State-dependent Chemical Reactivity of an Engineered Cysteine Reveals Conformational Changes in the Outer Vestibule of the Cystic Fibrosis Transmembrane Conductance Regulator

Zhi-Ren Zhang,Binlin Song,Nael A Mccarty

doi:10.1074/jbc.m510242200

Abstract

Cystic fibrosis transmembrane conductance regulator (CFTR) chloride channels are gated by binding and hydrolysis of ATP at the nucleotide-binding domains (NBDs). We used covalent modification of CFTR channels bearing a cysteine engineered at position 334 to investigate changes in pore conformation that might accompany channel gating. In single R334C-CFTR channels studied in excised patches, modification by [2-(trimethylammonium)ethyl] methanethiosulfonate (MTSET+), which increases conductance, occurred only during channel closed states. This suggests that the rate of reaction of the cysteine was greater in closed channels than in open channels. R334C-CFTR channels in outside-out macropatches activated by ATP alone were modified with first order kinetics upon rapid exposure to MTSET+. Modification was much slower when channels were locked open by the addition of nonhydrolyzable nucleotide or when the R334C mutation was coupled to a second mutation, K1250A, which greatly decreases channel closing rate. In contrast, modification was faster in R334C/K464A-CFTR channels, which exhibit prolonged interburst closed states. These data indicate that the reactivity of the engineered cysteine in R334C-CFTR is state-dependent, providing evidence of changes in pore conformation coupled to ATP binding and hydrolysis at the NBDs. The data also show that maneuvers that lock open R334C-CFTR do so by locking channels into the prominent s2 subconductance state, suggesting that the most stable conducting state of the pore reflects the fully occupied, prehydrolytic state of the NBDs.

Highlights

Quent channel closure [2]
We recently showed that single wild type CFTR (WT-CFTR) channels exhibit two subconductance states as well as the full-conductance state [17]; the stability and frequency of these substates are enhanced in some channels bearing mutations in the putative pore-lining regions
R334C Channels Are Modified by MTSETϩ Only in the Closed State—We have shown previously that the MTSETϩ-induced covalent modification of a cysteine engineered at CFTR position 334 increased single channel conductance without altering gating properties [17, 18]

Summary

EXPERIMENTAL PROCEDURES

Preparation of Oocytes and cRNA—For mutant R334C, site-directed mutagenesis used a nested PCR strategy in which the mutation was designed into antiparallel oligomers [18]. R334C-, R334C/ K464A-, and R334C/K1250A-CFTR channels were activated by excision into intracellular solution containing 300 mM NMDG-Cl, 1.1 mM MgCl2, 2 mM Tris-EGTA, 1 mM MgATP, 10 mM TES (pH 7.4), 50 units/ml PKA. For outside-out macropatch recordings, electrode tips were filled with a modified intracellular solution (150 mM NMDG-Cl, 1.1 mM MgCl2, 2 mM Tris-EGTA, 10 mM TES, pH 7.4) and backfilled with the same solution containing either 1 mM MgATP plus 100 units/ml PKA or 1 mM MgATP, 100 units/ml PKA, and 2.75 mM AMP-PNP. For outside-out macropatch experiments, we used pClamp 9.0 to fit the time course of covalent modification of CFTR currents using an exponential function to obtain the time constant, ␶, which was converted to a rate coefficient with units of MϪ1 sϪ1 by dividing by [MTSETϩ] or [MTSESϪ]. Statistical analysis was performed using the t test for unpaired or paired measurements by Sigma Stat 2.03 (Jandel Scientific; San Rafael, CA), with p Ͻ 0.05 considered indicative of significance

RESULTS

TABLE ONE

DISCUSSION