Eur J Neurosci. 2015 Aug;42(3):1984-2002.
Molecular architecture of the stria vascularis membrane transport system, which is essential for physiological functions of the mammalian cochlea.
- 1Department of Molecular Physiology, Niigata University School of Medicine, 1-757 Asahimachi-dori, Niigata, 951-8510, Japan.
- 2Center for Transdisciplinary Research, Niigata University, Niigata, Japan.
- 3Department of Otorhinolaryngology – Head and Neck Surgery, Graduate School of Medicine, Osaka University, Osaka, Japan.
- 4Division of Bio-system Pharmacology, Department of Pharmacology, Graduate School of Medicine, Osaka University, Osaka, Japan.
- 5Department of Otorhinolaryngology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
- 6Department of Otorhinolaryngology – Head and Neck Surgery, Nara Medical University, Nara, Japan.
- 7Mammalian Genetics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
Stria vascularis of the mammalian cochlea transports K(+) to establish the electrochemical property in the endolymph crucial for hearing. This epithelial tissue also transports various small molecules. To clarify the profile of proteins participating in the transport system in the stria vascularis, membrane components purified from the stria of adult rats were analysed by liquid chromatography tandem mass spectrometry. Of the 3236 proteins detected in the analysis, 1807 were membrane proteins. Ingenuity Knowledge Base and literature data identified 513 proteins as being expressed on the ‘plasma membrane’, these included 25 ion channels and 79 transporters. Sixteen of the former and 62 of the latter had not yet been identified in the stria. Unexpectedly, many Cl(-) and Ca(2+) transport systems were found, suggesting that the dynamics of these ions play multiple roles. Several transporters for organic substances were also detected. Network analysis demonstrated that a few kinases, including protein kinase A, and Ca(2+) were key regulators for the strial transports. In the library of channels and transporters, 19 new candidates for uncloned deafness-related genes were identified. These resources provide a platform for understanding the molecular mechanisms underlying the epithelial transport essential for cochlear function and the pathophysiological processes involved in hearing disorders.
KEYWORDS: ion channel; mass spectrometry; proteomics; rat; transporter
Deafness affects about 10% of the world’s population, thereby leading to serious social consequences (Stevens et al., 2013). This disorder is frequently caused by cochlear dysfunction in the inner ear, which is the peripheral organ for hearing. However, the molecular pathology of deafness has not been fully elucidated. Therefore, it is crucial to investigate the molecular mechanisms underlying the function of cochlea, to understand its role in diseases.
The cochlea is filled with a unique extracellular solution, known as the endolymph, which contains 150 mM K+ and has a highly positive endocochlear potential (EP) of +80 ~ +100 mV. Stereocilia of the sensory hair cells are exposed to the endolymph. In response to mechanical stimuli, cation channels present on the stereocilia open up, permitting the flow of endolymphatic K+ into the hair cells. This process triggers an electrical excitation of the cells, which, in turn, leads to the release of transmitter into the auditory neurons (Hudspeth, 1989). Highly positive EP, being the major driving force for K+ flow, sensitizes hearing (Chan & Hudspeth, 2005). Loss of the EP results in deafness. EP is maintained by ionic fluxes in the transport systems present on the lateral cochlear wall, which is composed of two types of tissues: stria vascularis and spiral ligament (Figure 1) (Wangemann, 2006; Hibino et al., 2010). Stria vascularis, an epithelial-like tissue, consists of three types of cells: marginal, intermediate, and basal. These cells not only serve as barriers separating the endolymphatic space from outer environment, but also function as filters for selective passage of particular ions, fluids, and nutrients. Furthermore, stria vascularis harbors a dense network of capillaries (Hinojosa & Rodriguez-Echandia, 1966); therefore, this tissue permits active transports of small molecules such as amino acids, hormones, glucose, and drugs inside the cochlea. Although previous studies have identified several proteins involved in the transport systems (Hibino & Kurachi, 2006; Hibino et al., 2010), more information is required for understanding the molecular elements and networks of the systems. Despite the fact that biochemical analysis of the membrane proteins is a technically difficult procedure, in the present study, a comprehensive proteomic analysis of the membrane fractions of stria vascularis was carried out for the first time, by liquid chromatography-tandem mass spectrometry (LC-MS/MS).
Figure 1. Structure of the cochlea. The panel depicts a cross-section of one turn of the cochlea (inset). The perilymph contained in the scala tympani and scala vestibuli is similar to a regular extracellular solution. The potential and K+ concentration in each fluid are also shown. StV; stria vascularis, SL; spiral ligament.
Isolation of stria vascularis
Stria vascularis is a small, complicated structure with a width of about 20-30 µm. Moreover, it is intertwined with its neighboring connective tissue, the spiral ligament. Therefore, it becomes difficult to isolate the epithelium with high purity for determination of its molecular properties by biochemical procedures. The lateral walls were dissociated from rat cochlea, followed by dissection of stria vascularis from the spiral ligament under the microscope, using a fine needle (Figure 2). Thereafter, expression analysis of marker genes from stria vascularis and spiral ligament was carried out by quantitative RT-PCR, to confirm the enrichment of stria vascularis and ensure minimal contamination of the ligament tissue in the samples.
Figure 2. Representative images of isolated StV and SL. The bony lateral wall (left panel), which was attached with stria vascularis (StV) and spiral ligament (SL), was separated from the cochlea. StV (surrounded with broken lines) was identified by a brown mark, representing the pigmented intermediate cells. The right panel shows the isolated StV and SL.
