Introduction

Ion channels are used by cells to regulate many cellular functions, from action potential conduction to water balance, which is sometimes achieved by using a single ion in the setting of different channels types. Although ion channels are described as transmembrane proteins that have a “pore” which allows for the diffusion of specific ions across a concentration gradient, other channels involved in ion transport include antiporters (exchange), symporters (cotransport in the same direction) and pumps (use energy from hydrolysis of ATP). Chloride channels are a remarkable example of this since they are involved in the control of transepithelial transport, membrane excitability, and the regulation of cell volume and intracellular and intraorganelle pH. All of this is achievable by the use of the many different types of chloride channels, of which there are three major families: the voltage-gated chloride channels, the cystic fibrosis transmembrane conductance regulator (CFTR) and related channels, and the ligand-gated channels activated by gamma-aminobutyric acid (GABA) and glycine.[1][2]

Fundamentals

The importance of chloride channels for cellular and organ function is exemplified by the fact that mutations in genes that encode them produce a range of pathologies in humans. Mutations in many of these channels have been identified and liked to disorders such as cystic fibrosis (CFTR), some forms of myotonia, osteopetrosis and inherited kidney stone diseases (voltage-gated chloride channels), epilepsy and startled disease (GABA and glycine receptor). The ability to understand and test for these mutations has led to the development of multiple screening tests and treatments for this channelopathies.[1]

Issues of Concern

Science has not identified several chloride channels at a molecular level, and those already identified are well less understood than cation channels. Thus, the lack of information on their location, structure, and function leads to their underutilization as therapeutic targets.

Cellular Level

Chloride channels are likely present in every cell type, with a variety of physiologic functions which are tightly regulated by various stimuli, such as transmembrane voltage, ligands, pH, and a range of intracellular messenger (like calcium and cyclic AMP). At the cellular level, chloride channels are in the plasma membrane or intracellular organelles. Plasma membrane chloride channels function to regulate cell volume and ionic homeostasis, electrical excitability, and transepithelial transport. Intracellular chloride channels are found throughout different organelles and are important in tightly regulating their pH, ionic hemostasis, and volume. Keeping homeostasis of ionic concentrations prevents disruptions in energy production within mitochondria, protein degradation within lysosomes, DNA replication in the nucleus, or cellular signaling at the endoplasmic reticulum.[3][4]

Molecular Level

Mechanism of activation has been the primary mode of categorization of chloride channels. However, this is problematic because of overlap and lack of information on structural similarities of the corresponding proteins. Molecular cloning has generated a considerable amount of data that will eventually help in classifying them structurally. However, this is far from being complete. Thus far, three major structural classes have been identified. Firstly, GABA and glycine receptors (ligand-gated Cl- channels) assemble as pentamers, and each monomer containing four transmembrane spans. CFTR is the only chloride channel member of the second class and has about 12 transmembrane spans, arranged in two domains, two nucleotide-binding folds (NBFs), and a regulatory R domain. Although CFTR belongs to the gene family of ATP-binding cassette (ABC) transporters, which generally function as active transporters driven by ATP hydrolysis. This Cl- channel works as a cyclic AMP-activated CI- channel. Last class includes the gene family of CLC Cl- channels, which have about 12 transmembrane domains, each monomer has a pore (double-barreled channels) and function as voltage-gated channels. There is proof that these three structural classes are Cl- channels beyond a reasonable doubt. However, there are suggestions that four other unrelated proteins may also be CI- channels (p64, Ca-CC, phospholemman, pICln).[3][4]

