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J. Biol. Chem., Vol. 277, Issue 3, 2125-2131, January 18, 2002
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From the The cystic fibrosis transmembrane conductance
regulator (CFTR) Cl
Mutations That Change the Position of the Putative
-Phosphate Linker in the Nucleotide Binding Domains of CFTR
Alter Channel Gating*
§,,
, and**
Howard Hughes Medical Institute, Departments
of Internal Medicine and Physiology and Biophysics, University of Iowa
College of Medicine, Iowa City, Iowa 52242, the ¶ Department of
Biological Sciences, Columbia University, New York, New York 10027, and
the Department of Physiology, University of Texas Southwestern
Medical Center, Dallas, Texas 75390
channel is an ATP-binding
cassette transporter that contains conserved nucleotide-binding
domains (NBDs). In CFTR, the NBDs bind and hydrolyze ATP to open and
close the channel. Crystal structures of related NBDs suggest a
structural model with an important signaling role for a 
-phosphate
linker peptide that couples bound nucleotide to movement of an
-helical subdomain. We mutated two residues in CFTR that the
structural model predicts will uncouple effects of nucleotide binding
from movement of the -helical subdomain. These residues are Gln-493
and Gln-1291, which may directly connect the ATP -phosphate to the
-phosphate linker, and residues Asn-505 and Asn-1303, which may form
hydrogen bonds that stabilize the linker. In NBD1, Q493A reduced
the frequency of channel opening, suggesting a role for this residue in
coupling ATP binding to channel opening. In contrast, N505C increased
the frequency of channel opening, consistent with a role for Asn-505 in
stabilizing the inactive state of the NBD. In NBD2, Q1291A decreased
the effects of pyrophosphate without altering other functions.
Mutations of Asn-1303 decreased the rate of channel opening and
closing, suggesting an important role for NBD2 in controlling channel
burst duration. These findings are consistent with both the bacterial
NBD structural model and gating models for CFTR. Our results
extend models of nucleotide-induced structural changes from
bacterial NBDs to a functional mammalian ATP-binding cassette transporter.
*
This work was supported by NHLBI, National Institutes of
Health (NIH) Grant HL29851; the Cystic Fibrosis Foundation; the Howard Hughes Medical Institute; the Diabetes and Endocrine Research Center,
Grant NIH DK25295; and the In Vitro Models Cell Culture Core, Grant NIH HL51670 and CFF.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
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