Demonstration of Phosphoryl Group Transfer Indicates That the ATP-binding Cassette (ABC) Transporter Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) Exhibits Adenylate Kinase Activity

Abstract

Background: Electrophysiological studies indicated that Cl− channel function of cystic fibrosis transmembrane conductance regulator (CFTR) can be coupled to adenylate kinase activity (ATP+AMP ⇆ 2 ADP).Results: CFTR catalyzes phosphoryl group transfer between a nucleotide triphosphate and a photoactivatable AMP analog.Conclusion: CFTR exhibits adenylate kinase activity.Significance: These data demonstrate biochemically that a membrane-bound ABC transporter can function as an adenylate kinase.

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

“…Cell membranes were prepared as described previously (22). The high speed membrane pellet (70,000 × g , 40 min, 4 °C) was resuspended in 20 m m Hepes (pH 7.5), 50 m m NaCl, 3 m m MgCl 2 , 2 μg/ml leupeptin, 100 μg/ml Pefabloc, and 7 μg/ml E-64.…”

“…Samples were stored at −80 °C overnight and thawed on ice before adding CFTR antibodies for immunoprecipitation. CFTR was immunoprecipitated as described (22) using monoclonal CFTR antibodies to its regulatory domain (13-1; 0.2 μg/sample) and NBD2 (M3A7; 1 μg/sample).…”

“…In this case, ATP is bound to both ATP-binding sites and hydrolyzed at ATP-binding site 2 (15–18). However, patch clamp and biochemical studies showed that in the presence of physiologically relevant concentrations of AMP, adenylate kinase activity regulates CFTR channel function (19–22). The data further suggested that AMP interacted with a binding site distinct from the two ATP-binding sites and that ATP at ATP-binding site 2 was involved in the adenylate kinase activity (19, 20, 22).…”

“…However, patch clamp and biochemical studies showed that in the presence of physiologically relevant concentrations of AMP, adenylate kinase activity regulates CFTR channel function (19–22). The data further suggested that AMP interacted with a binding site distinct from the two ATP-binding sites and that ATP at ATP-binding site 2 was involved in the adenylate kinase activity (19, 20, 22). Both ATPase and adenylate kinase activity have been reported for two other ABC proteins, Rad50 (23) and SMC (structural maintenance of chromosome protein) (24).…”

AJRCCM: 100-Year Anniversary.Progress along the Pathway of Discovery Leading to Treatment and Cure of Cystic Fibrosis

“…Then the question became, how does it work? Physiological, biochemical, and structural studies have provided insight (33)(34)(35)(36). Twelve transmembrane helices form a pore (Figure 3).…”