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Chloride channel openers refer to a specific category of drugs which are involved in a wide variety of physiological functions and processes such as the regulation of neuroexcitation, transepithelial salt transport, and smooth muscle contraction ^[1][9]. Due to their distribution throughout the body, diversity, functionality, and associated pathology, chloride channels ^[2][10] represent an ideal target for the development of channel modulating drugs such as chloride channel openers.

Chloride channel modulators include chloride channel openers, and chloride channel blockers, both of which modulate the transport of chloride ions through chloride channels ^[3][4]. Chloride channel openers specifically work by either preventing the closure of chloride channels, or promoting their opening, thus helping to facilitate movement of chloride ions into a cell.

In general, mutations in various chloride channels throughout the human body can result in many pathologies, such as macular degeneration, myotonia, cystic fibrosis, and hyperekplexia^[3] [4]. Chloride channel openers have been proposed to treat a variety of such pathologies, most notably including cystic fibrosis, a genetic disorder in which a defect in the cystic fibrosis transmembrane conductance regulator protein impacts chloride ion transport across epithelial cells ^[4][11].

Chloride channels remain a somewhat under-explored target for drug development despite their biological significance, due to various issues associated with the development of successful chloride channel drugs. As a result, the development of chloride channel modulators such as chloride channel opening drugs has been limited. The relative structural complexity of chloride channels, their varied roles in biological processes, and the problems associated with the development of drugs with high specificity emphasize the necessity for further research in this area.

Chloride Channels

Ion Channels

Ion channels are pore-forming proteins which help facilitate the transport of ions across membranes, typically plasma membranes or the membranes of organelles within cells ^[5][14]. They are considered to be the second largest drug target for existing drugs, after G protein-coupled receptors ^[5][14]. There is a wide variety of ion channels, and they are typically characterized by their gating mechanism, such as ligand-gated channels or voltage-gated channels, and the ion being transported, such as sodium (Na), potassium (K), or chloride (Cl) ions ^[5][14].

Voltage-gated ion channels

Several different intracellular transport channels, with varying functions, exist throughout the human body to facilitate routine upkeep and maintenance ^[6][1]. Such transport channels, including ligand gated and voltage gated channels, regulate uptake of chemical stimulants that trigger neuronal function ^[7][2]. Chloride (Cl-), the most abundant anion in the human body^[6] ^[7][1][2], as well as Sodium (Na+) and Potassium (K+), determine the electrochemical potential across a cell ^[6] [1]. The difference in concentration of chloride determines if a cell will depolarize or hyperpolarize the plasma membrane, resulting in a neural response ^[6] [1]. Chloride channels are a type of voltage-gated ion channel, and are responsible for regulating chloride ion transport and therefore neural excitation or inhibition ^[7][2].

Physiological significance of chloride ion channels

Chloride channels are present throughout the body and have important roles in many physiological processes. Some functions of chloride channels include ion homeostasis, cell volume regulation, and modulation of electrical excitability ^[8][17]. In the central nervous system (CNS), chloride channels are responsible for both direct modulation of neuronal activity and indirect control of neuronal functions through release of gliotransmitters by astrocytes through gating of organic anions such as GABA ^[7][2]. Chloride channels also partake in cell apoptosis caused by endoplasmic reticulum (ER) stress, in addition to their roles in modulation of membrane potential and gating anions ^[7][2]. ER stress notably plays a role in neurological disorders such as Alzheimer’s (AD).

Chloride ion transport

Chloride ions stimulate chloride channels to open, and so the active and efficient transport of chloride ions is crucial to neural function. Chloride is transported across necessary cell membranes via chloride transporting proteins ^[7][2]. The function of these proteins is crucial for appropriate neural pathology and dysfunction of these proteins is characteristic of diseases and disorders such as epilepsy and autism ^[7][2].

Gene families of chloride channels

Although opinions vary among experts regarding classification of the different chloride channels, they can generally be classified into 4 families of chloride channels that have been identified: ClC channel proteins, CFTR (cystic fibrosis transmembrane conductance regulator) channels, calcium-activated chloride channels, and voltage-dependent anion selective channels ^[9][26]. Groups are assigned by molecular properties and variance in activation stimuli. CLC channel proteins, which are expressed on cell membranes, organelles, and vesicles, are of particular interest for the development of chloride channel openers due to their regulation of chloride ion transport and gradients for many cellular functions ^[7][2].

ClC Channel Proteins

Chloride channel voltage regulation which allows for Cl- transmembrane transport is mediated by ClC Channel Proteins. This class of proteins, discovered in the 1990s, is found in the plasma and intracellular membranes of the cells of living organisms ^[10][15]. Expressed as either anion channels or anion/protein exchangers, these proteins can join together to form homomeric or heteromeric dimers ^[10] ^[6][15,16]. These proteins have both individual pathways as well as advanced pathway options when incorporated into a dimer. Specific channel and exchanger biological function roles vary based on organism type.

Voltage gated chloride channel proteins (ClC family) are a recently investigated family of channel proteins that are present in plasma membranes, intracellular organelles,and vesicles ^[7][2]. The founding member of this protein family, ClC-1, sparked extraordinary scientific interest when it was found to contain two channels/gates that functioned and operated independently of one another^[11] [3]. This so-called “double-barreled” structure poses both a potential for new understanding and very complex drug design utilizing the surprising structure of the ClC-1 protein ^[7][2].

