). is that the ADP release is a rate-limiting step in the catalytic cycle of of the family of ATP-binding cassette We Gottesman and Pastan for and and and for on the