Time course of inner ear degeneration and deafness in mice lacking the Kir4.1 potassium channel subunit

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Abstract

The Kir4.1 gene (KCNJ10) encodes an inwardly rectifying K+ channel subunit abundantly expressed in the CNS. Its expression in the mammalian inner ear has been suggested but its function in vivo in the inner ear is unknown. Because diverse human hereditary deafness syndromes are associated with mutations in K+ channels, we examined auditory function and inner ear structure in mice with a genetically inactivated Kir4.1 K+ channel subunit. Startle response experiments suggest that Kir4.1−/− mice are profoundly deaf, whereas Kir4.1+/− mice react like wild-type mice to acoustic stimuli. In Kir4.1−/− mice, the Reissner membrane is collapsed, the tectorial membrane is swollen, and type I hair cells and spiral ganglion neurons as well as their central processes degenerate over the first postnatal weeks. In the vestibular ganglia, neuronal cell death with apoptotic features is also observed. Immunostaining reveals that Kir4.1 is strongly expressed in stria vascularis of wild-type but not Kir4.1−/− mice. Within the spiral ganglion, Kir4.1 labeling was detected on satellite cells surrounding spiral ganglion neurons and axons. We conclude that Kir4.1 is crucial for normal development of the cochlea and hearing, via two distinct aspects of extracellular K+ homeostasis: (1) in stria vascularis, Kir4.1 helps to generate the cochlear endolymph; and (2) in spiral and vestibular ganglia, Kir4.1 in surrounding glial cells helps to support the spiral and vestibular ganglion neurons and their projecting axons.

Introduction

Inwardly rectifying K+ channels regulate the resting membrane potential by (a) contributing much of the resting K+ conductance in many cells and (b) maintaining low extracellular K+ via spatial buffering mechanisms (Hille, 1992, Lagrutta et al., 1996, Isomoto et al., 1997, Reimann and Ashcroft, 1999). The Kir4.1 K+ channel subunit underlies the major K+ conductance in oligodendrocytes in the spinal cord and Müller cells in the retina (Ishii et al., 1997, Kusaka et al., 1999, Kofuji et al., 2000, Neusch et al., 2001). Kir4.1 is also the only inward rectifier known to be expressed in the stria vascularis of the inner ear to date (Hibino et al., 1997, Ando and Takeuchi, 1999). Immunohistochemical studies suggest that Kir4.1 is localized to regions of the stria vascularis near capillaries. An early report presented evidence for specific Kir4.1 expression on marginal cells (Hibino et al., 1997), although later reports show Kir4.1 expression in intermediate cells (Ando and Takeuchi, 1999, Takeuchi et al., 2001). Non-specific blockers of inwardly rectifying K+ channels decreased the endocochlear potential (EP) in vivo (Hibino et al., 1997); furthermore, Kir4.1 expression follows the time course of the developmental pattern of EP generation. These observations have led to the specific hypothesis that Kir4.1 expressed on the intermediate cells helps to generate the EP by spatially buffering K+ at a low level in a distinct intrastrial compartment that is transcellular from the much higher-K+ cochlear endolymph (Takeuchi et al., 2000, Takeuchi et al., 2001). Thus Kir4.1 would help to buffer K+ in a low-K+ extracellular compartment, similar to its function in the Müller cells of the retina and probably elsewhere on glia.

This postulated role implies that animals without Kir4.1 would be deaf. To investigate further the role of the Kir4.1 K+ channel subunit in the ear and in other areas of the CNS, a mouse strain was developed that has a highly specific loss of the Kir4.1 gene product. Impact of this deletion on other areas of the CNS has been published elsewhere (Kofuji et al., 2000, Neusch et al., 2001). Here we report consequences of the gene inactivation for the inner ear at a behavioral and anatomical level. The hypothesis of deafness has been confirmed; furthermore, the Kir4.1 knockout mouse has pronounced anatomical anomalies in the inner ear, showing that Kir4.1 is directly or indirectly required for proper development as well as for acute auditory function. In another recent report, Kir4.1 knockout mice lack EP and have reduced K+ concentration in cochlear endolymph (Marcus et al., 2002).

Section snippets

Materials and methods

Experimentation on animals has been approved by the California Institute of Technology’s Animal Care and Use Committee.

Gross behavioral characterization

Kir4.1−/− mice present a general underdevelopment: they gain less weight than their Kir4.1+/− and WT littermates. Death occurs at 9–21 postnatal days. On a behavioral level, 8–10 days postnatally, mice homozygous for the mutation develop a severe motor impairment with difficulties righting themselves. This behavior could be attributed to a severe defect in spinal cord and brainstem myelination as well as to vacuolation of deep cerebellar nuclei at early stages of development (Neusch et al., 2001

Discussion

Thus far human hereditary deafness syndromes have been largely associated with mutations in outwardly rectifying, voltage-gated (Kv, KvLQT) K+ channels (Vetter et al., 1996, Romey et al., 1997, Schulze-Bahr et al., 1997, Wollnik et al., 1997, Wang et al., 1998, Chen et al., 1999, Talebizadeh et al., 1999, Van Hauwe et al., 1999, Chouabe et al., 2000, Kharkovets et al., 2000). Here we report that disruption of a gene encoding an inwardly rectifying K+ channel can lead to deafness in a mammalian

Acknowledgements

We thank Sami Barghshoon for help with animals. This work was supported by grants from the National Institutes of Health (GM-29836, EY12949), and the Deutsche Forschungsgemeinschaft (NE-767/1-1).

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