GABA receptors

GABAergic receptors, making up around 20% of all receptors, are the most abundant inhibitory neurotransmitter type found in the human CNS ^[12][27]. GABaergic receptors are classed into GABA-A, GABA-B, and GABA-C subclasses. The GABA-A and GABA–C receptors are ionotropic while the GABA-B receptor is metabotropic ^[12][27]. Due to their differing ability to form endogenous heteromeric and homomeric receptors, GABA-A and GABA–C receptors have unique pharmacological and physiological properties.

GABA-A receptor inhibition

GABA-A receptors are GABA-gated anion channels which are involved in the function of rapid inhibitory synaptic transmission through the vertebrate CNS ^[13][28]. These receptors are coupled with intrinsic chloride channels that are triggered to open through the binding of GABA, which is an inhibitory neurotransmitter ^[11][3]. When activated, GABAergic inhibition of two types, phasic and tonic, will occur ^[12][27]. Phasic GABA-A receptor-mediated inhibition is a result of a brief exposure of postsynaptic GABA-A receptors to high concentrations of GABA. Alternatively, tonic GABA-A receptor-mediated inhibition results from an activation of extrasynaptic receptors by low concentrations of ambient GABA ^[12][27]. Somewhere between 75% and 90% of GABA-A inhibition in the CNS is tonic ^[12][27].

GABA-A receptor structure and disease

GABA-A receptors are part of the cys-loop pentameric ligand-gated ion channel family, which includes multiple neurotransmitter-gated channels ^[11][3]. GABAA receptors are assembled from five subunits. Such subunits and splice variants can be distinguished as α1-α6, β1-β3, γ1-γ3, δ, ε, π and θ ^[12][27]. GABA-A receptor subunit mutations are believed to be a potential cause for many neurological and CNS disorders. For example, epilepsy related sleep disturbances are believed to be partially caused by improper activation of the β3 GABA-A receptor subunit ^[12][27]. Various other pathological mood disorders including anxiety and schizophrenia are major therapeutic targets for GABA-A ^[12][27]. GABA-A therapeutics are a related therapy that could potentially be treated or targeted by chloride ion channel opener drugs.

Perception of chloride ion gated channels as drug targets

Chloride channels were largely overlooked as drug targets for many years with a greater emphasis placed on ligand gated channels due to the high selectivity (easy targeting) of ligands in comparison to chloride ions ^[7] ^[11][2][3]. Outside of the discovery of the GABA-A receptors, chloride channels have remained understudied in the world of drug therapeutics ^[6] ^[7] ^[11][1-3]. The discovery of GABA-A receptors has allowed the scientific community to see that chloride channels could have a direct link to central nervous system (CNS) cell operation ^[7][2]. However, the lack of a complete understanding of the exact specifics of chloride channel functioning has made it difficult for the development of chloride channel modulating drugs with a high level of specificity.

Channel Modulators Overview

Channel Modulators

Channel modulating drugs, otherwise known as ion channel modulators, belong to a category of drugs which control the operation of ion channels. Channel modulators can act as either blockers or openers of these channels, and can either directly or indirectly modulate ion channels. Ion channel modulation is of great significance in drug development, as ion channel modulators can be used to treat a wide variety of medical conditions, including diabetes and hypertension ^[14][6].

Ion Channel Targeting

Ion channels are significant drug targets due to their importance in a vast range of physiological processes ^[15][7]. However, the development of ion channel modulating drugs has historically been challenging due to certain factors such target specificity, structural complexity of ion channel proteins, identification of drug binding sites, and the drug screening methods. Regarding specificity, as ion channels have a variety of different functions, a lack of selectivity can cause undesirable side effects from channel modulators ^[16][8].

Mechanism of Action

There are a variety of factors that contribute to the activation of chloride channels. Some of the factors contributing to CLC activation would be cellular swelling, chloride imbalance, intracellular Ca2+ signaling, membrane potential changes, and intracellular pH changes, among others ^[17][24].

One example of a common chloride channel activator which is used to treat both constipation caused by IBS (irritable bowel syndrome) as well as cystic fibrosis would be Lubiprostone ^[18][25]. This drug is poorly absorbed following oral administration, until its eventual metabolization within the stomach and the small intestine (specifically the jejunum). After it is metabolized, Lubiprostone utilizes membrane stimulation to selectively stimulate CLC-2 (type 2 chloride channels) channels leading to a pathway that releases fluids, relieving symptoms. Another example would be the drug ivermectin which binds to glutamate-gated chloride channel receptors, triggering them to open and allow chloride ions to flow into a cell ^[19] ^[20][29, 30]. Ivermectin binds in the transmembrane domain of the glutamate-gated chloride channel receptors, allowing for an open-pore conformation ^[19][29].

