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Dissection of voltage-gated sodium channels in developing cochlear sensory epithelia
Dear Editor,
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 Ca2+ 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 tissue- or 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 identication 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, housey and mosquito), were used for detecting the homologous sequence of cochlear VGSCs. All the resultant sequences were matched to the known mammalian VGSC subtypes (Nav1.11.9, data not shown), indicating that no novel sodium channel subtype could be found in cochlea.
Subtype-specic primers corresponding to conserved regions of Nav1.11.9 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 identied 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 (Eck-rich 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, specic primers were designed to clone the coding sequences (CDS) of all the VGSCs identied (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 RNA-editing 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 signicant 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
The Author(s) 2015. This article is published with open access at Springerlink.com and journal.hep.com.cn
Protein&Cell
Molecular information of sodium channels expressed in developing cochlea LETTER
A
B
Relative expression level of VGSCs in cochlear sensory epithelia
0.009
0.006
0.003
0.000
Na v1.1
Na v1.2
Na v1.3
Na v1.4
Na v1.5
Na v1.6
Na v1.7
Na v1.8
Na v1.9
Ca v1.3
F(1363)L
N(209)D
W(357)G
D(522)G
F(724)S
G(824)S
V(1290)M
E(1386)G
D(208)S
M(801)V
D(488)E
Q(859)R
F(1619)S
F(259)L
V(379)L
I(627)V
E(1439)G
I(1636)T
N(212)D
L(207)V
II(4
V(924)D
VS(206)TT
NIK(209)FVD, L(215)V
N(206)D
L(201)V
1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6 1 2 3 4 5 6
M(783)K
Y(1029)C
N(782)I F(777)S
K(805)R
(
F(1140)L
L(1229)P
D(322)G
NH2
F(1643)S
P(234)S
(625)VSQ
K(494)R F(1765)S
(647657)
V(1580)A
F(1134)L
COOH
Protein&Cell
Q(513)L
I(417)V
A(709)V
R(608)G L(1967)P
E(1904)G
N(983)D
D(586)G
I(1037)T
C(934)R
( E(1062)G
(1031)Q
1)Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q E(971)G
(10801133)
N(1047)D
CbmNav1.2 CbmNav1.3 CbmNav1.5 CbmNav1.6 CbmNav1.7 CbmNav1.8 CbmNav1.9
C Genomic contig of VGSC gene (SCN2A/3A/5A/8A/9A)
D
E
F
Genomic contig of VGSC gene (SCN5A/8A/10A)
G
H
mNav1.7NM 001290674.1
CbmNav1.7a/b
n
o
x
E Intron
3
1
n
o
x
E
4
1
mNav1.7NM_001205321.1
CbmNav1.8a
CbmNav1.8b
mNav1.3NM_018732.3
n
o
x
E Intron
7
1
n
o
x
E
8
1
n
o
x
E Intron
CbmNav1.3a/b
2
1
o
x
E
n
3
1
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LETTER You Zhou et al.
Figure 1. Molecular characteristics of sodium channels expressed in cochlear sensory epithelia. (A) Quantitative analysis of nine subtypes of sodium channels expressed in cochlear sensory epithelia. The mRNA encoding for TTX-R Nav channels (mNav1.5, mNav1.8 and mNav1.9) were detected of lower expression level, the mRNA copy numbers of TTX-S Nav channels were shown at high level. (B) The schematic diagram of the sodium channel topology showed RNA editing and alternative splicing sites of CbmNavs. RNA editing sites were illustrated with circles icon; and alternative splicing sites were highlighted with square icon. See Table 1 for details on these mutations. (CH) Alternative splicing events occured in cochlear sodium channels.
s
observed in hNav1.2adu (Xu et al., 2007). Likewise, cochlear hair cells expressing alternatively splicing variant of CbmNav1.2 may display more excitable ability. R608G,
A709V, Q513L and D586G identied in CbmNav1.2 locating
at intracellular loop between DI and DII were potentially adjacent to the phosphorylation sites may greatly inuence
the channel activity (Chahine and OLeary, 2014). RNA-editing sites I417V, E1439G and F1765S were located in extracellular loop between S5 and S6 of CbmNav1.2, relating
to ionic permeability and selectivity (Fig. S1).
