Characterization of the adenosinetriphosphatase and transport activities of purified cystic fibrosis transmembrane conductance regulator.
The cystic fibrosis transmembrane conductance regulator (CFTR) functions in vivo as a cAMP-activated 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[alpha-(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 lysophosphatidylglycerol (1%) and diheptanoylphosphatidylcholine (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(-1) (mg of protein)(-1), K(M)(MgATP) of 0.15 mM]. In further work, we found that neither nucleotide binding nor ATPase activity was altered by phosphorylation (using protein kinase A) or dephosphorylation (with protein phosphatase 2B); we also observed inhibition (approximately 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 CFTR-dependent transport of (125)I that could be inhibited by known chloride channel blockers; no significant CFTR-dependent transport of [alpha-(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.
Ketchum
Christian J
CJ
Department of Physiology, Johns Hopkins Medical School, Baltimore, Maryland 21205, USA.
Rajendrakumar
Garnepudi V
GV
Maloney
Peter C
PC
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D001386:Azides / Q000378:metabolism; D001692:Biological Transport / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification* / Q000502:physiology*; D006801:Humans; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism; D014316:Tritium
D001386:Azides; C505032:CFTR protein, human; D007457:Iodine Radioisotopes; D011510:Proteolipids; D011994:Recombinant Proteins; C020485:proteoliposomes; D014316:Tritium; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases