BDNF profoundly and specifically increases KCNQ4 expression in neurons derived from embryonic stem cells.

Neurons resembling the spiral ganglion neurons (SGNs) of the auditory nerve can be generated from embryonic stem cells through induced overexpression of the transcription factor Neurogenin-1 (Neurog1). While recapitulating this developmental pathway produces glutamatergic, bipolar neurons reminiscent of SGNs, these neurons are functionally immature, being characterized by a depolarized resting potential and limited excitability. We explored the effects of two neurotrophins known to be present in the inner ear, brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3), on the electrophysiology of neurons following Neurog1 induction. Our data reveal a significant reduction in resting membrane potential (RMP) following neurotrophin exposure, with BDNF producing a more robust effect than NT-3. This effect was accompanied by a profound and specific upregulation of the KCNQ4 subtype, where a 9-fold increase was observed with quantitative PCR. The other neuronally expressed KCNQ subtypes (2, 3, and 5) exhibited upregulation which was 3-fold or less in magnitude. Quantitative immunohistochemistry confirmed the increase in KCNQ4 expression at the protein level. The present data show a novel link between BDNF and KCNQ4 expression, yielding insight into the restricted expression pattern of a channel known to play special roles in setting the resting potential of auditory cells and in the etiology of progressive high-frequency hearing loss.

Developmental expression of BK channels in chick cochlear hair cells.

BACKGROUND: Cochlear hair cells are high-frequency sensory receptors. At the onset of hearing, hair cells acquire fast, calcium-activated potassium (BK) currents, turning immature spiking cells into functional receptors. In non-mammalian vertebrates, the number and kinetics of BK channels are varied systematically along the frequency-axis of the cochlea giving rise to an intrinsic electrical tuning mechanism. The processes that control the appearance and heterogeneity of hair cell BK currents remain unclear. RESULTS: Quantitative PCR results showed a non-monotonic increase in BK alpha subunit expression throughout embryonic development of the chick auditory organ (i.e. basilar papilla). Expression peaked near embryonic day (E) 19 with six times the transcript level of E11 sensory epithelia. The steady increase in gene expression from E11 to E19 could not explain the sudden acquisition of currents at E18-19, implicating post-transcriptional mechanisms. Protein expression also preceded function but progressed in a sequence from diffuse cytoplasmic staining at early ages to punctate membrane-bound clusters at E18. Electrophysiology data confirmed a continued refinement of BK trafficking from E18 to E20, indicating a translocation of BK clusters from supranuclear to subnuclear domains over this critical developmental age. CONCLUSIONS: Gene products encoding BK alpha subunits are detected up to 8 days before the acquisition of anti-BK clusters and functional BK currents. Therefore, post-transcriptional mechanisms seem to play a key role in the delayed emergence of calcium-sensitive currents. We suggest that regulation of translation and trafficking of functional alpha subunits, near voltage-gated calcium channels, leads to functional BK currents at the onset of hearing.