Tissue lysates of stria vascularis were ultra-centrifuged for enrichment of membrane fractions. The pellet thus obtained was divided into two fractions. The first fraction was treated with a chaotropic agent, urea, for removal of membrane-bound soluble proteins, followed by re-suspension in lysis buffer (Shimohata et al., 2007). This fraction was termed as ‘urea wash’. The other fraction was directly re-suspended in the lysis buffer and was termed as ‘no urea wash’. Thereafter, both the samples were subjected to LC-MS/MS. As a result, 2569 proteins from ‘urea wash’ and 2923 proteins from ‘no urea wash’ samples were identified, respectively. In these two series of assays, 2246 proteins were overlapped; therefore, 3236 proteins were identified at the end of this process. The proteins obtained were analyzed using Ingenuity Knowledge Base. Out of all the proteins examined, 511 proteins were annotated as plasma membrane proteins. The annotations were then manually assessed using the available literatures. Consequently, 513 plasma membrane proteins, including 25 ion channels and 79 transporter proteins were identified. These proteins are likely localized on the plasma membrane of strial cells (Figure 3). Of these proteins, 16 ion channels and 62 transporters are novel, and have not been identified in the stria until date.
Of the fluxes of different ions in stria vascularis, K+ transport seems to play a key role in generation of the EP. As expected, several K+ channels and K+ transporters were identified in our experiments. Notably, the library generated included an inwardly rectifying K+ channel (KCNJ13) and a Ca2+-activated K+ channel (KCNN4). Their precise localization and function, however, remains uncertain. In addition, a variety of proteins mediating Ca2+, Cl–, and H+ transport were identified in the present analysis, suggesting their crucial involvement in the strial ionic homeostasis. Next, network analysis of the identified strial proteins was performed using Ingenuity Pathway Analysis program. Only the marginal cells of stria vascularis were selected for the assay, because their molecular architecture has been characterized extensively. The proteins selected for analysis from marginal-cells were ATP1A1 (Na+,K+-ATPase α1), ATP1B1 (Na+,K+-ATPase β1), ATP1B2 (Na+,K+-ATPase β2), ATP4A (H+,K+-ATPase α1), SLC9A1 (NHE1), SLC12A2 (NKCC1), BSND (Barttin), CLCNKA (CLC-Ka), KCNQ1, TRPM4 and TRPV4 (Sakagami et al., 1991; McGuirt & Schulte, 1994; Schulte & Steel, 1994; Crouch et al., 1997; Goto et al., 1997; Mizuta et al., 1997; Bond et al., 1998; Liedtke et al., 2000; Estevez et al., 2001; Sage & Marcus, 2001; Takumida et al., 2005; Shibata et al., 2006; Takumida et al., 2009; Sakuraba et al., 2014). The network analysis predicted four hub proteins, namely, amyloid-β precursor protein (APP), AKT, ERK1/2, and protein kinase A (PKA), which interact with a number of other proteins. Every predicted connectome contained at least three channels/ transporters listed above. Moreover, it was observed that introduction of Ca2+ to the network led to its linkage with 14 proteins, including the four hub proteins and five of the channels/transporters of marginal cells mentioned above. Taken together, it was concluded that Ca2+ may play important regulatory role in the function of stria vascularis.
In various tissues and organs, amino acid transporters LAT1 and LAT2 are co–expressed and contribute to the establishment of a permeability barrier (del Amo et al., 2008). In cochlea, LAT1 occurs in the endothelial cells of strial capillaries (Sharlin et al., 2011). Mass spectrometric analysis identified LAT2 and its accessory subunit, CD98hc, in stria vascularis. They are likely to be involved in the formation of a blood–labyrinth barrier and may play a role in transport of amino acids crucial for maintenance of the stria vascularis. Moreover, transporters for certain organic substances including carbohydrates, phospholipids, fatty acids, nucleotides, hormones, and drugs were also identified in the study. These observations indicate that stria vascularis forms an active platform for exchange of various molecules.
Figure 3. Profiles of ion channels and transporters identified in our proteomic analysis. These proteins are classified by their transporting substances according to Ingenuity Knowledge Base and literature citations. Some of the transporters that carry multiple ions or substances were counted multiple times.
Identification of candidate genes involved in deafness
Disruption of the ion transport systems in stria vascularis leads to hearing loss (Zdebik et al., 2009). The proteome library of the stria was referred to the database in the Jackson Laboratory (http://hearingimpairment.jax.org), which is a repository for mouse deafness genes. Of the 295 genes listed in the database, 57 encoded the proteins listed in our library. Using the data warehouse TargetMine (http://targetmine.nibio.go.jp/targetmine/begin.do), it was shown that 97% of these genes had human homologs. The human genes were further compared with the gene lists available in the Hereditary Hearing Loss Homepage (HHLH; http://hereditaryhearingloss.org, updated on 19 May 2014) and Online Mendelian Inheritance in Man (OMIM) webpage (http://www.omim.org). It was observed that 19 genes of the predicted proteins in our library mapped to 16 deafness loci that have not been cloned yet.
The catalog of membrane proteins of the stria vascularis generated during the present study provides useful and important information for understanding the molecular elements involved in maintenance of cochlear functions and their relevance to deafness. We believe that our database will contribute towards advancement of the current research on hearing and development of effective treatments for such diseases.
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