ADP inhibits function of the ABC transporter cystic fibrosis transmembrane conductance regulator via its adenylate kinase activity

In the presence of ATP, genistein, like the ATP analogue adenosine 5ʹ-[β,γ-imido]triphosphate (pp[NH]pA), increases cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents by prolonging open times. As pp[NH]pA is thought to increase CFTR currents by interfering with ATP hydrolysis at the second nucleotide-binding fold (NBF-2), the present study was undertaken to investigate the effects of genistein on a fusion protein comprising maltose-binding protein (MBP) and NBF-2 (MBP-NBF-2). MBP-NBF-2 exhibited ATPase, GTPase and adenylate kinase activities that were inhibited by genistein in a partial non-competitive manner with respect to ATP or GTP. Ki values for competitive and uncompetitive inhibition were respectively 20 μM and 63 μM for ATPase, 15 μM and 54 μM for GTPase, and 46 μM and 142 μM for adenylate kinase. For ATPase activity, genistein reduced Vmax by 29% and Vmax/Km by 77%. Additional evidence for complex-formation between genistein and MBP-NBF-2 was obtained by the detection of genistein-dependent alterations in the CD spectrum of MBP-NBF-2 that were consistent with the formation of a higher-ordered state. Addition of MBP-NBF-2 increased the fluorescence intensity of genistein, consistent with a change to a less polar environment. pp[NH]pA partially eliminated this enhanced fluorescence of genistein. These observations provide the first direct biochemical evidence that genistein interacts with CFTR, thus inhibiting NBF-2 activity, and suggest a similar mechanism for genistein-dependent stimulation of CFTR chloride currents.

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Nonequilibrium Gating of CFTR on an Equilibrium Theme

Physiology ◽

10.1152/physiol.00026.2012 ◽

2012 ◽

Vol 27 (6) ◽

pp. 351-361 ◽

Cited By ~ 26

Author(s):

Kang-Yang Jih ◽

Tzyh-Chang Hwang

Keyword(s):

Cystic Fibrosis ◽

Abc Transporter ◽

Chloride Channel ◽

Genetic Disease ◽

Clinical Relevance ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Abc Protein ◽

Gating Mechanism ◽

Cystic Fibrosis Transmembrane

Malfunction of cystic fibrosis transmembrane conductance regulator (CFTR), a member of the ABC protein superfamily that functions as an ATP-gated chloride channel, causes the lethal genetic disease, cystic fibrosis. This review focuses on the most recent findings on the gating mechanism of CFTR. Potential clinical relevance and implications to ABC transporter function are also discussed.

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ATP and AMP Mutually Influence Their Interaction with the ATP-binding Cassette (ABC) Adenylate Kinase Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) at Separate Binding Sites

Journal of Biological Chemistry ◽

10.1074/jbc.m113.479675 ◽

2013 ◽

Vol 288 (38) ◽

pp. 27692-27701 ◽

Cited By ~ 4

Author(s):

Christoph O. Randak ◽

Qian Dong ◽

Amanda R. Ver Heul ◽

Adrian H. Elcock ◽

Michael J. Welsh

Keyword(s):

Binding Site ◽

Binding Sites ◽

Adenylate Kinase ◽

Kinase Activity ◽

Atp Binding ◽

Transmembrane Conductance Regulator ◽

Transmembrane Conductance ◽

Atp Binding Site ◽

Adenylate Kinases ◽

Cystic Fibrosis Transmembrane

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel in the ATP-binding cassette (ABC) transporter protein family. In the presence of ATP and physiologically relevant concentrations of AMP, CFTR exhibits adenylate kinase activity (ATP + AMP ⇆ 2 ADP). Previous studies suggested that the interaction of nucleotide triphosphate with CFTR at ATP-binding site 2 is required for this activity. Two other ABC proteins, Rad50 and a structural maintenance of chromosome protein, also have adenylate kinase activity. All three ABC adenylate kinases bind and hydrolyze ATP in the absence of other nucleotides. However, little is known about how an ABC adenylate kinase interacts with ATP and AMP when both are present. Based on data from non-ABC adenylate kinases, we hypothesized that ATP and AMP mutually influence their interaction with CFTR at separate binding sites. We further hypothesized that only one of the two CFTR ATP-binding sites is involved in the adenylate kinase reaction. We found that 8-azidoadenosine 5′-triphosphate (8-N3-ATP) and 8-azidoadenosine 5′-monophosphate (8-N3-AMP) photolabeled separate sites in CFTR. Labeling of the AMP-binding site with 8-N3-AMP required the presence of ATP. Conversely, AMP enhanced photolabeling with 8-N3-ATP at ATP-binding site 2. The adenylate kinase active center probe P1,P5-di(adenosine-5′) pentaphosphate interacted simultaneously with an AMP-binding site and ATP-binding site 2. These results show that ATP and AMP interact with separate binding sites but mutually influence their interaction with the ABC adenylate kinase CFTR. They further indicate that the active center of the adenylate kinase comprises ATP-binding site 2.

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