Medical Use/Treatment

The development of drug targets for anion gated channels such as chloride channels has lagged behind the development of cation gated channels due to technical challenges pertaining to the screening methods for chloride-channel modulators^[14] [6]. As the technology used for the development of cation and anion gated channel targets is largely similar, many of the strategies used to do so can be applied interchangeably. The major hurdle to the development of chloride gated channel drugs is therefore the screening method. There are an abundance of known toxins that are capable of modulating cations, allowing for molecular characterization of cation gated channels ^[14][6]. While it is widely accepted that anion channels are present in every mammalian cell, it has been more difficult to characterize them ^[22][19].

Applications

Treating genetic mutations which cause Cystic Fibrosis

Chloride gated channel opener mutations have been a major pharmacologic target, as dysfunction of this receptor results in common muscular diseases such as hyperekplexia and even depressive disorders ^[3][4]. One such musculoskeletal disorder that is also influenced by mutations in chloride gated channels is cystic fibrosis ^[23][5]. A mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the most common mutation causing cystic fibrosis and has, therefore, been a logical target for therapeutics ^[23][5]. The specific mutation is a deletion of the amino acid residue phenylalanine at position 508 (DeltaF508). A class of natural coumarin compounds, identified by scientists, is capable of correcting the defective chloride channel gating ^[23][5]. The compound, which is a Chinese medicinal herb, was mixed with a cAMP (cyclic adenosine monophosphate) agonist and exposed to a mutation in question ^[23][5]. The DeltaF508-CFTR was activated by the coumarin and cAMP agonist mixture. Upon washing the coumarin from the assay, the activation of the DeltaF508-CFTR was terminated ^[23][5]. This innovation indicates that natural lead compounds could be used as chloride channel opener drugs.

Treating CNS/Neurological Disorders

Many of the functions of the Central Nervous System (CNS) rely heavily upon the homeostasis of chloride ion levels. While chloride channels (CLC) and ion transporters are both useful in regulating this homeostasis, chloride channels provide a quicker response to changes in molecular levels, making them essential to CNS function ^[7][20]. In particular, CLC-1 and CLC-2 channels play essential roles in the function of the CNS; their dysfunction or mutation can lead to neurological disorders. CLC-1 is present in the hippocampus, frontal neocortex, and nuclei of the brainstem and thalamus. Due to its role in physiological processes such as neuronal network maturation and excitability, manipulation of the chloride channel has been linked to diseases such as epilepsy ^[24][21]. Multiple companies have created chloride channel targeting drugs with the intent of modulating/altering CLC-1 function, such as Acetazolamide (a drug that elevates chloride conductance, acting as a chloride channel opener) and NMD670, a CLC-1 inhibitor ^[7] ^[25] [20,22]. CLC-2 is more commonly expressed in the CNS and thus contributes to a wide array of functions that can lead to disease states if mutated. In particular, CLC-2 mediates chloride currents and aids with blood flow and neuroprotection of the hippocampus. CLC-2 channel dimers have protopores that can be opened individually or together via a common gating process activated by hyperpolarization [23]. Drugs such as Omeprazole and Lubistiprone seek to activate CLC-2 channels in specific areas utilizing their ability to be activated by methods such as extracellular pH shift. CLC-2 has also been linked to neurological diseases such as epilepsy.

Treating Epilepsy

As one of the most common neurological disorders, epilepsy is an advantageous target for treatment ^[12][27]. It is believed that epilepsy is associated with extremely high levels of Calcium, up to 2-5 times the normal physiological levels of calcium ^[12][27]. Propositions have been made to utilize the relationship between extracellular and intracellular ions to regulate high and persistently high levels of calcium when applicable. Though this approach could, in theory, be a solution, the lack of success in studies targeting calcium channels for this expressed purpose have been mostly abandoned ^[12][27]. Thus, the likelihood of further investment in chloride ion channel openers as opposed to calcium ion channel openers is low without a significant scientific discovery or innovation.

Future Perspectives

Future research on chloride channel opener drugs will likely involve the complete translation of chloride channel opener research from concept to incorporation into human medicine. While chloride channel openers, and modulators in general, are gaining traction as a promising treatment target for diseases such as cystic fibrosis, there are very few treatments that have progressed past animal models ^[26][12]. Indeed, Lubistiprone (trade name: Amitiza) is one of the only commercially available uses of chloride channel openers in humans ^[27][13].

One of the major issues preventing the development of not only chloride channel openers, but any channel modulators specifically targeting chloride channels would be the relatively poor understanding of certain aspects of chloride channels themselves. Although a vast range of information is available regarding chloride channel function and physiological roles, there are gaps in the present literature, such as the molecular identification of volume-sensitive chloride channels ^[3][4]. The relatively intricate and complex structures of chloride channel proteins and the somewhat limited knowledge of certain types of chloride channels creates obstacles for the development of chloride channel modulators. Most notably, this lack of understanding results in difficulties in developing chloride channel modulators with a high level of specificity. Further research regarding the functioning and properties of chloride channels would be necessary in order to overcome this barrier to the development of drugs targeting them.

Regarding the future direction of chloride channel opener drug development, research will likely proceed to the exploration of applications outside of the realm of cystic fibrosis. It has been observed that mutations in chloride channel proteins can result in a multitude of diseases and disorders beyond cystic fibrosis due to their importance in several biological processes. For example, mutated chloride channels can cause osteoporosis, kidney stones, muscle disorder myotonia, and more ^[28][18]. Due to the diverse set of conditions that are linked to chloride channel proteins, there is great potential for future research into chloride channel openers for conditions outside of cystic fibrosis such as epilepsy and other neurological diseases affected by CLCs.

