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Figure 2. Effects of genetic mutations. This diagram illustrates the four specific genes studied and the effects of their mutation. Each gene mutation causes malfunctions of a particular area within the inner ear. Alterations of any one of these four genes can lead to a defect in specific proteins. These proteins then lead to the deformation of cell structure, resulting in nonsyndromic deafness.

Put if in Park: The Anchor Mechanism

In mutants, the microvilli must have another anchoring mechanism that is sufficient for early development, but cannot prevent membrane fusion after they have become stereocilia. It is critical for the microvilli to be present in organisms during early development, as they are essential for the function of many organs (Self, 1999). Eventual loss of stereocilia has less impact than complete lack of microvilli, thus enabling the mutants to survive, providing they initially possess microvilli.

Where Do We Go From Here?

Myosin VI is a motor protein that anchors and stabilizes the stereocilia. When functioning normally, Myosin VI prevents fusion of the stereocilia, allowing them to open the membrane channels and enacting an action potential. As the potential moves down the cell, Ca²⁺ must enter to release the chemical signal. The PMCA2 gene plays a critical role in the formation and function of the Ca²⁺ channels.

PMCA2

Go With the Flow: Ion Trafficking

The influx of Ca²⁺ ions is necessary for vesicles to bind to the presynaptic membrane and neurotransmitters to be released. The surge of calcium ions is controlled by the plasma membrane Ca²⁺ PMCA (ATPase), a transport protein in the plasma membrane of cells that brings calcium into the cell. There are four basic isoforms of PMCA pump: PMCA1, PMCA2, PMCA3 and PMCA4 (Grati et al, 2006). PMCA1 and PMCA4 are expressed in many different tissues and cell types, whereas PMCA2 (also known as ATP2b2) and PMCA3 operate in specialized tissues, such as brain and muscle tissues (Grati et al, 2006). PMCA2 demonstrates a highly restricted tissue distribution, suggesting that PMCA2 serves a more specialized and physiological function than the other isoforms.

Specifically, PMCA2 is thought to be present in high levels on the cell membrane of the outer hair cells (OHC). PMCA2 plays an essential role for hearing in relation to its function in both the vestibular and auditory systems. The main function of the PMCA2 protein is to bring Ca²⁺ ions into the cell. Thus, a mutation in this protein could cause a Ca²⁺ shortage in the cell, which would disable the vesicles from binding to the presynaptic membrane and would cause a significant loss of hearing (Figure 1C).

Mutation: Takes Its Toll

A point mutation in the PMCA2 gene, in which adenine is substituted with guanine, causes a glycine-to-serine substitution (Street et al, 1998). In mice that have this missense mutation, PMCA2 is no longer localized in the stereocilia and the basolateral membrane of hair cells, as normally seen in wild-type mice. This indicates that a glycine-to-serine substitution on the PMCA2 protein may cause deafness and imbalance (Street et al, 1998).

The particular role of PMCA2 in hearing was examined by observing PMCA2 deficient mice. At five weeks of age, these mice demonstrated imbalance of movement (Kozel et al, 1998). They also showed an underdevelopment of the organ of Corti canal, as well as the hair and the pillar cells (Kozel et al, 1998). This suggests that a PMCA2 deficiency is connected to structural changes in the organ of Corti tunnel (Figure 2).

Additionally, the PMCA2 deficient mice were examined by an Auditory Brainstem Response (ABR) test, which demonstrated that the PMCA2 deficient mice showed no response to varying intensities of sound (Kozel et al, 1998). However, the wild-type mice demonstrated a normal response to varying intensities of sound (Kozel et al, 1998). This indicates that a lack of PMCA2 causes severe imbalance as well as a significant loss of hearing.

Lastly, the effects of ablation on the pump were examined. There are two locations at which the ablations could occur, site A and site C. Site A is located in the cytosolic loop and site C is located in the C-Terminal, or the tail of the pump (Ficarella et al, 2007). When the cells were perfused with calcium chloride, mutations at Site A and Site C caused a dramatic decrease in the intake of Ca²⁺ ions (Ficarella et al, 2007). Thus, it can be deduced that PMCA2 plays a critical role in the intake of Ca²⁺ ions, which implies that a decrease in the ion concentration leads to a significant loss of hearing. 

Expressway to Deafness

Recent research and experimentation have shown that malfunction of the PMCA2 leads to a decrease in Ca entering the cell, significant imbalance, and a loss of hearing. However, if PMCA2 gene is functioning normally, then a Ca influx causes the vesicle to bind to the presynaptic membrane and to release the neurotransmitter. 

At this point, the electrical signal has departed the cell. The sensory cells need to prepare for the next signal by returning to resting potential. The cell does this by forming ion channels that remove the K+ buildup. KCNQ4 is essential for the formation of these types of ion channels.