Nav1.3 is mainly expressed in CNS during the early development stage (Hains et al., 2003). In rNav1.3 or hNav1.3, extensive alternative splicing sites, such as 12v1, 12v2 and 12v3 (rat), as well as 12v1, 12v2, 12v3 and 12v4 (human) in DI-II intracellular loop were identied to be important for channel kinetics (Thimmapaya et al., 2005). Alternative splicing site of 12v2 was also detected in CbmNav1.3 (Fig. 1F). A missense mutation, K494R, was found in intracellular loop between DI and DII in both variant CbmNav1.3a and CbmNav1.3b. Similar to rNav1.2, a mis-sense mutation D208S of CbmNav1.3 at extracellular loop between S3 and S4 of the DI was caused by alternative splicing on Exon 6A or Exon 6B (Fig. 1C and 1D). All of these mutations might have substantial impact on phosphorylation and gating properties of the channel.
To date, seven alternative splicing variants of Nav1.5 including Nav1.5a (exon 18 deletion), Nav1.5b (exon 17 and
Protein&Cell
Table 1. Common and unique amino acid changes in fteen CbmNav variants
CbmNav1.2 CbmNav1.3 CbmNav1.5 CbmNav1.6 CbmNav1.7 CbmNav1.8 CbmNav1.9
t-a V924Da M783Kabc
t-g D488Eb W357Gab
t-c V1580Aa F1765Sb
F259La F777Sa F1363La I1636Ta F1619Sb L1967Pb
C934Rab
I1037Tab
F1643Sab
F1134Lab F724Sabc F1140Lb L1229Pb
K494Ra D322Ga Q859Ra M801Vb E1062Gb E1386Gb E1904Gb
a-g I417Vb
D586Gb R608Ga N1047Db E1439Gb
D522Gab
K805Rab
I627Vabc
E971Gc N983Dc Y1029Cb
a-t Q513Lb N782Ia
g-a V1290Mb G824Sab
g-t V379Labc
c-t A709Va
Substitution N209Dab D208Sb VS206TTb NIK209 FVDb L215Vb P234Sb
I207Vb N212Db
L201Vab
N206Dab
Deletion 10801133ab 12721312b 12661306b
647657ab
Insertion 625VSQab 1031Qb
Amino acid sequences of these variants (a, b and c represent different variant of each VGSC subtype) were compared with the genome sequence of corresponding subtypes. Nucleotide bases of RNA editing events that correspond to these in the mNav genomic sequence were in bold. Alternative splicing events included exons mutual repulsion resulting amino acid changes, exons kipping resulting amino acid deletion, alternative 3 splicing and alternatives 5 splicing resulting amino acid deletion and insertion.
460 The Author(s) 2015. This article is published with open access at Springerlink.com and journal.hep.com.cn
Molecular information of sodium channels expressed in developing cochlea LETTER
exon 18 deletion), Nav1.5c (CAG inclusive variant), Nav1.5d (exon 17 splicing), Nav1.5e (exon 6 splicing), Nav1.5f (exon 24 deletion) and C-terminal splice variants, have been reported (Schroeter et al., 2010). According to the splicing pattern, distribution and expressive proportion, these variants showed diversities in different species and different tissues of same species. Of all the VGSC subtypes found in mouse cochlear sensory epithelia, CbmNav1.5 was subjected
to the most intensive RNA-editing modication, among which T-to-C and A-to-G were the two common nucleotide substitution patterns, containing six editing sites each. Surprisingly, CbmNav1.5 was substantially modied by alternative splicing as well, resulting in two alternative splicing variants having signicant deletion at functionally important regions of the channel: to have seven amino acid residues substitution at DI S3S4 extracellular loop were found in CbmNav1.5b. In
addition, CbmNav1.5a and CbmNav1.5b lost the entire exon 18 at DI-DIII intracellular loop which contained 54 amino acid residues (Fig. 1E, Table 1). Notably, exon 18 deletion, encoding for 53 amino acids at the DIIDIII linker (residues 10781130 in hNav1.5), was the rst reported splice variant in the cardiac Na+ channel. This splicing variant showed a pronounced species-specic expression pattern in mammals (Blechschmidt et al., 2008).