References

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^ Goetz, T., Arslan, A., Wisden, W., & Wulff, P. (2007). "GABA(A) receptors: structure and function in the basal ganglia". Progress in brain research. 160: 21–41. doi:10.1016/S0079-6123(06)60003-4.{{cite journal}}: CS1 maint: multiple names: authors list (link)
^ ^a ^b ^c Birch, P. (May 2004). "Strategies to identify ion channel modulators: Current and novel approaches to target neuropathic pain". Drug Discovery Today. 9 (9): 410–418. doi:10.1016/s1359-6446(04)03043-0.
^ Gregory J. Kaczorowski, Owen B. McManus, Birgit T. Priest, Maria L. Garcia (April 2008). "Ion Channels as Drug Targets: The Next GPCRs". Journal of General Physiology. 131 (5): 399–405. doi:10.1085/jgp.200709946.{{cite journal}}: CS1 maint: multiple names: authors list (link)
^ Liszewski, Kathy. "Opening the Gates on Ion Channel Drugs". Genetic Engineering & Biotechnology News. Genetic Engineering & Biotechnology News.
^ Rahmati, N., Hoebeek, F. E., Peter, S., & De Zeeuw, C. I. (May 2018). "Chloride homeostasis in neurons with special emphasis on the olivocerebellar system: Differential roles for transporters and channels". Frontiers in Cellular Neuroscience. 12. doi:10.3389/fncel.2018.00101.{{cite journal}}: CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link)
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^ Atif, M., Estrada-Mondragon, A., Nguyen, B., Lynch, J. W., & Keramidas, A. (October 2017). "ffects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus". PLoS pathogens. 13 (10). doi:10.1371/journal.ppat.1006663.{{cite journal}}: CS1 maint: multiple names: authors list (link) CS1 maint: unflagged free DOI (link)
^ Atif M, Estrada-Mondragon A, Nguyen B, Lynch JW, Keramidas A (October 2017). "Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus". PLOS Pathog. 13 (10): e1006663. doi:10.1371/journal.ppat.1006663. PMC 5638611. PMID 28968469.
^ Lambert, S., & Oberwinkler, J. (2005). "Characterization of a proton-activated, outwardly rectifying anion channel". The Journal of physiology. 567: 191–213. doi:10.1113/jphysiol.2005.089888.{{cite journal}}: CS1 maint: multiple names: authors list (link)
^ ^a ^b ^c ^d ^e Xu, L., Na, W., Liu, X., Hou, S., Lin, S., Yang, H., & Ma, T. (July 2008). "Identification of natural coumarin compounds that rescue defective ΔF508‐CFTR Chloride Channel gating". Clinical and Experimental Pharmacology and Physiology. 35 (8): 878–883. doi:10.1111/j.1440-1681.2008.04943.x.{{cite journal}}: CS1 maint: multiple names: authors list (link)
^ Chen, T. T., Klassen, T. L., Goldman, A. M., Marini, C., Guerrini, R., & Noebels, J. L. (March 2013). "Novel brain expression of ClC-1 chloride channels and enrichment of CLCN1 variants in epilepsy". Neurology. 80 (12): 1078–1085. doi:10.1212/WNL.0b013e31828868e7.{{cite journal}}: CS1 maint: multiple names: authors list (link)
^ Eguchi, H., Tsujino, A., Kaibara, M., Hayashi, H., Shirabe, S., Taniyama, K., & Eguchi, K. (June 2006). "Acetazolamide acts directly on the human skeletal muscle chloride channel". Muscle & nerve. 34 (3): 292–297. doi:10.1002/mus.20585.{{cite journal}}: CS1 maint: multiple names: authors list (link)
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@@ Line 33: / Line 33: @@
 === GABA-A receptor inhibition ===
-GABA-A receptors are GABA-gated anion channels which are involved in the function of rapid inhibitory synaptic transmission through the vertebrate CNS [28]. These receptors are coupled with intrinsic chloride channels that are triggered to open through the binding of GABA, which is an inhibitory neurotransmitter <ref name = "Three" />[3]. When activated, GABAergic inhibition of two types, phasic and tonic, will occur <ref name = "Twenty-seven" />[27]. Phasic GABA-A receptor-mediated inhibition is a result of a brief exposure of postsynaptic GABA-A receptors to high concentrations of GABA. Alternatively, tonic GABA-A receptor-mediated inhibition results from an activation of extrasynaptic receptors by low concentrations of ambient GABA <ref name = "Twenty-seven" />[27]. Somewhere between 75% and 90% of GABA-A inhibition in the CNS is tonic <ref name = "Twenty-seven" />[27].
+GABA-A receptors are GABA-gated anion channels which are involved in the function of rapid inhibitory synaptic transmission through the vertebrate CNS <ref>{{cite journal |last1=Goetz, T., Arslan, A., Wisden, W., & Wulff, P. |title=GABA(A) receptors: structure and function in the basal ganglia |journal=Progress in brain research |date=2007 |volume=160 |pages=21-41 |doi=10.1016/S0079-6123(06)60003-4 |url=https://www.sciencedirect.com/science/article/pii/S0079612306600034?via%3Dihub}}</ref>[28]. These receptors are coupled with intrinsic chloride channels that are triggered to open through the binding of GABA, which is an inhibitory neurotransmitter <ref name = "Three" />[3]. When activated, GABAergic inhibition of two types, phasic and tonic, will occur <ref name = "Twenty-seven" />[27]. Phasic GABA-A receptor-mediated inhibition is a result of a brief exposure of postsynaptic GABA-A receptors to high concentrations of GABA. Alternatively, tonic GABA-A receptor-mediated inhibition results from an activation of extrasynaptic receptors by low concentrations of ambient GABA <ref name = "Twenty-seven" />[27]. Somewhere between 75% and 90% of GABA-A inhibition in the CNS is tonic <ref name = "Twenty-seven" />[27].
