Intracellular Proton-Transfer Mutants in a CLC Cl−/H+ Exchanger

Abstract

CLC-ec1, a bacterial homologue of the CLC family’s transporter subclass, catalyzes transmembrane exchange of Cl− and H+. Mutational analysis based on the known structure reveals several key residues required for coupling H+ to the stoichiometric countermovement of Cl−. E148 (Gluex) transfers protons between extracellular water and the protein interior, and E203 (Gluin) is thought to function analogously on the intracellular face of the protein. Mutation of either residue eliminates H+ transport while preserving Cl− transport. We tested the role of Gluin by examining structural and functional properties of mutants at this position. Certain dissociable side chains (E, D, H, K, R, but not C and Y) retain H+/Cl− exchanger activity to varying degrees, while other mutations (V, I, or C) abolish H+ coupling and severely inhibit Cl− flux. Transporters substituted with other nonprotonatable side chains (Q, S, and A) show highly impaired H+ transport with substantial Cl− transport. Influence on H+ transport of side chain length and acidity was assessed using a single-cysteine mutant to introduce non-natural side chains. Crystal structures of both coupled (E203H) and uncoupled (E203V) mutants are similar to wild type. The results support the idea that Gluin is the internal proton-transfer residue that delivers protons from intracellular solution to the protein interior, where they couple to Cl− movements to bring about Cl−/H+ exchange.

Water access points and hydration pathways in CLC H + /Cl − transporters

“…An unexplained detail of the CLC transport mechanism is how protons are translocated across a 15-Å central hydrophobic region between the established proton sites, Glu ex and Glu in (3,9,22,28). To resolve this key mechanistic detail, we performed extended equilibrium MD simulations and complementary experimental analysis to evaluate whether water wires might mediate proton transport between the two sites and to characterize factors that support water wire formation.…”

“…ClC-ec1, a CLC from Escherichia coli, has served as the prototype CLC for biophysical studies because of its known crystal structures (10, 11), its tractable biochemical behavior, and its structural and mechanistic similarities to mammalian CLC transporters (3)(4)(5)(6)(7)(8)(12)(13)(14)(15)(16)(17). Detailed structural and functional studies of 11,[18][19][20][21][22][23][24][25][26][27] have shed light on some of its key mechanistic aspects. Most prominently, these studies have characterized the Cl − permeation pathway and its lining residues (10,18,25) and established the role of E148, also known as Glu ex , as the extracellular gate for the Cl − pathway (9, 11).…”

“…Detailed structural and functional studies of 11,[18][19][20][21][22][23][24][25][26][27] have shed light on some of its key mechanistic aspects. Most prominently, these studies have characterized the Cl − permeation pathway and its lining residues (10,18,25) and established the role of E148, also known as Glu ex , as the extracellular gate for the Cl − pathway (9, 11).Although much less is known about the H + translocation pathway (and mechanism), experimental studies have provided key information on the involvement of specific residues in H + transport (9,13,14,20,22,27,28). Extensive site-directed mutagenesis studies have zeroed in on two glutamate residues essential for H + transport (Fig.…”

“…Although much less is known about the H + translocation pathway (and mechanism), experimental studies have provided key information on the involvement of specific residues in H + transport (9,13,14,20,22,27,28). Extensive site-directed mutagenesis studies have zeroed in on two glutamate residues essential for H + transport (Fig.…”

“…Extensive site-directed mutagenesis studies have zeroed in on two glutamate residues essential for H + transport (Fig. 1A): E148 (Glu ex ), which acts as the main extracellular H + binding site (9,11,27), and E203 (Glu in ), which plays a similar role on the cytoplasmic side (20,22,28). Neutralization of either glutamate eliminates H + translocation by ClC-ec1 (9,28).…”