Gating of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels by Adenosine Triphosphate Hydrolysis Quantitative Analysis of a Cyclic Gating Scheme

Gating of the cystic fibrosis transmembrane conductance regulator (CFTR) involves a coordinated action of ATP on two nucleotide binding domains (NBD1 and NBD2). Previous studies using nonhydrolyzable ATP analogues and NBD mutant CFTR have suggested that nucleotide hydrolysis at NBD1 is required for opening of the channel, while hydrolysis of nucleotides at NBD2 controls channel closing. We studied ATP-dependent gating of CFTR in excised inside-out patches from stably transfected NIH3T3 cells. Single channel kinetics of CFTR gating at different [ATP] were analyzed. The closed time constant ( t c) decreased with increasing [ATP] to a minimum value of z 0.43 s at [ATP] . 1.00 mM. The open time constant ( t o) increased with increasing [ATP] with a minimal t o of z 260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBD2, reveals the presence of two open states. A short open state with a time constant of z 250 ms is dominant at low ATP concentrations (10 m M) and a much longer open state with a time constant of z 3 min is present at millimolar ATP. These data suggest that nucleotide binding and hydrolysis at NBD1 is coupled to channel opening and that the channel can close without nucleotide interaction with NBD2. A quantitative cyclic gating scheme with microscopic irreversibility was constructed based on the kinetic parameters derived from single-channel analysis. The estimated values of the kinetic parameters suggest that NBD1 and NBD2 are neither functionally nor biochemically equivalent. key words: ATP-binding cassette transporter • cystic fibrosis • patch-clamp • gating mechanism • single-channel kinetics i n t r o d u c t i o n The cystic fibrosis transmembrane conductance regulator (CFTR) 1 (Riordan et al., 1989) is a member of the ATP-binding cassette (Riordan et al., 1989) or traffic ATPase (Ames and Lecar, 1992) superfamily. Proteins in this superfamily harvest the energy from the hydrolysis of ATP to transport a variety of substrates across the cell membrane (Kuchler and Thorner, 1992). Like other members in this family, CFTR is predicted to contain two membrane spanning domains, each composed of six putative membrane-spanning segments and two nucleotide binding domains (NBD1 and NBD2). However, unlike other members, CFTR has a regulatory domain containing multiple consensus sites for phosphorylation via PKA; functionally, CFTR itself is a plasma membrane chloride channel (e.g., Anderson et al., 1991a; Bear et al., 1992). It is thought that regulation of CFTR channel activity is through PKA-dependent phosphorylation of the regulatory (R) domain. Although a prerequisite for activation, PKA phosphorylation of the R domain in itself is not sufficient for opening of the channel. Once the R domain is phosphorylated, nucleotide interaction with the NBDs is coupled to the opening and closing (i.e., gating) of the channel. The NBDs contain a highly conserved region known as the Walker A and Walker B motifs. It is believed that this region forms a close association with the phosphates of bound nucleoside triphosphates (Saraste et al., 1990). From the sequence of the NBDs, it is predicted that they not only bind, but hydrolyze nucleotides. Biochemical experiments using purified CFTR have demonstrated that the protein functions as an ATPase (Bear et al., 1992). Molecular modeling by sequence comparison and functional studies of CFTR have suggested that the function of NBDs (especially NBD2) in CFTR parallels that of a G protein (Hwang et al., 1994; Carson and Welsh, 1995; ManavaAddress correspondence to Tzyh-Chang Hwang, DCRC Research Park, University of Missouri-Columbia, Columbia, MO 65211. Fax: 573-884-4232; E-mail: [email protected] 1 Abbreviations used in this paper: CFTR, cystic fibrosis transmembrane conductance regulator; NMDG, N -methyld -glucamine; pdf, probability density function; wt, wild type. on M ay 2, 2017 D ow nladed fom Published April 1, 1999