Gating of cystic fibrosis transmembrane conductance regulator chloride channels by adenosine triphosphate hydrolysis - Quantitative analysis of a cyclic gating scheme

被引:88
作者
Zeltwanger, S
Wang, F
Wang, GT
Gillis, KD
Hwang, TC
机构
[1] Univ Missouri, Dalton Cardiovasc Res Ctr, Dept Physiol, Columbia, MO 65211 USA
[2] Univ Missouri, Dalton Cardiovasc Res Ctr, Dept Elect Engn, Columbia, MO 65211 USA
关键词
ATP-binding cassette transporter; cystic fibrosis; patch-clamp; gating mechanism; single-channel kinetics;
D O I
10.1085/jgp.113.4.541
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
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 ( rc) decreased with increasing [ATP] to a minimum value of similar to 0.43 s at [ATP] >1.00 mM. The open time constant (tau o) increased with increasing [ATP] with a minimal tau o of similar to 260 ms. Kinetic analysis of K1250A-CFTR, a mutant that abolishes ATP hydrolysis at NBDP2, reveals the presence of two open states. A short open state with a time constant of similar to 250 ms is dominant at low ATP concentrations (10 mu M) and a much longer open state with a time constant of similar to 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.
引用
收藏
页码:541 / 554
页数:14
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