Differential Expression of KCNQ4 in Inner Hair Cells and Sensory Neurons Is the Basis of Progressive High-Frequency Hearing Loss

Abstract

Human KCNQ4 mutations known as DFNA2 cause non-syndromic, autosomal-dominant, progressive high-frequency hearing loss in which the cellular and molecular basis is unclear. We provide immunofluorescence data showing that Kcnq4 expression in the adult cochlea has both longitudinal (base to apex) and radial (inner to outer hair cells) gradients. The most intense labeling is in outer hair cells at the apex and in inner hair cells as well as spiral ganglion neurons at the base. Spatiotemporal expression studies show increasing intensity of KCNQ4 protein labeling from postnatal day 21 (P21) to P120 mice that is most apparent in inner hair cells of the middle turn. We have identified four alternative splice variants of Kcnq4 in mice. The alternative use of exons 9 -11 produces three transcript variants (v1-v3), whereas the fourth variant (v4) skips all three exons; all variants have the same amino acid sequence at the C termini. Both reverse transcription-PCR and quantitative PCR analyses demonstrate that these variants have differential expression patterns along the length of the mouse organ of Corti and spiral ganglion neurons. Our expression data suggest that the primary defect leading to highfrequency loss in DFNA2 patients may be attributable to high levels of the dysfunctional Kcnq4_v3 variant in the spiral ganglion and inner hair cells in the basal hook region. Progressive hearing loss associated with aging may result from an increasing mutational load expansion toward the apex in inner hair cells and spiral ganglion neurons.

Dominant-Negative Inhibition of M-Like Potassium Conductances in Hair Cells of the Mouse Inner Ear

“…Similar expression patterns have been noted for KCNQ4 (Beisel et al, 2005) and prestin (Judice et al, 2002;Zheng et al, 2002) in cochlear hair cells, which suggests that the turnover rate may be slower for some hair-cell proteins than the corresponding mRNA turnover rate. Four alternative splice variants of KCNQ4 have been identified previously in tissue harvested from mouse cochleas and vestibular organs (Beisel et al, 2005;Rocha-Sanchez et al, 2007). Three splice forms result from the alternate use of exons 9 through 11, and the fourth lacks all three exons.…”

“…Furthermore, Beisel et al (2000Beisel et al ( , 2005 have noted a temporal correlation between the physiological expression of G K,n and the molecular expression of KCNQ4, both following an apical to basal expression pattern. Previously, Kharkovets et al (2006) also showed a lack of G K,n expression in KCNQ4 knock-out mice.…”

“…We began by excising mouse cochleae at P0, 8 d before the onset of G K,n (Marcotti and Kros, 1999) and endogenous KCNQ4 expression (Beisel et al, 2005). To confirm transfection and expression of GFP and hKCNQ4-G285S, we applied Ad1-cmvhKCNQ4-G285S with a titer of 5 ϫ 10 9 viral particles/ml for 4 h. Two days later, the tissue was fixed and stained with an anti-KCNQ4 antibody and phalloidin (Fig.…”

“…As a result, identification of the molecular substrate for each conductance has not been trivial. For example, although there is substantial evidence for KCNQ4 expression in hair cells (Kubisch et al, 1999;Beisel et al, 2000Beisel et al, , 2005Kharkovets et al, 2000Kharkovets et al, , 2006Hurley et al, 2006;RochaSanchez et al, 2007), identification of its physiological correlate has been confounded by the significant difference between the biophysical properties of KCNQ4 expressed in heterologous cells and those of hair-cell potassium conductances. In heterologous cells, KCNQ4 expression results in large, slowly activating, noninactivating currents with broad voltage dependence centered around Ϫ20 to Ϫ30 mV (Kubisch et al, 1999;Schroder et al, 2001;Sogaard et al, 2001;Hougaard et al, 2004;Chambard and Ashmore, 2005).…”

Functional Significance of K+ Channel β-Subunit KCNE3 in Auditory Neurons

“…Moreover, the same study revealed that some Kv channels, such as K Ca , Kv3.1, and Kv1.1 subunits, were distributed predominantly in the basal SGNs and Kv4.2 predominantly in the apical SGNs (36). Later studies revealed other channels, such as Kv7.4 (25,47), also demonstrate an expression gradient along the cochlear axis. Therefore, specialized electrophysiological signatures along the cochlear axis are governed by differential ion channel distributions in the cochleae.…”