Elsevier

Gene

Volume 386, Issues 1–2, 15 January 2007, Pages 11-23
Gene

Diversity of Ca2+-activated K+ channel transcripts in inner ear hair cells

https://doi.org/10.1016/j.gene.2006.07.023Get rights and content

Abstract

Hair cells express a complement of ion channels, representing shared and distinct channels that confer distinct electrophysiological signatures for each cell. This diversity is generated by the use of alternative splicing in the α subunit, formation of heterotetrameric channels, and combinatorial association with β subunits. These channels are thought to play a role in the tonotopic gradient observed in the mammalian cochlea. Mouse Kcnma1 transcripts, 5′ and 3′ ESTs, and genomic sequences were examined for the utilization of alternative splicing in the mouse transcriptome. Comparative genomic analyses investigated the conservation of KCNMA1 splice sites. Genomes of mouse, rat, human, opossum, chicken, frog and zebrafish established that the exon–intron structure and mechanism of KCNMA1 alternative splicing were highly conserved with 6–7 splice sites being utilized. The murine Kcnma1 utilized 6 out of 7 potential splice sites. RT-PCR experiments using murine gene-specific oligonucleotide primers analyzed the scope and variety of Kcnma1 and Kcnmb1–4 expression profiles in the cochlea and inner ear hair cells. In the cochlea splice variants were present representing sites 3, 4, 6, and 7, while site 1 was insertionless and site 2 utilized only exon 10. However, site 5 was not present. Detection of KCNMA1 transcripts and protein exhibited a quantitative longitudinal gradient with a reciprocal gradient found between inner and outer hair cells. Differential expression was also observed in the usage of the long form of the carboxy-terminus tail. These results suggest that a diversity of splice variants exist in rodent cochlear hair cells and this diversity is similar to that observed for non-mammalian vertebrate hair cells, such as chicken and turtle.

Introduction

The peripheral auditory system is able to detect a wide range of sound frequencies generated from complex stimuli that are resolved topographically and are spatially encoded along the length of the auditory organ. Both the intensities and frequencies of this tonotopic information are transmitted by neuronal impulses and then relayed through a series of neurons to the auditory cortex of the brain. Numerous anatomical, mechanical and physiological specializations are graded along the length of the cochlea and are required for establishing a tonotopic map. These graded mechanical and structural features include changes in the width and stiffness of the basilar membrane, on which sits the sensory receptors (i.e., hair cells), differences in hair cell size and stereocilia length. Frequency specificity also extends to the intrinsic electrical signatures of both hair cells and spiral ganglion neurons, the initial neural component in the auditory system.

Presence or absence of ion channels as well as quantitative differences in ion channels is observed in mammalian cochlear hair cells. These longitudinal and radial ion channel variations may contribute to the tonotopic map (Beisel and Fritzsch, 2003, Eatock and Hurley, 2003). A variety of inwardly and outwardly rectifying K+ channels are found in mammalian hair cells and spiral ganglion neurons (Adamson et al., 2002, Geleoc et al., 2004, Hotchkiss et al., 2005). Outwardly rectifying potassium channels can be categorized as a delayed rectifier, A-type and Ca2+-dependent. The two types of calcium-dependent potassium channels, BKCa and SKCa, are thought to participate in the hair cell's synaptic transmission. Cation influx through the hair cell nicotinic acetylcholine receptors activates SKCa channels, which leads to a net hyperpolarization (Marcotti et al., 2004b). There is a close association of large BKCa channels (also described as large KCa, MaxiK, Slo, or BK) with the afferent synaptic active zone. Hair cell BKCa is suggested to permit both the rapid activation of K+ currents, required for electrical resonance in lower vertebrates, and the transmission of information about the phase of high-frequency stimuli (Fettiplace and Fuchs, 1999, Ramanathan and Fuchs, 2002, Duncan and Fuchs, 2003). In general, BKCa channels are important modulators of cellular excitability. These channels are characterized by large single-channel conductance, intrinsic voltage-dependence, Ca2+ modulation and blockage by charybdotoxin (CTX) and iberiotoxin (IBTX) (Magleby, 2003, Wu, 2003).