 === GABA-A receptor structure and disease ===
@@ Line 44: / Line 44: @@
 === Channel Modulators ===
-Channel modulating drugs, otherwise known as ion channel modulators, belong to a category of drugs which control the operation of ion channels. Channel modulators can act as either blockers or openers of these channels, and can either directly or indirectly modulate ion channels. Ion channel modulation is of great significance in drug development, as ion channel modulators can be used to treat a wide variety of medical conditions, including diabetes and hypertension [6].
+Channel modulating drugs, otherwise known as ion channel modulators, belong to a category of drugs which control the operation of ion channels. Channel modulators can act as either blockers or openers of these channels, and can either directly or indirectly modulate ion channels. Ion channel modulation is of great significance in drug development, as ion channel modulators can be used to treat a wide variety of medical conditions, including diabetes and hypertension <ref name = "six" >{{cite journal |last1=Birch, P. |title=Strategies to identify ion channel modulators: Current and novel approaches to target neuropathic pain |journal=Drug Discovery Today |date=May 2004 |volume=9 |issue=9 |pages=410-418 |doi=10.1016/s1359-6446(04)03043-0 |url=https://www.sciencedirect.com/science/article/pii/S1359644604030430?via%3Dihub}}</ref>[6].
 === Ion Channel Targeting ===
-Ion channels are significant drug targets due to their importance in a vast range of physiological processes [7]. However, the development of ion channel modulating drugs has historically been challenging due to certain factors such target specificity, structural complexity of ion channel proteins, identification of drug binding sites, and the drug screening methods. Regarding specificity, as ion channels have a variety of different functions, a lack of selectivity can cause undesirable side effects from channel modulators [8].
+Ion channels are significant drug targets due to their importance in a vast range of physiological processes <ref name = " seven " >{{cite journal |last1=Gregory J. Kaczorowski,  Owen B. McManus,  Birgit T. Priest,  Maria L. Garcia |title=Ion Channels as Drug Targets: The Next GPCRs |journal=Journal of General Physiology |date=April 2008 |volume=131 |issue=5 |pages=399-405 |doi=10.1085/jgp.200709946 |url=https://rupress.org/jgp/article/131/5/399/130/Ion-Channels-as-Drug-Targets-The-Next-GPCRs}}</ref>[7]. However, the development of ion channel modulating drugs has historically been challenging due to certain factors such target specificity, structural complexity of ion channel proteins, identification of drug binding sites, and the drug screening methods. Regarding specificity, as ion channels have a variety of different functions, a lack of selectivity can cause undesirable side effects from channel modulators <ref name = "eight" >{{cite web |last1=Liszewski |first1=Kathy |title=Opening the Gates on Ion Channel Drugs |url=https://www.genengnews.com/insights/opening-the-gates-on-ion-channel-drugs/ |website=Genetic Engineering & Biotechnology News |publisher=Genetic Engineering & Biotechnology News}}</ref>[8].
 === Mechanism of Action ===
-There are a variety of factors that contribute to the activation of chloride channels. Some of the  factors contributing to CLC activation would be cellular swelling, chloride imbalance, intracellular Ca2+ signaling, membrane potential changes, and intracellular pH changes, among others [24].
+There are a variety of factors that contribute to the activation of chloride channels. Some of the  factors contributing to CLC activation would be cellular swelling, chloride imbalance, intracellular Ca2+ signaling, membrane potential changes, and intracellular pH changes, among others <ref name = "twenty-four" >{{cite journal |last1=Rahmati, N., Hoebeek, F. E., Peter, S., & De Zeeuw, C. I. |title=Chloride homeostasis in neurons with special emphasis on the olivocerebellar system: Differential roles for transporters and channels |journal=Frontiers in Cellular Neuroscience |date=May 2018 |volume=12 |doi=10.3389/fncel.2018.00101 |url=https://www.frontiersin.org/articles/10.3389/fncel.2018.00101/full}}</ref>[24].
-One example of a common chloride channel activator which is used to treat both constipation caused by IBS (irritable bowel syndrome) as well as cystic fibrosis would be [[Lubiprostone]] [25]. This drug is poorly absorbed following oral administration, until its eventual metabolization within the stomach and the small intestine (specifically the jejunum). After it is metabolized, Lubiprostone utilizes membrane stimulation to selectively stimulate CLC-2 (type 2 chloride channels) channels leading to a pathway that releases fluids, relieving symptoms.
+One example of a common chloride channel activator which is used to treat both constipation caused by IBS (irritable bowel syndrome) as well as cystic fibrosis would be [[Lubiprostone]] <ref name = "twenty-five">{{cite journal |last1=Lunsford, T. N., & Harris, L. A. |title=Lubiprostone: evaluation of the newest medication for the treatment of adult women with constipation-predominant irritable bowel syndrome |journal=International Journal of Women's Health |date=October 2010 |volume=2 |pages=361-374 |doi=10.2147/IJWH.S4537 |url=https://www.dovepress.com/lubiprostone-evaluation-of-the-newest-medication-for-the-treatment-of--peer-reviewed-fulltext-article-IJWH}}</ref>[25]. This drug is poorly absorbed following oral administration, until its eventual metabolization within the stomach and the small intestine (specifically the jejunum). After it is metabolized, Lubiprostone utilizes membrane stimulation to selectively stimulate CLC-2 (type 2 chloride channels) channels leading to a pathway that releases fluids, relieving symptoms.
