Dissection of voltage-gated sodium channels in developing cochlear sensory epithelia

Abstract

Hair cells (HCs) clustered in cochlear sensory epithelia produce spontaneous spike and evoked action potentials during the developmental stage of auditory, which rely on many ion channels, such as Ca channels, K channels and Na channels (Housley et al., 2006; Marcotti et al., 2003). As the dominant molecule acting in action potentials in most of excitable cells, Na channels (VGSCs) were deemed to be indispensable in the spontaneous and evoked action potentials of HCs (Marcotti et al., 2003). The inward Na currents recorded from hair cells, shorten the time to reach the action-potential threshold (Eckrich et al., 2012). However, little was known about the tissueor developmental expression and the molecular structural information of Na channel subtype in hair cells, which extremely restricts the understanding of physiological mechanisms related to hearing development. To examine whether there exist any novel sodium channel subtype in cochlear hair cells, the sensory epithelia locating in premature mouse cochlea was carefully dissected and used for the identification of VGSCs in cochlear hair cells. Three pairs of degenerate primers derived from conserved region of mammalian VGSCs (Table S2), as well as their invertebrate counterparts (from Drosophila, cockroach, housefly and mosquito), were used for detecting the homologous sequence of cochlear VGSCs. All the resultant sequences were matched to the known mammalian VGSC subtypes (Nav1.1–1.9, data not shown), indicating that no novel sodium channel subtype could be found in cochlea. Subtype-specific primers corresponding to conserved regions of Nav1.1–1.9 were designed and used to probe and quantify the abundance of VGSCs expressed in cochlea (Table S1). Analysis of these cDNA fragments ranging from 115 to 340 bp revealed that all the known VGSC subtypes could be detected in cochlea though in a less expression level than that of Cav1.3 (Fig. 1A). Co-expression of multiple Na channel isoforms has been described not only in neurons from the brain but also in primary sensory receptors of the mammalian cochlea. Nav1.7 has been suggested as the main carrier of INa in mouse inner hair cells because of the distinct biophysical and pharmacological properties (Marcotti et al., 2003). Both Nav1.1 and Nav1.6 were identified through immunohistochemistry on cell membrane of rat hair cells, while Nav1.2 was localized to the unmyelinated efferent axons and terminals (Eckrich et al., 2012). Nav1.6 with high expression in sensory epithelia is considered as the developmental regulator involved in the frequency of action potential activity and hair cell maturation. In this study, four sodium channels mentioned above were found with pronounced expression, providing further evidence of these subtypes in regulating action potential (Eckrich et al., 2012; Marcotti et al., 2003). Nevertheless, Nav1.4, Nav1.5, Nav1.8 and Nav1.9, were found to be expressed in relatively low levels in cochlear sensory epithelia (Fig. 1A). To obtain a clear view on the structural features of cochlear VGSCs, specific primers were designed to clone the coding sequences (CDS) of all the VGSCs identified (Table S2). The sequences of cochlear Nav1.1 and Nav1.4 were identical with that of counterparts in other mouse tissues. The other seven full-length VGSCs or structural variants comprised 5298 and 6021 bp (Genebank number: KM373687-373700), encoding for 1765 and 2006 amino acids. According to the generally acceptable nomenclature (Goldin et al., 2000), the cochlear VGSCs mentioned above were named as CbmNav1.x, in which the subscript letter ‘bm’ indicates the originated tissue of cochlear VGSCs and ‘1.x’ indicates the corresponded mammalian Nav counterparts. For instance, cochlear VGSC corresponding to Nav1.2 was named CbmNav1.2. Hereinafter, alternative splicing or RNAediting variants that belong to CbmNav1.2 were named as CbmNav1.2a, CbmNav1.2b..., respectively. When mapping these sequences of CbmNav1.x to the mouse genome, it was found that CbmNav1.x were subjected to diverse point mutations, fragment insertions and deletions brought by the alternative splicing and RNA-editing events, most of which sit in functionally significant regions (Fig. 1B). All the editing sites and alternative splicing sites were double checked by pyrosequencing to remove any interference of RT-PCR errors. The sites location and nucleotide substitution patterns of all the alternative splicing and RNA-editing variants were shown in Table 1. Two variants of CbmNav1.2 differed by a single exon 6 exhibited the same alternative splicing site (Fig. 1C and 1D), which maybe resulting in functional enhancement as

DOI: 10.1007/s13238-015-0157-1

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Cite this paper

@inproceedings{Zhou2015DissectionOV, title={Dissection of voltage-gated sodium channels in developing cochlear sensory epithelia}, author={You Zhou and F Fang and Zhi-Rui Liu and Yonghua Ji}, booktitle={Protein & Cell}, year={2015} }