Christine E. Bear

The two NBDs (nucleotide-binding domains) of ABC (ATP-binding-cassette) proteins function in a complex to mediate ATPase activity and this activity has been linked to their regulated transport activity. A similar model has been proposed for CFTR (cystic fibrosis transmembrane conductance regulator), the chloride channel defective in cystic fibrosis, wherein ATP binding and hydrolysis regulate the channel gate. Recently, it was shown that the individual NBDs isolated from CFTR primarily mediate adenylate kinase activity, raising the possibility that this activity may also contribute to gating of the CFTR channel. However, this present study shows that whereas the isolated NBDs exhibit adenylate kinase activity, the full-length purified and reconstituted CFTR protein functions as an ATPase, arguing that the enzymatic activity of the NBDs is dependent on their molecular context and appropriate domain–domain assembly. As expected, the disease-causing mutant bearing a mutation in the ABC signature motif, CFTR-G551D, exhibited a markedly reduced ATPase activity. Furthermore, mutation of the putative catalytic base in CFTR caused a reduction in ATPase activity, with the CFTR-E1371Q mutant supporting a low level of residual activity. Neither of these mutants exhibited detectable adenylate kinase activity. Together, these findings support the concept that the molecular mechanism of action of CFTR is dependent on ATP binding and hydrolysis, and that the structure of prokaryotic ABC ATPases provide a useful template for understanding their mechanism of action.

The chloride channel, ClC‐2 is expressed ubiquitously and participates in multiple physiological processes. In particular, ClC‐2 has been implicated in the regulation of neuronal chloride ion homeostasis and mutations in ClC‐2 are associated with idiopathic generalized epilepsy. Despite the physiological and pathophysiological significance of this channel, its regulation remains incompletely understood. The functional expression of ClC‐2 at the cell surface has been shown to be enhanced by depletion of cellular ATP, implicating its possible role in cellular energy sensing. In the present study, biochemical assays of cell surface expression suggest that this gain of function reflects, in part, an increase in channel number due to the reduction in ClC‐2 internalization by endocytosis. Cell surface expression of the disease‐causing mutant: G715E, thought to lack wild‐type nucleotide binding affinity, is similarly affected, suggesting that ATP‐depletion modifies the function of proteins in the endocytic pathway rather than ClC‐2 directly. Using a combination of immunofluorescence and biochemical studies, we confirmed that ClC‐2 is internalized via dynamin‐dependent endocytosis and that the change in surface expression evoked by ATP depletion is partially mimicked by inhibition of dynamin function using a dynamin dominant‐negative mutant (DynK44A). Furthermore, trafficking via the early endosomal compartment occurs in part through rab5‐associated vesicles and recycling of ClC‐2 to the cell surface occurs through a rab11 dependent pathway. In summary, we have determined that the internalization of ClC‐2 by endocytosis is inhibited by metabolic stress, highlighting the importance for understanding the molecular mechanisms mediating the endosomal trafficking of this channel. J. Cell. Physiol. 214:273–280, 2008.

CFTR (cystic fibrosis transmembrane conductance regulator), a member of the ABC (ATP-binding cassette) superfamily of membrane proteins, possesses two NBDs (nucleotide-binding domains) in addition to two MSDs (membrane spanning domains) and the regulatory ‘R’ domain. The two NBDs of CFTR have been modelled as a heterodimer, stabilized by ATP binding at two sites in the NBD interface. It has been suggested that ATP hydrolysis occurs at only one of these sites as the putative catalytic base is only conserved in NBD2 of CFTR (Glu1371), but not in NBD1 where the corresponding residue is a serine, Ser573. Previously, we showed that fragments of CFTR corresponding to NBD1 and NBD2 can be purified and co-reconstituted to form a heterodimer capable of ATPase activity. In the present study, we show that the two NBD fragments form a complex in vivo, supporting the utility of this model system to evaluate the role of Glu1371 in ATP binding and hydrolysis. The present studies revealed that a mutant NBD2 (E1371Q) retains wild-type nucleotide binding affinity of NBD2. On the other hand, this substitution abolished the ATPase activity formed by the co-purified complex. Interestingly, introduction of a glutamate residue in place of the non-conserved Ser573 in NBD1 did not confer additional ATPase activity by the heterodimer, implicating a vital role for multiple residues in formation of the catalytic site. These findings provide the first biochemical evidence suggesting that the Walker B residue: Glu1371, plays a primary role in the ATPase activity conferred by the NBD1–NBD2 heterodimer.

The ClC family of chloride channels and transporters includes several members in which mutations have been associated with human disease. Clearly, an understanding of the structure-function relationships of these proteins will be critical in defining the molecular mechanisms underlying disease pathogenesis. The X-ray crystal structure of prokaryotic ClC proteins provides an exquisite template with which to model molecular aspects of eukaryotic ClC protein function. The dimeric structure of these proteins highlights the pivotal importance of intermolecular interactions in the modulation of channel/transporter activity, while mutagenesis studies implicate a crucial role for intrinsic interdomain interactions in regulated function. In this review, we will initially focus on the channel forming members of this family and discuss interactions within homodimeric channel complexes important for gating. Finally, with regard to both channel and transporter family members, we will discuss the multiple heteromeric interactions which occur with cytosolic proteins, and the putative functional impact of these interactions.

Studies have shown that expression of cystic fibrosis transmembrane conductance regulator (CFTR) is associated with enhanced glutathione (GSH) efflux from airway epithelial cells, implicating a role for CFTR in the control of oxidative stress in the airways. To define the mechanism underlying CFTR‐associated GSH flux, we studied wild‐type and mutant CFTR proteins expressed in Sf9 membranes, as well as purified and reconstituted CFTR. We show that CFTR‐expressing membrane vesicles mediate nucleotide‐activated GSH flux, which is disrupted in the R347D pore mutant, and in the Walker A K464A and K1250A mutants. Further, we reveal that purified CFTR protein alone directly mediates nucleotide‐dependent GSH flux. Interestingly, although ATP supports GSH flux through CFTR, this activity is enhanced in the presence of the non‐hydrolyzable ATP analog AMP‐PNP. These findings corroborate previous suggestions that CFTR pore properties can vary with the nature of the nucleotide interaction. In conclusion, our data demonstrate that GSH flux is an intrinsic function of CFTR and prompt future examination of the role of this function in airway biology in health and disease.