Function

Members of the CLC channel family, are named as CIC-1, 2, 3, etc., and are voltage-gated channels that modulate a verity of cellular functions. CIC-1 is the major Cl- channel in skeletal muscle, maintaining the resting potential of the sarcoplasmic reticulum and regulating repolarization after contraction. CIC-2 is expressed ubiquitously and activated by cellular swelling, acidic pH and hyperpolarization, modulating intracellular chloride concentration, cell volume regulation, and modifying neuronal excitability. CIC-5 is predominantly expressed in the kidneys and may play a part of proximal tubule endocytosis. CIC-3 and CIC-4 function are not clearly understood and under investigation. ClC-K/Barttin channels are two members of the CLC family that function in basolateral Cl- recycling and are also predominantly expressed in the kidney (thick ascending loop of Henle and distal nephron) and inner ear. CFTR is a voltage-independent chloride channel located in the apical membrane of many epithelia, most predominantly in those of the airways, intestines, secretory glands, epididymis, and bile ducts. In some epithelial, such as that of the intestine, CFTR may account for the entire apical chloride conductance and thus plays a crucial role in salt and fluid secretion and absorption. CFTR may also be essential in the acidification of the Golgi bodies and endosomes and has been studied as a regulator of other ion channels such as ENaC. GABA- and glycine-gated chloride channels function to regulate membrane electrical excitability, specifically, by causing neuronal inhibition. GABA is predominantly used in the brain, while glycine is more common in the spinal cord and brain stem. In adult brains, binding of these neurotransmitters to their corresponding ligand-gated anion channel causes Cl- influx into the cell, which leads to hyperpolarization and neuronal activity inhibition. However, early in development, a more positive equilibrium potential of Cl- in undeveloped neurons causes excitability in response to GABA and glycine. Glycine receptors control motor rhythm generation, coordinate spinal reflex responses, and modulate processing by sensory neurons.[3][5]

Mechanism

The variety and versatility of chloride channels play a crucial function in maintaining cellular homeostasis, and implications exist of their dysfunction in multiple human disorders. Chloride channels alter the ionic composition of the cytoplasm and volume of cells by working closely with other ion transporters, such as pumps and cotransporters. One example is the strict fine-tuning of cellular pH, and the need to maintain electrochemical equilibrium when most cells are more alkaline than expected. This equilibrium mostly occurs via Na+/H+ exchangers and Na+/HCO3-/H+/Cl- exchangers that require a parallel Cl- shunt for recycling chloride. Additionally, similarly to the mechanism used for the acidification of some cellular organelles, some cells that have proton ATPases use Cl- channel to achieve electroneutrality.

Conversely, some cells are acid-loaded by Cl-/HCO3- exchangers and also need a separate pathway for chloride recycling. As well, chloride channels play a pivotal role in correcting cell volume when the opening of swelling-activated K+ and Cl- channels result in a net efflux of salts. Other cells, such as airway epithelia, acinar cells of multiple glands and intestinal cells take advantage of the polarized expression of Cl- channels and secondary active Cl- uptake mechanisms to transport water and salts in a particular direction across their apical membrane. This transport occurs by increasing the intracellular Cl- concertation above equilibrium by Na+/K+/2Cl- cotransporters, allowing for the passive diffusion of Cl- through apical Cl- channels. In the thick ascending loop of Henle, an apical cotransporter rises intracellular Cl- levels which then can leave via basolateral Cl- channels that are identical to the previously mentioned ClC-Kb/barttin. To contrast, intestinal crypt cells use the Na+/K+/2Cl- cotransporter, located in the basolateral membrane, and the CFTR Cl- channels, to secrete Cl- apically, a process that is stimulated by cAMP. In acinar cells, intracellular Ca+ concentration regulates Cl- secretion by direct activation of apical Cl- channels.

CFTR needs the presence of phosphate donating nucleosides to function efficiently. Thus it is tightly regulated by protein kinase C (PKC), protein kinase A (PKA), and a high ATP/ADP ratio. Opening of the channel requires phosphorylation by PKA, Mg+ and hydrolysis of ATP at the Nucleoside binding fold 1 (NFB1). Binding of ATP and NBF2 stabilized that opened channel and hydrolysis closes it. Also, cAMP can modulate the number of channels present in the plasma membrane by regulating exocytosis. Another significant function of chloride channels in the regulation of membrane electrical excitability. Voltage-gated Cl- channels open in response to depolarization with an inward flow at positive potentials, such as CIC-1 used in skeletal muscle, to maintain resting membrane potential. In smooth muscle, chloride channels activated by Ca+ or cellular swelling lead to depolarization that open voltage-gated Ca+ channels leading to an influx of calcium. GABA and glycine receptors are ligand-gated chloride channels that when activated cause influx or efflux of Cl-, depending on the electrochemical potential of Cl- for the cell, which leads to an inhibitory and sometimes excitatory response. These receptors hyperpolarize the postsynaptic membrane or stabilize its potential close to its resting levels (shunting inhibition), thus suppressing both the spatial and temporal summation of excitatory postsynaptic potentials.

