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Calcium-activated chloride channels (CaCC) are expressed on many cell types and a chloride (Clâˆ’) channel is activated by calcium (Ca2+) in the cytosol as shown in figure 1. Calcium is in low concentrations in the cytosol; normally of a range 0.2-5 ÂµM. Depolarization of the plasma membrane occurs when the concentration of calcium increases causing chloride to be released from the cell. There are a large majority of calcium-activated chloride channels in cells, since they control the apical flux of the chloride ions which are important in secretory organs (Yang et al.2008); some examples are in vascular smooth muscle, in the airways and neuronal calcium-activated chloride channels (Hartzell et al. 2005). The purpose of this essay is to discuss the some of the roles that calcium-activated chloride channels are involved in, and to explain the ways they operate in maintaining health and also to discuss their significance in disease.
Calcium-activated chloride channels were first identified in the oocytes of Xenopus and are further recognised in neurons, cardiac muscle cells, epithelial cells and in many other physiological processes. Accordingly, these channels are found in many other different types of various cells including neurons, sweat glands, olfactory transduction and amplification and also photo-receptors. These channels are anion selective and are activated by calcium concentrations in the cytosol (Eggermont, 2004). The size of the CaCC pore is thought to be quite big because ions such as C(CN)3 which is about 0.33 Ã-0.75 nm can manage to fit inside the pore (Hartzell et al. 2005).
Figure - this figure shows a calcium-activated chloride channel where calcium causes activation of the channel which results in the flow of chloride ions across the channel in the plasma membrane. (Image from Duran C, et al. 2010)
The role of calcium-activated chloride channels in health
Excitability of neurons
Several types of stimuli cause pain that arise in the dorsal root ganglia neurons as a result of calcium increase in the cytoplasm (Verkman & Galietta, 2009). Calcium-activated chloride channels are involved in the excitability of neurons and accordingly, these channels are expressed in are; dorsal root ganglion (DRG) neurons as well as neurons throughout the spinal cord and autonomic nervous system. In the somatosensory system, around 45-90% of neurons all convey calcium-activated chloride channels. These neurons are responsible for things like sensation of temperature and pain. Experiments have been undertaken in the spinal cords of mice and several other species that show when the calcium concentration increases in the cell, this causes the channels to open and calcium goes into or out of the cell. This would affect membrane conductance to ultimately cause substantial excitability (Hartzell et al. 2005).
Kidney chloride channels are regulated by calcium, acting as a second messenger, in response to an alteration in membrane potential. Calcium-activated chloride channels are found in the collecting ducts of the kidney. When the renal cells are open to ATP in the extracellular space, this causes the entry of calcium into the intracellular space (Veizis & Cotton, 2007). Alterations in calcium concentration intracellular space cause an influence of the activity of the chloride channel and the rate of renin secretion. When the chloride channels are blocked, there seems to be a reduction in the inhibition of renin release by calcium, this may suggest that calcium-activated chloride channels are possibly involved in blood pressure control. Experiments to prove that calcium concentrations cause an influence on the chloride channel activity and renin secretion showed that when 1mM of calcium was exposed to glomeruli, the release of renin was increased by the calcium reduction as shown in figure 2. Subsequently, when calcium was increased again, this resulted in decreased quantities of renin release; therefore, when calcium is increased and the chloride channels blocked by a substance that acts as a chloride channel blocker, such as 4,4'-diisothiocyanostilbene- 2,2'-disulfonic acid (DIDS), does not cause an inhibition of renin secretion (Jensen & Skott 1996).
Addition of 0.5mM DIDS
Without addition of DIDS
Figure - This figure demonstrates that after the chloride channels were blocked by DIDS- renin inhibition is reduced by the presence of calcium. At a time of 35 minutes, calcium is removed until 109 minutes where 1mM of calcium was added again in both experiments. The * signifies a considerable variance (Image from Jensen & Skott 1996).
