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© The Rockefeller University Press, 0022-1295/1999//541/ $5.00
Journal of General Physiology, Volume 113, Number 4, 1999

Article

Gating of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channels by Adenosine Triphosphate Hydrolysis

Quantitative Analysis of a Cyclic Gating Scheme



Shawn Zeltwanger*, Fei Wang*, Guo-Tang Wang*, Kevin D. Gillis{ddagger}, and Tzyh-Chang Hwang*

From the * Department of Physiology and {ddagger} Department of Electrical Engineering, Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, Missouri 65211

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 ({tau}c) decreased with increasing [ATP] to a minimum value of ~0.43 s at [ATP] >1.00 mM. The open time constant ({tau}o) increased with increasing [ATP] with a minimal {tau}o of ~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 ~250 ms is dominant at low ATP concentrations (10 µM) and a much longer open state with a time constant of ~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

Address correspondence to Tzyh-Chang Hwang, DCRC Research Park, University of Missouri-Columbia, Columbia, MO 65211. Fax: 573-884-4232; E-mail: dcrctch{at}showme.missouri.edu

Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; NMDG, N-methyl-D-glucamine; pdf, probability density function; wt, wild type


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