When the endogenous ligand binds GABA, it promotes the channel’s pore opening, allowing Cl- and HCO3- to flow down their electrochemical gradients. Within organelles, Cl- is used for volume regulation, transport of anionic substrates, and to maintain electroneutrality. Mitochondria undergo volume changes, depending on the metabolic state of the cells, which is mediated by the transport of K+ and Cl- across its inner membrane. Lysosomes and the Golgi apparatus use chloride channels to help get rid of sulfate and phosphate. The import of chloride neutralizes the excess positive charge of organelles that must have a high calcium or proton gradient (endoplasmic and sarcoplasmic reticulum, endosomal and synaptic vesicles, etc.).[3][4]

Testing

Newborn screening algorithms for CF involves measurement of immunoreactive trypsinogen in the dried blood spot and genetic testing with a panel of CFTR mutations.  The sweat chloride test may be the most common test used to diagnose chloride channel dysfunction, CFTR specifically. A sweat chloride test measures the level of chloride in the sweat using an electric current, and it is positive when it is greater than 60mmol/L. For inconclusive results, CFTR can be diagnosed using other studies such as a nasal potential difference measurement or intestinal current measurement. Chloride channel functional assays are under development to test for other pathologies involving chloride channels such as myotonia, kidney stone, etc.[6][7]

Pathophysiology

Several human inherited diseases are associated with chloride channel mutations. Out of all of these, cystic fibrosis (CF) is probably the most popular and studied one. However, several others are being researched to find targeted therapies. CF is caused by abnormal salt and water transport across epithelia that affects multiple organ systems, most predominantly the lungs, pancreas, liver, and intestines. Altered composition of fluid films covering epithelia leads to obstruction of ducts, bacterial overgrowth, and decreased reabsorption. GABA and glycine receptors in the brain have an associated inhibitory chloride current and defects in these channels carry links with various symptoms of neuronal excitability. Mutations in genes that code for subunits of the GABA receptor correlate to different monogenic epilepsy syndromes, such as genetic epilepsy with febrile seizure plus (GEFS+) and its milder forms of febrile seizures, childhood absence epilepsy, autosomal dominant juvenile myoclonic epilepsy (ADJME), among others.

Glycine receptor mutations cause hereditary hyperekplexia (HPX), also known as ‘stiff baby syndrome,’ a neurological disorder typically causing an exaggerated startle reflex with mild auditory, tactile, or visual stimuli. Mutations in the CLC channel and transports are associated with a variety of genetic diseases such as myotonia, kidney stones, renal salt loss, deafness, osteopetrosis, and lysosomal storage disease. CIC-1 channel mutations cause different types of myotonia depending on the degree of chloride conductance impairment. CIC-2 is important to establish the low intraneural Cl- concentration needed for inhibitory response to GABA and glycine, and mutations of these channels have links to epilepsy. ClC-K/barttin chloride channels mutations cause some types of Bartter syndrome with renal salt wasting and congenital deafness due to lack of basolateral chloride currents needed for NaCl- reabsorption, and maintenance of electrochemical potentials. Dent’s disease, a rare X-linked kidney stone disorder with proteinuria, is due to a mutation in CIC-5 leading to impaired endocytosis of proteins and hormones. CIC-7 is an H+/Cl- exchanger that is important in pH regulation and expressed in lysosome and osteoclast, and mutations lead to osteoporosis and lysosomal storage disorders.[1][8]

Clinical Significance

Besides being linked to numerous genetic disorders, chloride channels are also the target of medications that treat a variety of conditions. For example, GABA and glycine receptors are targets of a wide range of clinically important drugs, such as antiepileptic agents, anxiolytics, sedatives, hypnotics, muscle relaxants, and anesthetics. Additionally, medications that directly target chloride channels known as CFTR modulators are being developed to treat CF. Chloride channels modulators also have the potential of treating other disorders such as secretory diarrheas, polycystic kidney disease, osteoporosis, and hypertension. Furthermore, mutations in specific chloride channels that cause known human diseases have been identified and are useful for screening, diagnosis, and targeted therapies. As an example, pulmonary gene therapy for cystic fibrosis patients has been developed where corrected copies of the CFTR gene are delivered using viral and non-viral vectors.[9][10]