Calcium-activated chloride channels are also involved in olfactory transduction which gives us our capability to smell. The binding of odours to olfactory receptors causes an increase of calcium into the cilia of the neurons involved in olfactory sensation, called olfactory sensory neurons (OSNs), their structure is shown in figure 3. Calcium entry causes the chloride channels to become activated and this leads to the chloride transport from the cilia and this results in the depolarization of the olfactory sensory neurons. Experiments involving reverse-transcription polymerase chain reaction (RT-PCR) in mice showed that their olfactory epithelium expressed bestrophin-2 in the olfactory cilia as shown in figure 4. Accordingly, OSNs are accountable for sensing compounds that have a smell, also known as, odorant molecules which occur in the atmosphere and ultimately, these sensory neurons transmit the signals to the brain. When the odorant molecules bind to the receptor responsible for olfactory transduction, this causes G protein and adenylate cyclase to be activated which thus results in the cyclic adenosine monophosphate (cAMP) concentration to increase in the cilia. The CaCC's are located in the ciliary membrane and due to activation by cAMP, this results in the concentration of calcium ions being increased in the cilia and this goes on to cause and activation of the chloride ions (Pifferi S, et al. 2006).
Olfactory neuron axon
Olfactory sensory neuron
Figure - an olfactory sensory neuron (OSN) is involved in sensing odorant molecules in the environment resulting in these sensory neurons transmitting the signal to the brain (Image from Siegel GJ. et al. 1999).
A protein called bestrophin 2, found in mice, is originated to form a CaCC from one species to another. From identification of this protein in mice, it has been possible to determine the function of these channels in greater detail (Pusch, 2004). Studies involving patch-clamp techniques identify bestrophin-2 from mice and show that it is involved as a CaCC and a part of the ion-conducting pore. The protein mBest2 is situated on the cilia and this is where olfactory transduction occurs. In the olfactory epithelium, experiments were undertaken to show the expression of Best-2 by RT-PCR. Results show the expression of mBest2 mRNA, but not any of the mBest1 and mBest4 mRNA which are other coding bestrophin genes in the mouse. (Pifferi S, et al. 2006).
Figure - this shows results from an experiment involving a RT-PCR in which primers were used precisely for mBest 1, 2 and 4 and the cDNA from the olfactory epithelium RNA was amplified. The expression of mRNA was only detected on mBest2. (Image from Pifferi S, et al. 2006).
Block of Polyspermy
Calcium-activated chloride channels play an important part in the blockage of polyspermy, which are eggs that have been fertilised by greater than one sperm, this can obviously become problematic for the zygote survival as the egg fertilised may comprise an abnormal chromosome count. The blockage of polyspermy causes the membrane potential to alter from around -40mV which is the known resting value to become depolarized to around +20mV for a short period of time. The increase in membrane conductance results in the depolarization and the increase of calcium ions. Consequently, during the process of fertilization, there is an increase in calcium ions by inositol 1,4,5-triphosphate (IP3) which is involved in calcium release. IP3 or Ca2+ cause depolarization of the membrane and activate the egg, in addition, these are quite comparable to the fertilization process (Hartzell et al. 2005).
The role of calcium-activated chloride channels in disease
Best vitelliform macular dystrophy
Best vitelliform macular dystrophy, also known as Best disease, is named after a German ophthalmologist; Friedrich Best who discovered this disease is 1905. Thought to be caused by a mutation of the Vitelliform Macular Degeneration Type 2 gene (VMD2) of which Bestrophin 1 is the encoded protein of this diseased gene; consequently, this disease follows an autosomal dominant inheritance and characteristics associated with it occur when lipofuscin, a fatty yellow pigment, builds up in and near to the pigment epithelium which is located in the retina (Pusch, 2004) as shown in figure 5 by the means of ophthalmoscopy a comparison of a normal retina (A) and a retina that has Best vitelliform macular dystrophy (B). The disease appears as an accumulation of this yellow substance in the macula area and accordingly, patients with this disease complain of their vision being quite blurred. It develops slowly and gradually resulting in the wasting of retinal pigment epithelium (RPE), where the bestrophins are distributed, which can subsequently result in damage to the photoreceptors in the retina and eventually lead to the central vision becoming weakened and ultimate, macular degeneration (Planells-Cases & Jentsch, 2009).
Arteries and veins of the retina
B. Best vitelliform macular dystrophy
Figure - this figure displays an ophthalmoscopic picture of two retinas build up of lipofuscin situated in the retinal pigment epithelium. (Image from Hartzell C, et al. 2005).
Initially, it was thought that the only role that bestrophin proteins had was to up-regulate the chloride current that was being carried by some other protein channel (Pusch 2004) but however; it is now understandable that bestrophin proteins may be involved in the pore-containing channel structure because point mutations in the bestrophins result in a different phenotype which means that bestrophins are involved in channel components (Eggermont, 2004). The mutations in homologous residues were tested and results indicate that two mutations called W93C and G299E caused the channels to lose their function (Zhiqiang Q, et al. 2003). Bestrophin proteins have been extracted from model organisms including humans, mice, xenopus and many others in which they have demonstrated that they function as chloride channels when expressed from different species and subsequently, most mammals are said to have four bestrophin genes (Duran C et al. 2010). There is a large amount of scepticism about the structure of bestrophin, it is occasionally reported that it has six transmembrane domains (Zhiqiang Q, et al. 2003) but some places state that there are four (Sun H, et al. 2002).; therefore, the molecular identity is still uncertain.