KCNQ4

The Beginning of the Roadtrip: Background on Voltage-Gated Potassium Channels

In the mid 1990’s, only two genes were known to encode K+ channels, KCNQ1 and KCNE1 (Kubisch et al, 1999). KCNQ1 is a voltage-gated K+ channel with six transmembrane domains, found in the cochlea and other locations, including the heart and brain cells. KCNQ1 helps in the secretion of K+ into the endolymph from the stria vascularis. KCNE1 further enhances the currents from KCNQ1. Mutations in either protein halts the K+ recirculation (Beisel et al., 2005) and can lead to a disease known as long QT syndrome. This disease is characterized by arrhythmias due to repolarization anomalies of the cardiac action potential, and can lead to sudden death (Kubisch et al, 1999). Two other genes, KCNQ2 and KCNQ3, also create K+ channels, but are not specific to the ear. Neonatal convulsions result from mutations in either of these proteins (Kubisch et al., 1999). Recently, the KCNQ protein family has been found in many more electrically excitable tissues, including skeletal muscle and the retina (Beisel et al., 2005).

An Unknown Route: The Identification of KCNQ4

In 1999, a new gene was discovered that belonged in the KCNQ family of K+ channels. This gene, KCNQ4, was specifically found in the outer hair cells of the cochlea and was mapped on the DFNA2 locus (Kubisch et al, 1999). This area also encodes many other proteins, which, when mutated, cause diseases associated with dominant progressive hearing loss (Figure 2). Therefore, KCNQ4 was a likely candidate for autosomal dominant high-frequency hearing loss. 

A Well-Oiled Vehicle: The Function of KCNQ4

In non-mutated genes, KCNQ4 forms tetramers in which four proteins join to form the pore. In each of these proteins, a region called the P-loop forms the narrowest part. This narrow section allows the pore to be selective for K+ ions (Kubisch et al, 1999). After the influx of calcium, the voltage-gated KCNQ4 channels open to allow the passive diffusion of K+ out into the perilymph, repolarizing the cell (Wangemann, 2006). This K+ current out of the hair cells is then picked up by the surrounding cells and recycled back into the endolymph.

Road Construction Ahead: A Mutation in the Genetic Sequence

In a French family with high-frequency deafness, a mutation (G285S) was found in the sequence for the P-loop for the K+ pore (Kubisch et al, 1999). The normal amino acid sequence contains glycine, which is highly conserved in all the KCNQ proteins (Kubisch et al., 1999). However, the mutant gene sequence had a change of one specific nucleotide, replacing guanine with adenine. From this, the glycine amino acid is converted to a serine (Kubisch et al, 1999). This mutation disrupts the selectivity of the pore, and causes it to function improperly (figure 1D). When the wild-type (WT) and mutant proteins were expressed in Xenopus oocytes, the K+ current out of the cell drastically decreased in mutant channels compared to the WT channels (Kubisch et al, 1999). From these results, it was believed that KCNQ4 was important in the removal of K+ for keeping the electrical excitation conditions (the concentration of K+ in the endolymph) constant for the next round of stimulation.

Triple A: Developing the Mouse Model

At that time, the only models available to study channels were frog eggs, which are very different from sensory transduction cells. Therefore, an advanced model was needed to better understand the function of the proteins on the OHC, and a mouse model was created. Researchers were able to produce knockout mice (KO) which deleted the P-loop region, and knock-in mice (KI) that had the specific mutation in the genome (dominant negative allele). It was found that the knockout and dominant negative allele mice had the same reduction for the K+ current out of the cells at 3 weeks of age. Over a longer period of time, both types of mice had the OHC completely degraded (Kharkovets et al., 2006). By missing an essential part of the hearing pathway (the OHC), the signal was not able to be passed on, and thus deafness occurred. Currently, it is believed that KCNQ4 is essential in the OHC to remove the K+ ions in order to repolarize the cell, not to replenish the endolymph K+ concentration as originally thought.

A mutation in the KCNQ4 gene leads to an amino acid change in the K+ pore and results in a nonselective ion channel (Figure 1D). The cell is unable to repolarize, and degration of the cell occurs over time. If the KCNQ4 gene is normal, the K+ channel is able to return the cell to resting potential, and the next signal can be generated.

Another important part in generating the next signal is the recycling of K+. While returning to resting potential, the hair cells excrete the K+ ions into the perilymph. However, the ions must go back to the endolymph to be used in a future action potential. The use of intercellular connections made by connexins is critical for this movement.

Connexin 26

Conjunction Junction, What's Your Function: Location and Normal Function of Connexin 26

Connexin 26, a gap junction protein found in the cochlea and epidermis, is created with instructions found on the GJB2 gene. More specifically, connexin 26 is found in the stria vascularis, the spiral limbus, and the supporting cells found in the cochlea (Figure 1A).

Connexins are a family of transmembrane proteins that form channels allowing for rapid transport of ions and small molecules. Intercellular communication relies on gap junctions created by proteins like connexin 26. Channels formed with connexin 26 are largely involved in the transport of potassium and other small molecules (Casalotti et al., 1999). Potassium is required for the transformation of sound waves into electrical impulses that can be sent from cell to cell. The importance of potassium in hearing and influence of connexin 26 on potassium levels has lead researchers to explore the link between connexin 26 and deafness.