-Another example would be the drug [[ivermectin]] which binds to glutamate-gated chloride channel receptors, triggering them to open and allow chloride ions to flow into a cell[29, 30]. Ivermectin binds in the transmembrane domain of the glutamate-gated chloride channel receptors, allowing for an open-pore conformation [29].
+Another example would be the drug [[ivermectin]] which binds to glutamate-gated chloride channel receptors, triggering them to open and allow chloride ions to flow into a cell <ref name = "twenty-nine" /> <ref name = "thirty" >{{cite journal |last1=Atif, M., Estrada-Mondragon, A., Nguyen, B., Lynch, J. W., & Keramidas, A. |title=ffects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus |journal=PLoS pathogens |date=October 2017 |volume=13 |issue=10 |doi=10.1371/journal.ppat.1006663 |url=https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006663}}</ref>[29, 30]. Ivermectin binds in the transmembrane domain of the glutamate-gated chloride channel receptors, allowing for an open-pore conformation <ref name = "twenty-nine" >{{cite journal |last1=Hibbs, R. E., & Gouaux, E. |title=Principles of activation and permeation in an anion-selective Cys-loop receptor |journal=Nature |date=May 2011 |volume=474 |pages=54-60 |doi=10.1038/nature10139 |url=https://www.nature.com/articles/nature10139}}</ref>[29].
 [[File:Glutamate-gated chloride channel receptor with bound ivermectin 3RHW.png|thumb|ivermectin (shown in dark blue) bound to the glutamate-gated chloride channel receptor (shown in turquoise).<ref>{{cite journal |vauthors=Atif M, Estrada-Mondragon A, Nguyen B, Lynch JW, Keramidas A |title=Effects of glutamate and ivermectin on single glutamate-gated chloride channels of the parasitic nematode H. contortus |journal=PLOS Pathog |volume=13 |issue=10 |pages=e1006663 |date=October 2017 |pmid=28968469 |pmc=5638611 |doi=10.1371/journal.ppat.1006663 |doi-access=free }}</ref> From {{PDB|3RHW}}.]]
@@ Line 59: / Line 59: @@
 === Medical Use/Treatment ===
-The development of drug targets for anion gated channels such as chloride channels has lagged behind the development of cation gated channels due to technical challenges pertaining to the screening methods for chloride-channel modulators [6]. As the technology used for the development of cation and anion gated channel targets is largely similar, many of the strategies used to do so can be applied interchangeably. The major hurdle to the development of chloride gated channel drugs is therefore the screening method. There are an abundance of known toxins that are capable of modulating cations, allowing for molecular characterization of cation gated channels [6]. While it is widely accepted that anion channels are present in every mammalian cell, it has been more difficult to characterize them [19].
+The development of drug targets for anion gated channels such as chloride channels has lagged behind the development of cation gated channels due to technical challenges pertaining to the screening methods for chloride-channel modulators<ref name = "six" /> [6]. As the technology used for the development of cation and anion gated channel targets is largely similar, many of the strategies used to do so can be applied interchangeably. The major hurdle to the development of chloride gated channel drugs is therefore the screening method. There are an abundance of known toxins that are capable of modulating cations, allowing for molecular characterization of cation gated channels <ref name = "six" />[6]. While it is widely accepted that anion channels are present in every mammalian cell, it has been more difficult to characterize them <ref name = "nineteen" >{{cite journal |last1=Lambert, S., & Oberwinkler, J. |title=Characterization of a proton-activated, outwardly rectifying anion channel |journal=The Journal of physiology |date=2005 |volume=567 |pages=191-213 |doi=10.1113/jphysiol.2005.089888 |url=https://physoc.onlinelibrary.wiley.com/doi/epdf/10.1113/jphysiol.2005.089888}}</ref>[19].
 == Applications ==
 === Treating genetic mutations which cause Cystic Fibrosis ===
-Chloride gated channel opener mutations have been a major pharmacologic target, as dysfunction of this receptor results in common muscular diseases such as hyperekplexia and even depressive disorders <ref name = "Verkman" />[4]. One such musculoskeletal disorder that is also influenced by mutations in chloride gated channels is cystic fibrosis [5]. A mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the most common mutation causing cystic fibrosis and has, therefore, been a logical target for therapeutics [5]. The specific mutation is a deletion of the amino acid residue phenylalanine at position 508 (DeltaF508). A class of natural coumarin compounds, identified by scientists, is capable of correcting the defective chloride channel gating [5]. The compound, which is a Chinese medicinal herb, was mixed with a cAMP (cyclic adenosine monophosphate) agonist and exposed to a mutation in question [5]. The DeltaF508-CFTR was activated by the coumarin and cAMP agonist mixture. Upon washing the coumarin from the assay, the activation of the DeltaF508-CFTR was terminated [5]. This innovation indicates that natural lead compounds could be used as chloride channel opener drugs.