Electro-oculography shows the functioning of the retina (Brown M, et al. 2006) and accordingly, conductance of chloride channels are displayed as a light peak on the electro-oculogram (EOG) as shown in figure 6, and this is a useful diagnostic feature. Any faults of light peaks as shown in figure 7 are a specific trait of Best disease and subsequently, likely mutations in the VMD2 gene. The role of Best-1 is as a calcium-activated chloride channel and in turn, causes a light peak thought to have a relationship with the chloride conductance regulator within the basolateral membrane, which is where Best1 is located, of the retinal pigment epithelium (Hartzell C, et al. 2005). The idea of Best-1 functioning as a CaCC is also from studies involving the Patch clamp method (Marmorsteina & Kinnicka, 2007).
Figure - this figure shows results from an electro-oculogram from a normal patient. The EOG was taken place from a reaction to an illumination pattern. In this diagram, the light peak lasts for around 10 minutes. The LP:DT ratio is >1.8 (Image from Hartzell C, et al.2005).
Figure - this figure shows an electro-oculogram output from a patient suffering with Best vitelliform macular dystrophy. The light peak is diminished than the normal representation in figure 5. Faults in light peaks are a characteristic trait of this disease (Image from Hartzell C, et al.2005).
The tansmembrane protein (TMEM16) family, also known as the anoctamin family, have a structure of eight transmembrane domains and the family have ten known members. Out of the ten known members, two are identified as CaCC's; however, the purpose of the other members is still unidentified (Duran C, et al. 2010). TMEM16A was studied by cloning a Xenopus oocyte and TMEM16A and TMEM16B are responsible for calcium-activated chloride channels in the oocytes of large aquatic salamanders (Schroeder B.C, et al. 2008). A member of the TMEM16 family called TMEM16E is mutated in Gnathodiaphyseal dysplasia. Consequently, TMEM16E is also found in cardiac as well as skeletal muscle tissues and TMEM16A is usually expressed in epithelia but when it is mutated, it can cause damage in the tracheal cartilage, the 8TM domain structure is shown in figure 8 (Planells-Cases & Jentsch, 2009). Gnathodiaphyseal dysplasia (GDD) is an autosomal dominant syndrome resulting in fragile bones, tubular bone sclerosis and cemento-osseous dysplasia which causes abnormal jawbone tissue (Riminucci M, et al. 2001). Gnathodiaphyseal dysplasia also displays facial deformities which may include jaw lesions and the tubular bones are thickened in upper and lower limbs.
Patients with GDD were screened and genetic material was collected; subsequently, during DNA screening, a missense mutation was detected on people affected with this disease. Samples were taken from people of Asian and African-American descent. The mutation is situated on the 11th exon and the cysteine residue is implicated at the 356th amino acid and additionally, those unaffected with GDD were without the mutation. The GDD1 protein responsible for Gnathodiaphyseal dysplasia has 8 transmembrane domains with the N and C terminus positioned in the cytosol. TMEM16E (GDD1) is associated with Gnathodiaphyseal dysplasia. (Tsutsumi S, et al. 2004).
Figure - this figure shows the predicted structure of TMEM16A with the 8 transmembrane domains and the amino and carboxyl termini are in the cytosol (Image from Verkman & Galietta, 2009).
According to the National Eye Institute research, mutations in the bestrophin gene that are involved with the expression of the chloride channel are responsible for the retinal pigment epithelium. There are still many disagreements about this notion and it seems one of the best ways to solve this controversy would be to test pharmacologic blockers on the bestrophins that express mutations that result in disease (National Eye Institute, 2010). It is still unknown how a mutated chloride channel in the RPE causes Best vitelliform macular dystrophy and more research needs to be undertaken in this field. (Pusch, 2004). There is strong authentication that bestrophins function as calcium-activated chloride channels but however, there is some data that doesn't seem consistent. Studies that determine how the function of calcium-activated chloride channels result in retinopathies needs to be taken into consideration as do the functions of anoctamins and their role as calcium-activated chloride channels (Duran C, et al. 2010).
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