Nav1.6 is widely expressed in both PNS and CNS (Caldwell et al., 2000). A variant of hNav1.6 lost a part of S3 and the whole transmembrane S4 at DIII (Zubovic et al., 2012a). Alternative splicing sites previously found in rNav1.6 and hNav1.6 were also appeared in CbmNav1.6b variants which appeared as a loss of exon 21, taking away partial sequence of DIIIS3 and the entire DIIIS4 (Fig. 1E). Our nding identied the same deletion in CbmNav1.6b of mouse
cochlea (Fig. 1E). Deletion of exon 21 caused the loss of voltage sensor S4, it may be a fail-safe mechanism that produces a non-functional truncated protein (Plummer et al., 1997).
Alternative splicing variants of Nav1.7 were found in dorsal root ganglia (DRG) neurons under neuropathic pain conditions of rats (Dib-Hajj et al., 2013). In this study, CbmNav1.7a and CbmNav1.7b were characterized by the existence of exon 6B, differing at two amino acids (201V, 206D) in the D1/S3S4 linker comparing to mNav1.7 (201L, 206N) (Fig. 1C and 1D). Previous study revealed that the variant containing exon 6B had slower kinetics of inactivation for negative potentials than that of the variant containing exon 6A (Chatelier et al., 2008; Jarecki et al., 2009). Meanwhile, exon 22 deletion was rst observed in CbmNav1.7b which was lacking partial sequence of DIIIS3 and the entire DIIIS4 (Fig. 1E), suggested that the truncated protein maybe bear a similar protection mechanism to Nav1.6 (Plummer et al., 1997; Zubovic et al., 2012b).
For CbmNav1.8, it was found ve RNA-editing sites located at the DI/DII pore region and intracellular loops. Nine novel RNA-editing sites were identied in two CbmNav1.9 variant, eight of which were caused by the nucleotide changes of adenine to guanine (Table 1). The alternative splicing event caused a deletion of Glu at position 1031 of CbmNav1.8b (Fig. 1H). Few cases have been approached as for the functional consequences of these mutations. Although several mutation sites in Nav1.8 and Nav1.9 variants were identied, whether these mutations may diversify the function of channels will still need further researches.
Groundbreakingly, all known subtypes of sodium channels were identied in mouse cochlear sensory epithelia before hearing onset, strongly indicating the indispensible role in hearing development and formation. Moreover, some structural variations of CbmNav were found, possibly endowing distinct functions in order to adapt to the physiologically distinct properties for the cochlear VGSCs. This work lays a foundation for further studies to understand the roles of diverse CbmNav variants in shaping action potentials in cochlea and regulating hearing development.
FOOTNOTES
The authors gratefully acknowledge Prof. Lin Chen for his interests in this study and providing valuable help with cochlear microdis-section. This study was supported by the National Basic Research Program (973 Program) (No. 2010CB529806), partially by grants from National Natural Science Foundation of China (Grant No. 31171064) and Key Research Program of Science and Technology Commissions of Shanghai Municipality (11JC1404300, 13DJ1 400300).
You Zhou, Fang-Hao Fang, Zhi-Rui Liu and Yong-Hua Ji declare that they have no conict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed.
You Zhou, Fang-Hao Fang, Zhi-Rui Liu, Yong-Hua Ji&
Laboratory of Neuropharmacology and Neurotoxicology, Shanghai University, Shanghai 200436, China& Correspondence: [email protected] (Y.-H. Ji)
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LETTER You Zhou et al.
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