Severed molecules functionally define the boundaries of the cystic fibrosis transmembrane conductance regulator's NH2-terminal nucleotide binding domain

The cystic fibrosis transmembrane conductance regulator is a Cl- channel th at belongs to the family of ATP-binding cassette proteins. The CFTR polypep tide comprises two transmembrane domains, two nucleotide binding domains (N BD1 and NBDP), and a regulatory (R) domain. Gating of the channel is contro lled 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 amin o 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 bo undaries of NBD1. CFTR channel activity, assayed using two-microelectrode v oltage 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 act ivity was taken as an indication that NBD1 was functionally intact. This ap proach revealed that the COOH terminus of NBD1 extends beyond aa 590 and li es 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 C FTR segments truncated before the normal COOH terminus (aa 1480). The funct ionally identified NBD1 boundaries are supported by Western blotting, coimm unoprecipitation, and deglycosylation studies, which showed that an NH2-ter minal segment representing aa's 3-622 (Flag3-622) or 3-633 (Flag3-633) coul d physically associate with a COOH-terminal fragment representing aa's 634- 1480 (634-1480); however; the latter fragment was glycosylated to the matur e form only in the presence of Flag3-633. Similarly, 433-1480 could physica lly associate with Flag3-432 and was glycosylated to the mature form; howev er, 449-1480 protein seemed unstable and could hardly be detected even when expressed with Flag3-432. In excised-patch recordings, all functional seve red CFTR channels displayed the hallmark characteristics of CFTR, including the requirement of phosphorylation and exposure to MgATP for gating, abili ty to be locked open by pyrophosphate or AMP-PNP, small single channel cond uctances, and high apparent affinity of channel opening by MgATP. Our defin itions of the boundaries of the NBD1 domain in CFTR are supported by compar ison with the solved NBD structures of HisP and RbsA.