Basis of substrate binding and conservation of selectivity in the CLC family of channels and transporters

Abstract

Ion binding to secondary active transporters triggers a cascade of conformational rearrangements resulting in substrate translocation across cellular membranes. Despite the fundamental role of this step, direct measurements of binding to transporters are rare. We investigated ion binding and selectivity in CLC-ec1, a H+/Cl− exchanger of the CLC family of channels and transporters. Cl− affinity depends on the conformation of the protein: it is highest with the extracellular gate removed, and weakens as the transporter adopts the occluded configuration and with the intracellular gate removed. The central ion-binding site determines selectivity in CLC transporters and channels, a serine to proline substitution at this site confers NO3− selectivity upon the Cl− specific CLC-ec1 transporter and CLC-0 channel. We propose that CLC-ec1 operates through an affinity-switch mechanism and that the bases of substrate specificity are conserved in the CLC channels and transporters.

A Single-Pore Residue Renders the Arabidopsis Root Anion Channel SLAH2 Highly Nitrate Selective

“…CLC channels are gated by the side chain carboxylate group of a central highly conserved glutamate that closes the pore by mimicking a chloride ion. The anion selectivity with the order chloride > bromide > nitrate > iodide found for animal CLC channels confirmed the existence of a high-field-strength anion binding site (Wright and Diamond, 1977;Picollo et al, 2009).…”

“…Here, we used Xenopus laevis oocytes to study the anion selectivity of SLAH2 and compared it to that of SLAC1. In this way, we were able to establish SLAH2, in contrast to the NO 3 2 and Cl 2 permeable SLAC1, and homologs from other kingdoms, as an anion channel that transports nitrate exclusively (Chen and Hwang, 2008;Jentsch, 2008;Picollo et al, 2009;Accardi and Picollo, 2010;Hedrich, 2012;Stauber et al, 2012). Based on homology models for SLAC1 and SLAH2, structure-guided sitedirected mutations were used to explore the molecular basis of the extraordinarily high nitrate selectivity of SLAH2.…”

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

“…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.…”

“…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 ClC-ec1 (9,11,(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).…”