At least four other connexins are also found in the ear, some of which are also associated with deafness. Connexin 26 mutations, however, are the most prominent cause of nonsyndromic forms of deafness. Deafness related to connexin 26 occurs in an autosomal recessive manner (Ahmad et al., 2005). Different mutations in the GJB2 gene lead to various problems involving function, but the outcome is the same in all cases: profound deafness. While researchers know that GJB2 mutations lead to deafness, the way in which GJB2 mutations affect hearing remains largely unknown. Several factors have made studying GJB2 mutations difficult. The biggest hindrance to research is the fact that GJB2 deficient mice are embryonically lethal (Aoki et al., 2003). Thus, transgenic mice are the key to understanding deafness as caused by GJB2 mutations.

Potholes in the Road: The Effects of Mutations

Mutation of the GJB2 gene was found to generate significant changes in the inner ear of mutant mice. Hearing development in mice differs from that of humans, in that humans have fully developed hearing capabilities at birth, while mice are not fully matured in this area until about two weeks after birth (Aoki et al., 2003). Examination of the inner ear of the genetically mutated mice at two weeks revealed that the tunnel of Corti had collapsed and that sensory hair cells were deformed. By seven weeks the organ of Corti had begun to degenerate as had the spiral ganglia and hair cells. However, components responsible for the generation of endocochlear potential, such as the spiral ligament and stria vascularis, showed no alteration (Aoki et al., 2003).

The sustained structural integrity of these components suggests that the conditions within the endolymph have been maintained. This led researchers to believe that the epithelial gap junction system as the malfunctioning system. Disruption of the epithelial gap junction system hinders potassium intake which is necessary for transmission of signals by the hair cells (Aoki et al., 2003).

In addition to destroying or damaging many of the necessary inner ear components, various GJB2 mutations have been seen to impair intercellular coupling. Gap junctions are thought to be involved in the circulation of potassium between the fluids of the inner ear (Ahmad et al., 2005). Thus, understanding how and where mutations interfere with the formation of gap junctions is an important step in moving towards treatment. Connexin 26 is comprised of two transmembrane domains and extracellular loops. Alterations in these respective areas produced different results (Casalotti et al, 1999). Mutations in the transmembrane domains prevented oligomerization of proteins, thereby hindering the formation of connexons. Other mutations disrupt the alignment of the connexons or the structure of the gap. Problems with connexons or their formations interfere with intercellular coupling (Figure 2). Certain mutations affect the flow of biochemicals while leaving the flow of ions unharmed (Ahmad, 2005).

Around the Bend: Future Studies in Deafness

Deafness is a pathological condition that causes partial or complete loss of hearing. Many genes responsible for this disease were identified and four specific genes were investigated. Although current studies on deafness have helped to fill some of the gaps in knowledge, the precise mechanisms involved in hearing are still not completely understood. The inner ear is a complex organ involving many cells with varying functions. While individual cells and their components seem like small tasks to investigate, their effects on the system as a whole are much more difficult to understand.

This review examined four genes, but over thirty different genes have been linked to deafness. The complexity of the four genes reviewed helps illustrate how complicated the picture truly is. Mutations on any of these thirty genes lead to profound changes that hinder other processes as well, leading to the degradation of sound perception.

Despite the fact that recent research has discovered much about these four genes, many questions still remain. The rate step of the ATP hydrolysis mechanism and its effects on hearing have many gaps that need to be explored to truly understand the role of Myosin VI in the auditory system. Additional studies could examine the overall understanding of the mechanism underlying the histopathology of the organ of Corti and how PMCA2 is dependent on other ion channels. It is still not clear how the build up of K+ in the hair cells leads to the degradation.

Further understanding of how and why the cell responds to the high K+ concentration is key to understanding another step in deafness.  Also, the mouse model created in recent years can provide a useful tool for testing drugs (like K+ channel openers) to slow the progress of deafness (Kharkovets et al., 2006). One recent study illustrated how Connexin30-linked deafness could be combated by increasing the expression of Connexin 26 (Ahmad, 2007). Future studies could reverse this experiment and explore correcting Connexin 26-linked deafness through the use of other connexins, such as connexin30.

The Final Destination

Many genes play a critical role in auditory system function. The four genes described each play a different role in hearing, but share a common theme. Mutation of each gene causes a significant disruption of hearing (Figure 2). Myosin VI gene mutations affect the mobility of proteins, which in turn affects the stability of the stereocilia. Mutations in the PMCA2 pump cause a significant loss of hearing, imbalance, and a dramatic decrease in calcium’s entrance into the cell. KCNQ4 mutations reduce the ability to repolarize the hair cells resulting in the cells degradation. Connexin 26 is prevalent throughout the inner ear and plays a vital role in the formation of gap junctions, which allow for the transport of potassium ions. While each gene performs a very different function, they all play a critical role in the auditory system.

Acknowledgements