Manipulation of BK channel expression is sufficient to alter auditory hair cell thresholds in larval zebrafish

Abstract

Non-mammalian vertebrates rely on electrical resonance for frequency tuning in auditory hair cells. A key component of the resonance exhibited by these cells is an outward calcium-activated potassium current that flows through large-conductance calcium-activated potassium (BK) channels. Previous work in midshipman fish (Porichthys notatus) has shown that BK expression correlates with seasonal changes in hearing sensitivity and that pharmacologically blocking these channels replicates the natural decreases in sensitivity during the winter non-reproductive season. To test the hypothesis that reducing BK channel function is sufficient to change auditory thresholds in fish, morpholino oligonucleotides (MOs) were used in larval zebrafish (Danio rerio) to alter expression of slo1a and slo1b, duplicate genes coding for the pore-forming α-subunits of BK channels. Following MO injection, microphonic potentials were recorded from the inner ear of larvae. Quantitative real-time PCR was then used to determine the MO effect on slo1a and slo1b expression in these same fish. Knockdown of either slo1a or slo1b resulted in disrupted gene expression and increased auditory thresholds across the same range of frequencies of natural auditory plasticity observed in midshipman. We conclude that interference with the normal expression of individual slo1 genes is sufficient to increase auditory thresholds in zebrafish larvae and that changes in BK channel expression are a direct mechanism for regulation of peripheral hearing sensitivity among fishes.

Keywords: Auditory thresholds; Hair cell; Potassium channels; Saccule.

Fig. 1.

slo1a and slo1b splice-blocking morpholino and qPCR design. (A) A splice-blocking morpholino (MO) was designed targeting exon 9 (e9) of slo1a. This MO causes activation of a cryptic splice site resulting in a truncation of the protein encoded by e9 by 25 amino acids (AA, hatched box). Primers (arrows) were designed to measure transcript abundance of both intact (L) and truncated (S) slo1a as well as slo1b. A splice-blocking MO was designed targeting exon 9 (e9) of slo1b. This MO caused inclusion of the intron between exons 8 and 9 resulting in the introduction of stop codons between e8 and e9, causing a loss of pore, S6 and intracellular C-terminal tail domains. (B) The 25 amino acid region of e9 removed by the slo1a MO corresponds to the final third (8 of 24 amino acids) of the BK channel pore and approximately the first half (11 of 23 amino acids) of the S6 transmembrane domain. Amino acid numbering is just for scale and does not reflect amino acid number within the entire BK channel protein. (C) Schematic diagram of the BK channel α-subunit encoded by each slo1 gene.

Fig. 2.

slo1a MO causes a dose-dependent increase in auditory threshold. Only at the highest dose (250 μmol l⁻¹) does slo1a MO cause a change in auditory threshold compared with non-manipulated wild-type controls. Error bars indicate 95% confidence intervals.

Fig. 3.

slo1a MO increases auditory threshold. (A) Auditory hair cell thresholds were significantly increased by slo1a MO treatment compared with both control MO and non-manipulated wild-type controls. Error bars indicate 95% confidence intervals. (B) There is no significant relationship between frequency and difference in threshold between slo1a and control MO treatments.

Fig. 4.

slo1b MO increases auditory threshold. (A) Auditory hair cell thresholds were significantly increased by slo1b MO treatment compared with both control MO and non-manipulated wild-type animals. Error bars indicate 95% confidence intervals. (B) There is no significant relationship between frequency and difference in threshold between slo1b and control MO treatments.

Fig. 5.

slo1a/slo1b combined MO does not significantly increase auditory thresholds compared with control MO-injected animals. Both slo1a/slo1b MO- and slo1a/slo1b control-injected animals had auditory thresholds that were significantly higher than those of non-manipulated wild-type animals. Error bars indicate 95% confidence intervals.

Fig. 6.

slo1a MO treatment significantly alters slo1 transcript abundance. slo1a MO treatment (N=10) caused significant increases in both MO-truncated slo1a (slo1a S) and intact slo1a (slo1a L) transcript abundance compared with control MO groups (N=9, N=10, respectively). slo1a MO treatment significantly decreased slo1b transcript abundance. *P=0.0086, **P=0.0012, ***P=0.0008. Error bars indicate standard errors.

Fig. 7.

slo1b MO treatment significantly alters slo1 transcript abundance. slo1b MO treatment (N=16) caused significant increases in both total slo1a and total slo1b transcript abundance compared with the control MO group (N=15). slo1b MO treatment significantly increased the abundance of a slo1b transcript containing an intron inclusion between exons 8 and 9 (slo1b intron). *P<0.0001 Welch ANOVA. Error bars indicate standard errors.

Fig. 8.

slo1a/slo1b combined MO treatment does not significantly alter slo1 transcript abundance. MO treatment (N=12) did not have a significant effect on the abundance of slo1b, slo1b transcript containing an intron inclusion (slo1b intron), intact slo1a (slo1a L) or MO-truncated slo1a (slo1a S) compared with control MO-injected (N=6) or wild-type (N=9) groups. Error bars indicate standard errors.