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

Abstract

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 ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in <em>Saccharomyces cerevisiae</em>

“…As such it indicates a greater range of purification conditions for CFTR than have previously been reported [10][11][12][13] . In addition milligram quantities of purified CFTR can be obtained using these procedures when combined with a high volume yeast growth system such as a 20 L fermenter and a high capacity cell harvesting system such as a 6 L low speed centrifuge rotor.…”

“…On the basis of its primary structure, CFTR is classified as a C-family member of the ABC transporter family, but there is no strong evidence for a residual functional linkage to this sub-family. There have been some reports of glutathione transport activity for CFTR [5][6][7] , which would be consistent with the roles of other C-family members 8,9 , although other reports suggest that reduced glutathione may inhibit the CFTR ATPase activity, rather than showing the substrate-induced stimulation that characterize the ABC transporters 10 . Measurement of ion conductance is sufficiently sensitive to allow the channel activity of single CFTR molecules to be studied 1 and CFTR channel properties have been monitored as a function of time, temperature, ATP concentration, membrane potential, and phosphorylation state, as well as in the presence of a host of small molecule inhibitors, potentiators, and modifiers.…”

“…These studies have also added significantly to our knowledge of how ABC transporters function. Nevertheless, expression of CFTR in significant amounts and its subsequent purification has proven to be particularly challenging and success has been limited to a few laboratories [10][11][12][13] .…”

“…We have previously reported a protocol for the large-scale expression of GFP-and His-tagged murine CFTR in S. cerevisiae [15][16][17][18][19][20][21] . The ionic detergent 1-tetradecanoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (LPG-14) is highly efficient in the solubilization of CFTR and has previously been used in the purification of functional membrane proteins 10,22,23 , including purification of CFTR from S. cerevisiae 24 .…”

Modulation of the hepatocyte rough endoplasmic reticulum single chloride channel by nucleotide–Mg2+ interaction

“…This chloride conductance has properties different from those attributed to CLIC, because the CLICs are insensitive to chloride channel blocker DIDS [19,29,35]. When reconstituted in a planar lipid bilayer, the properties of CFTR [14,20,34] are also distinct from the present Cl − channel. They differ markedly in single channel conductance and sensitivity to DIDS.…”