ATPase Activity of the Cystic Fibrosis Transmembrane Conductance Regulator

Li, Canhui; Ramjeesingh, Mohabir; Wang, Wei; Garami, Elizabeth; Hewryk, Marek; Lee, Daniel; Rommens, Johanna M.; Galley, Kevin; Bear, Christine E.

doi:10.1074/jbc.271.45.28463

Cited by 254 publications

(317 citation statements)

References 43 publications

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“…These values are consistent with previous reports for CFTR [16,40] and also for several other ABC transport proteins, including Ste6p, ABCR, MRP1 and MRP2 [33,[41][42][43]. Moreover, if one assumes that ATP hydrolysis is linked to channel gating, these values are sufficient to achieve the maximal rate of channel gating observed in vivo [16,17].Using the CFTR ATPase assay (Fig. 5), we next explored the question of whether (R domain) phosphorylation affects the kinetics of the ATP hydrolysis.…”

supporting

confidence: 94%

“…5), we next explored the question of whether (R domain) phosphorylation affects the kinetics of the ATP hydrolysis. This work failed to show an effect of either dephosphorylation (with PP2B) or with phosphorylation (using PKA) on the CFTR ATPase (Table II), in contrast to an early suggestion that phosphorylation might increase the affinity for MgATP [16,40]. Instead, these more recent tests (Fig.…”

Section: Atp Hydrolysis By Purified and Reconstituted Cftrmentioning

confidence: 57%

“…Treatment with Protein Phosphatase 2B (PP2B), a relevant phosphatase in vivo [39], had no apparent effect on the SDS-PAGE mobility of CFTR, while exposure to cAMP-dependent Protein Kinase (PKA) caused a clear retardation in CFTR mobility (Fig. 4fg) (see Table II and [16] for methods), coincident with phosphorylation as documented by labeling by [γ 32 P]ATP (data not shown). Moreover, the mobility shift induced by PKA was readily reversed by subsequent exposure to PP2B (Fig.…”

Section: Nucleotide Processing By Purified and Reconstituted Cftrmentioning

confidence: 95%

“…4) limits the analysis to a partial turnover of a binding domain. To study net ATP hydrolysis, we measured liberation of [α 32 P]ADP from [α 32 P]ATP [16] (Fig. 5).…”

Section: Atp Hydrolysis By Purified and Reconstituted Cftrmentioning

confidence: 99%

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Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

2004

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The cystic fibrosis transmembrane conductance regulator (CFTR) functions in vivo as a cAMPactivated chloride channel. A member of the ATP-binding cassette superfamily of membrane transporters, CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. It is presumed that CFTR couples ATP hydrolysis to channel gating, and as a first step in addressing this issue directly, we have established conditions for purification of biochemical quantities of human CFTR expressed in Sf9 insect cells. Use of an 8-azido-[α 32 P]-ATP binding and vanadate-trapping assay allowed us to devise conditions to preserve CFTR function during purification of a C-terminal His 10 -tagged variant after solubilization with lyso-phosphatidylglycerol (1%) and diheptanolylphosphatidylcholine (0.3%) in the presence of excess phospholipid. Study of purified and reconstituted CFTR showed that it binds nucleotide with an efficiency comparable to that of P-glycoprotein, and that it hydrolyzes ATP at rates sufficient to account for presumed in vivo activity (V Max of 58 ± 5 nmol/min/mg protein, K M (MgATP) of 0.15 mM). In further work, we found that neither nucleotide binding nor ATPase activity were altered by phosphorylation (using Protein Kinase A) or dephosphorylation (with Protein Phosphatase 2B); we also observed inhibition (∼40%) of ATP hydrolysis by reduced glutathione, but not by DTT. To evaluate CFTR function as an anion channel, we introduced an in vitro macroscopic assay based on the equilibrium exchange of proteoliposome-entrapped radioactive tracers. This revealed a CFTRdependent transport of 125 I that could be inhibited by known chloride channel blockers; no significant CFTR-dependent transport of [α 32 P]-ATP was observed. We conclude that heterologous expression of CFTR in Sf9 cells can support manufacture and purification of fully functional CFTR. This should aid in further biochemical characterization of this important molecule.The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is the cAMP-activated chloride channel encoded by the gene defective in patients with the disease [1,2]. As a member of the ATP-Binding Cassette (ABC) superfamily of membrane transporters, CFTR shares a conserved architecture consisting of two homologous halves, each containing a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). In CFTR, unlike other ABC transporters, a third domain, termed the regulatory (R) domain, is located between the two half molecules. Current evidence suggests that the TMDs define the CFTR chloride channel, while the NBDs and the R domain mediate channel gating [3][4][5][6][7][8][9][10][11] Although CFTR is glycosylated, there is currently no evidence indicating that the presence of carbohydrate affects CFTR structure or function [12]. Consistent with this presumption, expression of human CFTR in Sf9 insect cells results in appearance of the 140 kD core polypeptide -containing little or no glycosylation -that mediates a newly acquired anion ...

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supporting

confidence: 94%

Section: Atp Hydrolysis By Purified and Reconstituted Cftrmentioning

confidence: 57%

Section: Nucleotide Processing By Purified and Reconstituted Cftrmentioning

confidence: 95%

“…4) limits the analysis to a partial turnover of a binding domain. To study net ATP hydrolysis, we measured liberation of [α 32 P]ADP from [α 32 P]ATP [16] (Fig. 5).…”

Section: Atp Hydrolysis By Purified and Reconstituted Cftrmentioning

confidence: 99%

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Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

2004

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“…The transport follows Michaelis-Menten kinetics with a K m(ATP) of 17.6±2.4 μM for ATP (Wolters et al 2005), which is 4-20 fold lower than for TAP, P-glycoprotein, cystic fibrosis transmembrane conductance regulator, and maltose permease from E.coli (Sarkadi et al 1992;Li et al 1996;Landmesser et al 2002;Chen et al 2003). Based on the physiological ATP concentration, this K m(ATP) value ensures that TAPL is always fully active.…”

Section: Nucleotide-dependent Peptide Transportmentioning

confidence: 99%

TAP and TAP-like — Brothers in arms?

Zhao

Tampé

Abele

2006

Naunyn Schmied Arch Pharmacol

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The transporter associated with antigen processing like (TAPL, ABCB9) is a member of the ATP-binding cassette (ABC) transporter family. Moreover, TAPL belongs to the TAP family due to its high sequence homology to TAP1 and TAP2. TAPL forms a homodimer which is localized in lysosomes with a minor fraction in the ER. It functions as an ATP-dependent peptide transporter which shows a broad peptide specificity ranging from 6-mer up to 59-mer peptides. In contrast to TAP, TAPL transports peptides with low affinity but high efficiency. This review will briefly summarize current knowledge about the structural organization and possible physiological function of TAPL in antigen processing and presentation.

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Multidrug resistance mediated by the ATP-binding cassette transporter protein MRP

1998

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Resistance to multiple natural product drugs associated with reduced drug accumulation in human tumor cells may be conferred by either the 170 kDa P‐glycoprotein or the 190 kDa multidrug resistance protein, MRP. Both MRP and P‐glycoprotein belong to the large and ancient ATP‐binding cassette (ABC) superfamily of transport proteins but share only 15% amino acid identity. Unlike P‐glycoprotein, MRP actively transports conjugated organic anions such as the cysteinyl leukotriene C4 and glutathione‐conjugated aflatoxin B1. Transport of unconjugated chemotherapeutic agents appears to require cotransport of glutathione. MRP and several more recently discovered ABC proteins contain an additional NH2‐proximal membrane‐spanning domain not found in previously characterized ABC transporters. This domain, whose NH2‐terminus is extracytosolic, is essential for MRP‐mediated transport activity. This review summarizes current knowledge of the structural and transport characteristics of MRP which suggest that the physiologic functions of this protein could range from a protective role in chemical toxicity and oxidative stress to mediation of inflammatory responses involving cysteinyl leukotrienes. BioEssays 20:931–940, 1998. © 1998 John Wiley & Sons, Inc.

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ATPase Activity of the Cystic Fibrosis Transmembrane Conductance Regulator

Cited by 254 publications

References 43 publications

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

Characterization of the Adenosinetriphosphatase and Transport Activities of Purified Cystic Fibrosis Transmembrane Conductance Regulator

TAP and TAP-like — Brothers in arms?

Multidrug resistance mediated by the ATP-binding cassette transporter protein MRP

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