Severed Molecules Functionally Define the Boundaries of the Cystic Fibrosis Transmembrane Conductance Regulator's Nh2-Terminal Nucleotide Binding Domain

Abstract

The cystic fibrosis transmembrane conductance regulator is a Cl− channel that belongs to the family of ATP-binding cassette proteins. The CFTR polypeptide comprises two transmembrane domains, two nucleotide binding domains (NBD1 and NBD2), and a regulatory (R) domain. Gating of the channel is controlled by kinase-mediated phosphorylation of the R domain and by ATP binding, and, likely, hydrolysis at the NBDs. Exon 13 of the CFTR gene encodes amino acids (aa's) 590–830, which were originally ascribed to the R domain. In this study, CFTR channels were severed near likely NH2- or COOH-terminal boundaries of NBD1. CFTR channel activity, assayed using two-microelectrode voltage clamp and excised patch recordings, provided a sensitive measure of successful assembly of each pair of channel segments as the sever point was systematically shifted along the primary sequence. Substantial channel activity was taken as an indication that NBD1 was functionally intact. This approach revealed that the COOH terminus of NBD1 extends beyond aa 590 and lies between aa's 622 and 634, while the NH2 terminus of NBD1 lies between aa's 432 and 449. To facilitate biochemical studies of the expressed proteins, a Flag epitope was added to the NH2 termini of full length CFTR, and of CFTR segments truncated before the normal COOH terminus (aa 1480). The functionally identified NBD1 boundaries are supported by Western blotting, coimmunoprecipitation, and deglycosylation studies, which showed that an NH2-terminal segment representing aa's 3–622 (Flag3-622) or 3–633 (Flag3-633) could physically associate with a COOH-terminal fragment representing aa's 634–1480 (634-1480); however, the latter fragment was glycosylated to the mature form only in the presence of Flag3-633. Similarly, 433-1480 could physically associate with Flag3-432 and was glycosylated to the mature form; however, 449-1480 protein seemed unstable and could hardly be detected even when expressed with Flag3-432. In excised-patch recordings, all functional severed CFTR channels displayed the hallmark characteristics of CFTR, including the requirement of phosphorylation and exposure to MgATP for gating, ability to be locked open by pyrophosphate or AMP-PNP, small single channel conductances, and high apparent affinity of channel opening by MgATP. Our definitions of the boundaries of the NBD1 domain in CFTR are supported by comparison with the solved NBD structures of HisP and RbsA.

Cysteine Substitutions Reveal Dual Functions of the Amino-terminal Tail in Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

“…to increase channel closing rate). A role for the N-tail in modulating channel opening rate is also supported by the recent findings of Chan et al (15), who reported that CFTR channels that had been epitope-tagged at the amino terminus exhibited reduced opening rates in excised patches. Our prior failure to detect an obvious effect of the alanine and cysteine substitutions on channel opening rate may be due to the fact that absolute rates of channel opening can be overestimated in multichannel patches that contain mutant channels with low activity (i.e.…”

“…We observed previously that the isolated N-tail could bind in vitro to a peptide fragment of CFTR (residues 595-813) that included the distal portion of NBD1 followed by the R domain (13). (At the time of that study residues 595-813 were thought to constitute the R domain alone (4); however, the more recent functional data of Chan et al (15) indicate that NBD1 probably extends to between residues 622 and 634.) N-tail mutations that disrupt binding to this NBD1/R domain fragment have no effect on channel phosphorylation (14), but it is possible that R domain phosphorylation could be affected by chemical modification of these sites.…”

“…closed time) for D58N CFTR (14) as if this mutation also affected the ability of the channel to open. In addition, Chan et al (15) have recently reported that CFTR channels that have been epitope-tagged (FLAG-tagged) at the extreme amino terminus exhibit fewer channel openings in excised membrane patches (i.e. reduced opening rate) relative to the untagged channel.…”

Slc26a9 Is Inhibited by the R-region of the Cystic Fibrosis Transmembrane Conductance Regulator via the STAS Domain

“…2). As noted these boundaries were previously functionally designated by Chan and co-workers (17). As the Western blots in Fig.…”

Assembly and Misassembly of Cystic Fibrosis Transmembrane Conductance Regulator: Folding Defects Caused by Deletion of F508 Occur Before and After the Calnexin-dependent Association of Membrane Spanning Domain (MSD) 1 and MSD2

“…Calnexin may facilitate CFTR folding by stabilizing MSD2 and thereby promoting formation of proper contacts between MSD1 and MSD2. To test this model, we took advantage of the observation that split CFTR fragments that individually contain the N-and C-terminal subdomains assemble into an ion channel when expressed in trans (Chan et al, 2000). Nterminal CFTR fragments containing MSD1, NBD1, and the R domain fold to a conformation that has a long half-life and accumulates at high levels when expressed alone or in trans with CFTR 837-1480 (Ostedgaard et al, 1997;Xiong et al, 1997;Meacham et al, 1999).…”