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

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 ATPbinding cassette transporters. 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. Despite their diverse functions, they share a common basic architecture comprised of two transmembrane domains (TMDs) that function as a pathway for the permeation of substrates and two cytoplasmic nucleotide-binding domains (NBDs). The highly conserved NBDs are the molecular motors that transform the chemical potential energy of ATP into conformational changes that drive substrate molecules through the TMDs (1). Recent biochemical, structural, and genetic studies have led to a common mechanism in which ATP binding and hydrolysis induce the formation and dissociation of an NBD dimer, respectively. This regulated switch induces conformational changes in the TMDs to mediate vectorial transport of substrates across cell membranes (2-4). However, the structural bases for the propagation of conformational changes in the NBDs to the TMDs, and the conformational changes in TMDs are not well understood.Cystic fibrosis transmembrane conductance regulator (CFTR; ATP-binding cassette transporter subfamily C member 7), the product of the cystic fibrosis gene, is unique among ABC transporters in that its TMDs provide a conductive channel for anions. Phosphorylation of serines in the regulatory domain by cAMP-dependent protein kinase activates CFTR (see Fig. 1A). Once phosphorylated, ATP-induced dimerization of NBDs opens the channel, and their subsequent dissociation upon ATP hydrolysis closes the channel (5). Despite extensive biochemical, structural, and functional studies, the nature of conformational changes in TMDs associated with CFTR channel gating remains elusive.The ...

Relative Movements of Transmembrane Regions at the Outer Mouth of the Cystic Fibrosis Transmembrane Conductance Regulator Channel Pore during Channel Gating

“…Channel Inhibition by External Cd 2ϩ Ions-Cysteine side chains can bind metal ions like Cd 2ϩ , and indeed, current inhibition by externally applied Cd 2ϩ ions has previously been used to identify individual pore-lining cysteine side chains in CFTR (23). Furthermore, coordination of Cd 2ϩ ions by multiple cysteine side chains can lead to stronger binding if these side chains are separated by Ͻ10 Å (see "Experimental Procedures").…”

“…In TM6, we selected three functionally important residues: Arg-334 and Lys-335, which act to attract extracellular Cl Ϫ ions electrostatically to the pore (17,29), and Thr-338, which lines the narrowest part of the pore (6) and contributes to a region of high resistance to Cl Ϫ flux (30). Each of these three residues have been proposed to show conformation-dependent access from the extracellular solution (9,22,23). The three TM6 mutants R334C, K335C and T338C were combined with cysteine substitution of five externally accessible TM11-ECL6-TM12 residues (S1118C, T1121C, T1122C, G1127C, and T1134C) (15,25) to give a total of 15 cysteine pair mutants (see Figs.…”

“…1 and 7). In some cases, cysteine mutants were combined with the NBD2 mutation E1371Q, which we (8,9) and others (7,23) have used to abolish ATP-dependent channel gating and lock CFTR channels in the open state (see supplemental materials).…”

“…The functional consequences of both CuPhe-induced cross-linking and Cd 2ϩ coordination have previously been used to infer distances between pairs of cysteine side chains of Ͻ10 Å in other channel types (31)(32)(33)(34)(35). Furthermore, both CuPhe-induced cysteine cross-linking (18,19) and Cd 2ϩ inhibition of single cysteine mutants (23) have been investigated in the CFTR channel pore using electrophysiological recording.…”

“…The outer mouth of the CFTR pore is lined both by TM6 (6,23,25) and the TM11-ECL6-TM12 loop (15). In the present study, we have sought to understand the three-dimensional arrangement of these two parts of the protein by engineering disulfide cross-links between introduced cysteine residues.…”