Chloride channel

The CLC channel structure has not yet been resolved, however the structure of the CLC exchangers has been resolved by x-ray crystallography. Because the primary structure of the channels and exchangers are so similar, most assumptions about the structure of the channels are based on the structure established for the bacterial exchangers.[5]

A cartoon representation of a CLC chloride channel. The arrows indicate the orientation of each half of the individual subunit. Each CLC channel is formed from two monomers, each monomer containing the antiparallel transmembrane domain. Each monomer has its own pore through which chloride and other anions may be conducted.

Each channel or exchanger is composed of two similar subunits—a dimer—each subunit containing one pore. The proteins are formed from two copies of the same protein—a homodimer—though scientists have artificially combined subunits from different channels to form heterodimers. Each subunit binds ions independently of the other, meaning conduction or exchange occur independently in each subunit.[3]

A cartoon representation of a CLC channel monomer. Two of these subunits come together to form the CLC channel. Each monomer has three binding sites for anions, Sext, Scen, and Sint. The two CBS domains bind adenosine nucleotides to alter channel function

Each subunit consists of two related halves oriented in opposite directions, forming an ‘antiparallel’ structure. These halves come together to form the anion pore.[5] The pore has a filter through which chloride and other anions can pass, but lets little else through. These water-filled pores filter anions via three binding sites—S_int, S_cen, and S_ext—which bind chloride and other anions. The names of these binding sites correspond to their positions within the membrane. S_int is exposed to intracellular fluid, S_cen lies inside the membrane or in the center of the filter, and S_ext is exposed to extracellular fluid.^[4] Each binding site binds different chloride anions simultaneously. In the exchangers, these chloride ions do not interact strongly with one another, due to compensating interactions with the protein. In the channels, the protein does not shield chloride ions at one binding site from the neighboring negatively charged chlorides.[6] Each negative charge exerts a repulsive force on the negative charges next to it. Researchers have suggested that this mutual repulsion contributes to the high rate of conduction through the pore.[5]

CLC transporters shuttle H⁺ across the membrane. The H⁺ pathway in CLC transporters utilizes two glutamate residues—one on the extracellular side, Glu_ex, and one on the intracellular side, Glu_in. Glu_ex also serves to regulate chloride exchange between the protein and extracellular solution. This means that the chloride and the proton share a common pathway on the extracellular side, but diverge on the intracellular side.[6]

CLC channels also have dependence on H⁺, but for gating rather than Cl⁻ exchange. Instead of utilizing gradients to exchange two Cl⁻ for one H⁺, the CLC channels transport one H⁺ while simultaneously transporting millions of anions.[6] This corresponds with one cycle of the slow gate.

Eukaryotic CLC channels also contain cytoplasmic domains. These domains have a pair of CBS motifs, whose function is not fully characterized yet.[5] Though the precise function of these domains is not fully characterized, their importance is illustrated by the pathologies resulting from their mutation. Thomsen's disease, Dent's disease, infantile malignant osteopetrosis, and Bartter's syndrome are all genetic disorders due to such mutations.

At least one role of the cytoplasmic CBS domains regards regulation via adenosine nucleotides. Particular CLC transporters and proteins have modulated activity when bound with ATP, ADP, AMP, or adenosine at the CBS domains. The specific effect is unique to each protein, but the implication is that certain CLC transporters and proteins are sensitive to the metabolic state of the cell.[6]

The S_cen acts as the primary selectivity filter for most CLC proteins, allowing the following anions to pass through, from most selected to least: SCN⁻, Cl⁻, Br⁻, NO⁻
₃, I⁻. Altering a serine residue at the selectivity filter, labeled Ser_cen, to a different amino acid alters the selectivity.[6]

Gating occurs through two mechanisms: protopore or fast gating and common or slow gating. Common gating involves both protein subunits closing their pores at the same time (cooperation), while protopore gating involves independent opening and closing of each pore.[5] As the names imply, fast gating occur at a much faster rate than slow gating. Precise molecular mechanisms for gating are still being studied.

For the channels, when the slow gate is closed, no ions permeate through the pore. When the slow gate is open, the fast gates open spontaneously and independently of one another. Thus, the protein could have both gates open, or both gates closed, or just one of the two gates open. Single-channel patch-clamp studies demonstrated this biophysical property even before the dual-pore structure of CLC channels had been resolved. Each fast gate opens independently of the other and the ion conductance measured during these studies reflects a binomial distribution.[3]

H⁺ transport promotes opening of the common gate in CLC channels. For every opening and closing of the common gate, one H⁺ is transported across the membrane. The common gate is also affected by the bonding of adenosine nucleotides to the intracellular CBS domains. Inhibition or activation of the protein by these domains is specific to each protein.[6]

The CLC channels allow chloride to flow down its electrochemical gradient, when open. These channels are expressed on the cell membrane. CLC channels contribute to the excitability of these membranes as well as transport ions across the membrane.[3]

The CLC exchangers are localized to intracellular components like endosomes or lysosomes and help regulate the pH of their compartments.[3]

Bartter's syndrome, which is associated with renal salt wasting and hypokalemic alkalosis, is due to the defective transport of chloride ions and associated ions in the thick ascending loop of Henle. CLCNKB has been implicated.[7]

Another inherited disease that affects the kidney organs is Dent's Disease, characterised by low molecular weight proteinuria and hypercalciuria where mutations in CLCN5 are implicated.[7]

Thomsen disease is associated with dominant mutations and Becker disease with recessive mutations in CLCN1.[7]

• CLCN1, CLCN2, CLCN3, CLCN4, CLCN5, CLCN6, CLCN7, CLCNKA, CLCNKB
• BSND - encodes barttin, accessory subunit beta for CLCNKA and CLCNKB

• CLCA1, CLCA2, CLCA3, CLCA4

They possess one or two putative transmembrane α-helical segments (TMSs). The bovine p64 protein is 437 amino acyl residues in length and has the two putative TMSs at positions 223-239 and 367-385. The N- and C-termini are cytoplasmic, and the large central luminal loop may be glycosylated. The human nuclear protein (CLIC1 or NCC27) is much smaller (241 residues) and has only one putative TMS at positions 30-36. It is homologous to the second half of p64.

Structural studies showed that in the soluble form, CLIC proteins adopt a GST fold with an active site exhibiting a conserved glutaredoxin monothiol motif, similar to the omega class GSTs. Al Khamici et al. demonstrated that CLIC proteins have glutaredoxin-like glutathione-dependent oxidoreductase enzymatic activity.[12] CLICs 1, 2 and 4 demonstrate typical glutaredoxin-like activity using 2-hydroxyethyl disulfide as a substrate. This activity may regulate CLIC ion channel function.[12]

The generalized transport reaction believed to be catalyzed chloride channels is:

Cl⁻ (cytoplasm) → Cl⁻ (intraorganellar space)

Cystic fibrosis is caused by mutations in the CFTR gene on chromosome 7, the most common mutation being deltaF508 (a deletion of a codon coding for phenylalanine, which occupies the 508th amino acid position in the normal CFTR polypeptide). Any of these mutations can prevent the proper folding of the protein and induce its subsequent degradation, resulting in decreased numbers of chloride channels in the body. This causes the buildup of mucus in the body and chronic infections.[13]