+Chloride gated channel opener mutations have been a major pharmacologic target, as dysfunction of this receptor results in common muscular diseases such as hyperekplexia and even depressive disorders <ref name = "Verkman" />[4]. One such musculoskeletal disorder that is also influenced by mutations in chloride gated channels is cystic fibrosis <ref name = "five" >{{cite journal |last1=Xu, L., Na, W., Liu, X., Hou, S., Lin, S., Yang, H., & Ma, T. |title=Identification of natural coumarin compounds that rescue defective ΔF508‐CFTR Chloride Channel gating |journal=Clinical and Experimental Pharmacology and Physiology |date=July 2008 |volume=35 |issue=8 |pages=878–883 |doi=10.1111/j.1440-1681.2008.04943.x |url=https://onlinelibrary.wiley.com/doi/10.1111/j.1440-1681.2008.04943.x}}</ref>[5]. A mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is the most common mutation causing cystic fibrosis and has, therefore, been a logical target for therapeutics <ref name = "five" />[5]. The specific mutation is a deletion of the amino acid residue phenylalanine at position 508 (DeltaF508). A class of natural coumarin compounds, identified by scientists, is capable of correcting the defective chloride channel gating <ref name = "five" />[5]. The compound, which is a Chinese medicinal herb, was mixed with a cAMP (cyclic adenosine monophosphate) agonist and exposed to a mutation in question <ref name = "five" />[5]. The DeltaF508-CFTR was activated by the coumarin and cAMP agonist mixture. Upon washing the coumarin from the assay, the activation of the DeltaF508-CFTR was terminated <ref name = "five" />[5]. This innovation indicates that natural lead compounds could be used as chloride channel opener drugs.
 === Treating CNS/Neurological Disorders ===
-Many of the functions of the Central Nervous System (CNS) rely heavily upon the homeostasis of chloride ion levels. While chloride channels (CLC) and ion transporters are both useful in regulating this homeostasis, chloride channels provide a quicker response to changes in molecular levels, making them essential to CNS function [20]. In particular, CLC-1 and CLC-2 channels play essential roles in the function of the CNS; their dysfunction or mutation can lead to neurological disorders. CLC-1 is present in the hippocampus, frontal neocortex, and nuclei of the brainstem and thalamus. Due to its role in physiological processes such as neuronal network maturation and excitability, manipulation of the chloride channel has been linked to diseases such as epilepsy [21]. Multiple companies have created chloride channel targeting drugs with the intent of modulating/altering CLC-1 function, such as Acetazolamide (a drug that elevates chloride conductance, acting as a chloride channel opener) and NMD670, a CLC-1 inhibitor [20,22]. CLC-2 is more commonly expressed in the CNS and thus contributes to a wide array of functions that can lead to disease states if mutated. In particular, CLC-2 mediates chloride currents and aids with blood flow and neuroprotection of the hippocampus. CLC-2 channel dimers have protopores that can be opened individually or together via a common gating process activated by hyperpolarization [23]. Drugs such as Omeprazole and Lubistiprone seek to activate CLC-2 channels in specific areas utilizing their ability to be activated by methods such as extracellular pH shift. CLC-2 has also been linked to neurological diseases such as epilepsy.
+Many of the functions of the Central Nervous System (CNS) rely heavily upon the homeostasis of chloride ion levels. While chloride channels (CLC) and ion transporters are both useful in regulating this homeostasis, chloride channels provide a quicker response to changes in molecular levels, making them essential to CNS function <ref name = "Wang" />[20]. In particular, CLC-1 and CLC-2 channels play essential roles in the function of the CNS; their dysfunction or mutation can lead to neurological disorders. CLC-1 is present in the hippocampus, frontal neocortex, and nuclei of the brainstem and thalamus. Due to its role in physiological processes such as neuronal network maturation and excitability, manipulation of the chloride channel has been linked to diseases such as epilepsy <ref name = "twenty-one" >{{cite journal |last1=Chen, T. T., Klassen, T. L., Goldman, A. M., Marini, C., Guerrini, R., & Noebels, J. L. |title=Novel brain expression of ClC-1 chloride channels and enrichment of CLCN1 variants in epilepsy |journal=Neurology |date=March 2013 |volume=80 |issue=12 |pages=1078-1085 |doi=10.1212/WNL.0b013e31828868e7 |url=https://www.neurology.org/doi/10.1212/WNL.0b013e31828868e7}}</ref>[21]. Multiple companies have created chloride channel targeting drugs with the intent of modulating/altering CLC-1 function, such as Acetazolamide (a drug that elevates chloride conductance, acting as a chloride channel opener) and NMD670, a CLC-1 inhibitor <ref name = "Wang" /> <ref>{{cite journal |last1=Eguchi, H., Tsujino, A., Kaibara, M., Hayashi, H., Shirabe, S., Taniyama, K., & Eguchi, K. |title=Acetazolamide acts directly on the human skeletal muscle chloride channel |journal=Muscle & nerve |date=June 2006 |volume=34 |issue=3 |pages=292-297 |doi=10.1002/mus.20585 |url=https://onlinelibrary.wiley.com/doi/10.1002/mus.20585}}</ref> [20,22]. CLC-2 is more commonly expressed in the CNS and thus contributes to a wide array of functions that can lead to disease states if mutated. In particular, CLC-2 mediates chloride currents and aids with blood flow and neuroprotection of the hippocampus. CLC-2 channel dimers have protopores that can be opened individually or together via a common gating process activated by hyperpolarization [23]. Drugs such as Omeprazole and Lubistiprone seek to activate CLC-2 channels in specific areas utilizing their ability to be activated by methods such as extracellular pH shift. CLC-2 has also been linked to neurological diseases such as epilepsy.
