Conformational Changes in a Pore-lining Helix Coupled to Cystic Fibrosis Transmembrane Conductance Regulator Channel Gating

Edward J Beck,Yu Yang,Sirin Yaemsiri,Viswanathan Raghuram

doi:10.1074/jbc.m702235200

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR), the protein dysfunctional in cystic fibrosis, is unique among ATP-binding cassette transporters in that it functions as an ion channel. In CFTR, ATP binding opens the channel, and its subsequent hydrolysis causes channel closure. We studied the conformational changes in the pore-lining sixth transmembrane segment upon ATP binding by measuring state-dependent changes in accessibility of substituted cysteines to methanethiosulfonate reagents. Modification rates of three residues (resides 331, 333, and 335) near the extracellular side were 10-1000-fold slower in the open state than in the closed state. Introduction of a charged residue by chemical modification at two of these positions (resides 331 and 333) affected CFTR single-channel gating. In contrast, modifications of pore-lining residues 334 and 338 were not state-dependent. Our results suggest that ATP binding induces a modest conformational change in the sixth transmembrane segment, and this conformational change is coupled to the gating mechanism that regulates ion conduction. These results may establish a structural basis of gating involving the dynamic rearrangement of transmembrane domains necessary for vectorial transport of substrates in ATP-binding cassette transporters.

Highlights

ATP-binding cassette (ABC)2 transporters are a large family of integral membrane proteins that actively transport a broad range of substrates across cell membranes
The substituted cysteine-accessibility method assumes that only cysteine residues at a water-accessible surface of the protein will react with hydrophilic MTS reagents and that the modification produces an irreversible change in channel function
We believe that K329C, whose whole cell conductance was stimulated by Cd2ϩ, is an example of one such residue that reacts with MTSEA, but the modification is without effect on channel function

Summary

EXPERIMENTAL PROCEDURES

Molecular Biology—Mutations were constructed in pSPCFTR [14] plasmid containing the cDNA of human CFTR, using the QuikChange site-directed mutagenesis kit from Stratagene (La Jolla, CA). The oocyte vitelline membranes were removed manually, and the oocytes were transferred to a recording chamber containing standard bath solution. Single CFTR channel currents were recorded in cellattached configuration, at a pipette potential of Ϯ100 mV via an Axopatch 200B amplifier, filtered at 100 Hz, digitized online at 500 Hz using an ITC-18 board, and recorded on disk by Pulse software. The lowest MTS concentration sufficient to produce a measurable change in conductance (within 3 min) was used to calculate the apparent second order reaction rate constant (k ϭ 1/([⌴TS]␶)). Statistical Methods—One-way analysis of variance with posthoc Donnet’s test (PrismGraphPad) was used to assess the statistical significance of any change in cAMP-activated conductance following exposure to Cd2ϩ and MTS reagents (see Fig. 1C). Student’s unpaired (two-tailed) t test was used to assess the statistical significance for all other experiments

RESULTS

IBMX μS μS

This analysis was carried out in both

DISCUSSION