The BKCa channels are comprised of four α subunits (Kcnma1) and accessory β subunits (Kcnmb1–4). One mechanism to generate diversity of BKCa is by the alternative splicing of the KCNMA1 gene. A number of transcript variants were identified in the chicken, turtle, and rat inner ears (Navaratnam et al., 1997, Jones et al., 1999, Langer et al., 2003). These alternatively spliced transcript variants of the BKCa α subunit appear to vary in expression along the tonotopic axis. Examination of both chick and turtle BKCa currents suggested that variations in BKCa channel kinetics determine the tuning frequencies of hair cells in these non-mammalian vertebrates (Fettiplace and Fuchs, 1999, Ramanathan and Fuchs, 2002). Utilization of the β subunits provides additional variations in BKCa conductances. In the cochlea graded expression of Kcnmb1 is associated with the low frequency end (Jones et al., 1999, Ramanathan and Fuchs, 2002, Langer et al., 2003). Thus, the molecular basis for BKCa channel heterogeneity arises from 1) variation in the usage of alternatively spliced transcripts of the α (KCNMA1) gene, 2) formation of heterotetrameric channels, and 3) combinatorial association with β subunits derived from the Kcnmb1–4 genes (Orio et al., 2002, Lippiat et al., 2003).

Comparison of KCNMA1 cDNAs from a number of different vertebrate species suggests that there are ∼ 6–7 splice sites, along with additional species-unique splice variants. In the present study, we examined genomic sequences and compared the genomic organization of mouse, rat, human, opossum, chicken, frog and zebrafish KCNMA1 orthologs. Data obtained from the derivation RIKEN mouse transcriptome encyclopedia and Cap-Analysis Gene Expression (CAGE) (Carninci et al., 2005, Kawaji et al., 2006, Kodzius et al., 2006, Maeda et al., 2006) along with RT-PCR analyses document the extent of Kcnma1 alternative splicing in the mouse. Expression profiles of the Kcnmb1–4 genes were also investigated to identity if additional alternatively spliced transcript variants were also generated. The final goal of our studies was to examine the variability and potential diversity of BKCa channel expression in mouse inner ear hair cells and relate this diversity to evolutionary conservation of the associated electrophysiological and tonotopic properties.

Section snippets

Tissue procurement tissue preparation and whole mount in situ hybridization and immunodetection

Animal use for these investigations was conducted with the approval of the Institutional Animal Care and Use Committee (Creighton University). As previously described (Beisel et al., 2000, Carninci et al., 2005), semi-outbred CF1 mice were anesthetized with 50 mg/kg pentobarbital and perfused transcardially with 4% paraformaldehyde (PFA) in saline. The inner ears were isolated and decalcified in 150 mM EDTA/PFA and subsequently the cochleae were separated from inner ear structures, hemisected,

Comparative genomic analysis of KCNMA1 genes

Comparisons of published KCNMA1 transcript variants from human, mouse, rat, chicken, and turtle suggested the existence of up to 8 alternatively spliced sites that result in two or more changes of amino acid sequence. These potential sites are located at both the amino- and carboxy-termini as well as internally (Fettiplace and Fuchs, 1999).

Discussion

BKCa channels are one of the predominant ion channels expressed in the sensory epithelium and afferent neurons of the inner ear. These channels in the auditory system, combined with voltage-dependent calcium channels, play a significant role in neurotransmitter release that sends neuronal impulses from the inner ear to the brain. Their importance in inner ear function is demonstrated by null mutations of the Kcnma1 (Ruttiger et al., 2004) and Cacna1d (Dou et al., 2004) genes that result in

Acknowledgements

This investigation was supported by research grants from the USPHS National Institute on Deafness and Other Communication Disorders grants R01 DC04279 (K.W.B.), R01 DC05009 (K.W.B.), and the National Organization for Hearing Research (S.M.R-S.), a research grant for the RIKEN Genome Exploration Research Project from the Ministry of Education, Culture, Sports, Science and Technology of the Japanese Government (Y.H.), and a grant of the Genome Network Project from the Ministry of Education,

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    Present address: Department of Obstetrics and Gynecology, University of Nebraska Medical Center, Omaha, Nebraska, United States of America.

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