 === Treating Epilepsy ===
@@ Line 73: / Line 73: @@
 == Future Perspectives ==
-Future research on chloride channel opener drugs will likely involve the complete translation of chloride channel opener research from concept to incorporation into human medicine. While chloride channel openers, and modulators in general, are gaining traction as a promising treatment target for diseases such as cystic fibrosis, there are very few treatments that have progressed past animal models [12]. Indeed, Lubistiprone (trade name: Amitiza) is one of the only commercially available uses of chloride channel openers in humans [13].
+Future research on chloride channel opener drugs will likely involve the complete translation of chloride channel opener research from concept to incorporation into human medicine. While chloride channel openers, and modulators in general, are gaining traction as a promising treatment target for diseases such as cystic fibrosis, there are very few treatments that have progressed past animal models <ref name = "twelve" >{{cite journal |last1=Verkman, A. S., & Galietta, L. J. |title=Chloride transport modulators as drug candidates |journal=American Journal of Physiology-Cell Physiology |date=November 2021 |volume=321 |issue=6 |doi=10.1152/ajpcell.00334.2021 |url=https://journals.physiology.org/doi/full/10.1152/ajpcell.00334.2021}}</ref>[12]. Indeed, Lubistiprone (trade name: Amitiza) is one of the only commercially available uses of chloride channel openers in humans <ref name = "thirteen" >{{cite web |last1=Shaffer |first1=Chad |title=Chloride channel activators: Uses, common brands, and safety info |url=https://www.singlecare.com/drug-classes/chloride-channel-activators |website=SingleCare |publisher=SingleCare}}</ref>[13].
 One of the major issues preventing the development of not only chloride channel openers, but any channel modulators specifically targeting chloride channels would be the relatively poor understanding of certain aspects of chloride channels themselves. Although a vast range of information is available regarding chloride channel function and physiological roles, there are gaps in the present literature, such as the molecular identification of volume-sensitive chloride channels <ref name = "Verkman" />[4].
 The relatively intricate and complex structures of chloride channel proteins and the somewhat  limited knowledge of certain types of chloride channels creates obstacles for the development of chloride channel modulators. Most notably, this lack of understanding results in difficulties in developing chloride channel modulators with a high level of specificity. Further research regarding the functioning and properties of chloride channels would be necessary in order to overcome this barrier to the development of drugs targeting them.
-Regarding the future direction of chloride channel opener drug development, research will likely proceed to the exploration of applications outside of the realm of cystic fibrosis. It has been observed that mutations in chloride channel proteins can result in a multitude of diseases and disorders beyond cystic fibrosis due to their importance in several biological processes. For example, mutated chloride channels can cause osteoporosis, kidney stones, muscle disorder myotonia, and more [18]. Due to the diverse set of conditions that are linked to chloride channel proteins, there is great potential for future research into chloride channel openers for conditions outside of cystic fibrosis such as epilepsy and other neurological diseases affected by CLCs.
+Regarding the future direction of chloride channel opener drug development, research will likely proceed to the exploration of applications outside of the realm of cystic fibrosis. It has been observed that mutations in chloride channel proteins can result in a multitude of diseases and disorders beyond cystic fibrosis due to their importance in several biological processes. For example, mutated chloride channels can cause osteoporosis, kidney stones, muscle disorder myotonia, and more <ref>{{cite journal |last1=Jentsch, T. J., Maritzen, T., & Zdebik, A. A. |title=Chloride channel diseases resulting from impaired transepithelial transport or vesicular function. |journal=The Journal of clinical investigation |date=August 2005 |volume=115 |issue=8 |doi=10.1172/JCI25470 |url=https://www.jci.org/articles/view/25470}}</ref>[18]. Due to the diverse set of conditions that are linked to chloride channel proteins, there is great potential for future research into chloride channel openers for conditions outside of cystic fibrosis such as epilepsy and other neurological diseases affected by CLCs.