The CFTR Chloride Channel: Nucleotide Interactions and Temperature-dependent Gating |
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Authors: | CJ Mathews JA Tabcharani JW Hanrahan |
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Institution: | (1) Department of Physiology, McGill University, Montréal, Québec, Canada, CA |
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Abstract: | The gating cycle of CFTR (Cystic Fibrosis Transmembrane conductance Regulator) chloride channels requires ATP hydrolysis
and can be interrupted by exposure to the nonhydrolyzable nucleotide AMP-PNP. To further characterize nucleotide interactions
and channel gating, we have studied the effects of AMP-PNP, protein kinase C (PKC) phosphorylation, and temperature on gating
kinetics. The rate of channel locking increased from 1.05 × 10−3 sec−1 to 58.7 × 10−3 sec−1 when AMP-PNP concentration was raised from 0.5 to 5 mm in the presence of 1 mm MgATP and 180 nm protein kinase A catalytic subunit (PKA). Although rapid locking precluded estimation of P
o
or opening rate immediately after the addition of AMP-PNP to wild-type channels, analysis of locking rates in the presence
of high AMP-PNP concentrations revealed two components. The appearance of a distinct, slow component at high AMP-PNP] is
evidence for AMP-PNP interactions at a second site, where competition with ATP would reduce P
o
and thereby delay locking. All channels exhibited locking when they were strongly phosphorylated by PKA, but not when exposed
to PKC alone. AMP-PNP increased P
o
at temperatures above 30°C but did not cause locking, evidence that the stabilizing interactions between domains, which have
been proposed to maintain CFTR in the open burst state, are relatively weak. The temperature dependence of normal CFTR gating
by ATP was strongly asymmetric, with the opening rate being much more temperature sensitive (Q
10= 9.6) than the closing rate (Q
10= 3.6). These results are consistent with a cyclic model for gating of phosphorylated CFTR.
Received: 28 August 1997/Revised: 4 February 1998 |
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Keywords: | : Cystic fibrosis — Anion channel — Nucleotide binding — Temperature coefficient — Patch clamp |
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