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Characterization of the ATP-dependent Gating of a Phosphorylation-independent CFTR Channel (
R-CFTR)
2 Dalton Cardiovascular Research Center, University of Missouri-Columbia, Columbia, MO 65211
3 Department of Physiology, Osaka Medical College, Takatsuki, Osaka 569-8686, Japan
Correspondence to Tzyh-Chang Hwang: hwangt{at}health.missouri.edu
The CFTR chloride channel is activated by phosphorylation of serine residues in the regulatory (R) domain and then gated by ATP binding and hydrolysis at the nucleotide binding domains (NBDs). Studies of the ATP-dependent gating process in excised inside-out patches are very often hampered by channel rundown partly caused by membrane-associated phosphatases. Since the severed R-CFTR, whose R domain is completely removed, can bypass the phosphorylation-dependent regulation, this mutant channel might be a useful tool to explore the gating mechanisms of CFTR. To this end, we investigated the regulation and gating of the R-CFTR expressed in Chinese hamster ovary cells. In the cell-attached mode, basal R-CFTR currents were always obtained in the absence of cAMP agonists. Application of cAMP agonists or PMA, a PKC activator, failed to affect the activity, indicating that the activity of R-CFTR channels is indeed phosphorylation independent. Consistent with this conclusion, in excised inside-out patches, application of the catalytic subunit of PKA did not affect ATP-induced currents. Similarities of ATP-dependent gating between wild type and R-CFTR make this phosphorylation-independent mutant a useful system to explore more extensively the gating mechanisms of CFTR. Using the R-CFTR construct, we studied the inhibitory effect of ADP on CFTR gating. The Ki for ADP increases as the [ATP] is increased, suggesting a competitive mechanism of inhibition. Single channel kinetic analysis reveals a new closed state in the presence of ADP, consistent with a kinetic mechanism by which ADP binds at the same site as ATP for channel opening. Moreover, we found that the open time of the channel is shortened by as much as 54% in the presence of ADP. This unexpected result suggests another ADP binding site that modulates channel closing.
Key Words: chloride channel • single-channel kinetics • ABC transporter • gating mode • phosphorylation
Abbreviations used in this paper: ABC, ATP-binding cassette; BIM, bisindolylmaleimide; CHO, Chinese hamster ovary; CFTR, cystic fibrosis transmembrane conductance regulator; CPT-cAMP, 8-(4-chlorophenylthio)-cAMP; NBD, nucleotide binding domain; NMDG-Cl, N-methyl-D-glucamine chloride; PKI, PKA inhibitor; R, regulatory; WT, wild type.
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