Joseph A. Mindell

The Cl - /H + antiporter ClC-7 is the primary chloride permeation pathway in lysosomes

Lysosomes are the stomachs of the cell—terminal organelles on the endocytic pathway where internalized macromolecules are degraded. Containing a wide range of hydrolytic enzymes, lysosomes depend on maintaining acidic luminal pH values for efficient function. Although acidification is mediated by a V-type proton ATPase, a parallel anion pathway is essential to allow bulk proton transport. The molecular identity of this anion transporter remains unknown. Recent results of knockout experiments raise the possibility that ClC-7, a member of the CLC family of anion channels and transporters, is a contributor to this pathway in an osteoclast lysosome-like compartment, with loss of ClC-7 function causing osteopetrosis. Several mammalian members of the CLC family have been characterized in detail; some (including ClC-0, ClC-1 and ClC-2) function as Cl--conducting ion channels, whereas others act as Cl-/H+antiporters (ClC-4 and ClC-5). However, previous attempts at heterologous expression of ClC-7 have failed to yield evidence of functional protein, so it is unclear whether ClC-7 has an important function in lysosomal biology, and also whether this protein functions as a Cl- channel, a Cl-/H+ antiporter, or as something else entirely. Here we directly demonstrate an anion transport pathway in lysosomes that has the defining characteristics of a CLC Cl-/H+ antiporter and show that this transporter is the predominant route for Cl- through the lysosomal membrane. Furthermore, knockdown of ClC-7 expression by short interfering RNA can essentially ablate this lysosomal Cl-/H+ antiport activity and can strongly diminish the ability of lysosomes to acidify in vivo, demonstrating that ClC-7 is a Cl-/H+ antiporter, that it constitutes the major Cl- permeability of lysosomes, and that it is important in lysosomal acidification.

Projection structure of a ClC-type chloride channel at 6.5 Å resolution

Virtually all cells in all eukaryotic organisms express ion channels of the ClC type, the only known molecular family of chloride-ion-selective channels. The diversity of ClC channels highlights the multitude and range of functions served by gated chloride-ion conduction in biological membranes, such as controlling electrical excitability in skeletal muscle, maintaining systemic blood pressure, acidifying endosomal compartments, and regulating electrical responses of GABA (γ-aminobutyric acid)-containing interneurons in the central nervous system. Previously, we expressed and purified a prokaryotic ClC channel homologue. Here we report the formation of two-dimensional crystals of this ClC channel protein reconstituted into phospholipid bilayer membranes. Cryo-electron microscopic analysis of these crystals yields a projection structure at 6.5u2009Å resolution, which shows off-axis water-filled pores within the dimeric channel complex.

The Poststructural Festivities Begin

ClC chloride channels orchestrate the movement of chloride necessary for proper neuronal, muscular, cardiovascular, and epithelial function. In this issue of Neuron, Jentsch, Pusch, and colleagues use the structure of a bacterial ClC homolog to guide a mutagenic analysis of inhibitor binding to ClC-0, ClC-1, and ClC-2.

It Runs in the Family: Determining the Transport Mechanism of Sodium/Dicarboxylate Symporter hNaDC3

Members of the divalent anion:Na(+) symporter (DASS) family play important roles in mammalian physiology, transporting divalent anions, including Krebs cycle intermediates and sulfate, across the plasma membrane. These transporters may be key contributors to determining urinary citrate levels, which, in turn, may affect kidney stone formation; they also have been implicated in metabolic regulation in both drosophila and mammals. It is therefore important to understand the relationships in these proteins between their structure and functional mechanisms. Though no structures are yet available for mammalian DASS family members, a crystal structure of a bacterial homolog, vcINDY, has been determined. We recently demonstrated that vcINDY, a Na+-coupled succinate transporter, utilizes a dramatic “elevator” mechanism to transport substrate, involving a large-scale vertical movement of a protein domain perpendicular to the plane of the lipid bilayer membrane. Here, we sought to determine whether a mammalian family member utilizes a similar transport mechanism, specifically human NaDC3. With ∼30% identity between the vcINDY and hNaDC3, we hypothesized that a similar mechanism could be responsible for the functioning of hNADC3. Homology modeling with vcINDY suggested that several pairs of residues are brought into proximity upon substrate translocation. We prepared a series of double mutants introducing cysteines at positions predicted to be brought together in the outward-facing state of the protein and expressed them in Xenopus laevis oocytes. Using two-electrode voltage clamp and disulfide cross-linking, we investigated whether inhibition of transport activity observed upon formation of disulfides is consistent with the proposed mechanism of transport.

Dissection of Proton Inhibition Mechanism in the H+/Cl− Exchanger, CLC-7

ClC-7 is a voltage-activated 1H+:2Cl− exchanger that serves a critical yet somewhat uncertain role in lysosomal membranes of diverse mammalian cell types. Despite trafficking to acidic vesicles in the cell, proton inhibition is a functional characteristic of mammalian ClC H+/Cl− exchangers in general and of ClC-7 in particular. We sought to dissect the mechanism of proton inhibition in ClC-7 function. Leisle L, Ludwig CF, et. al recently utilized a method to robustly redirect ClC-7 to the plasma membrane by mutating lysosomal sorting motifs at the amino terminus of ClC-7 and by co-expressing an essential auxiliary subunit called Ostm-1 (Leisle, Ludwig, et al, 2011 EMBO J, 30,2140). using this strategy, we describe extracellular [H+]-dependent inhibition of ClC-7 mediated ionic current in comparison with published data for ClC-5 from experiments by Picollo, Malvezzi, and Accardi (2010, JGP, 135, 653). In the tested conditions, ClC-7 is more sensitive than ClC-5 to extracellular H+ (pK(0 mV) = 7.9 estimated for ClC-7/Ostm-1 and pK(0 mV) = 6.6 reported for ClC-5 expressed in oocytes). Further, we evaluate effects of increasing extracellular [H+] and varying intracellular [Cl−] on voltage-dependent transient current intrinsic to a transport deficient mutant of ClC-7 (E314A). Increasing [H+]ext shifts the half-activating voltage of voltage-dependent charge movement in ClC-7 (mean V1/2 ≥ +20 mV shift per pH unit increase), but does not drastically change the apparent charge valence (mean z = 0.85 to 0.95 qe at all [H+]ext tested). This result suggests that the voltage-dependent transient current does not arise from protonation of ClC-7, but that extracellular pH changes the work required for the electric field to elicit charge movement.