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Developmental expression of BK channels in chick cochlear hair cells.

Li Y, Atkin GM, Morales MM, Liu LQ, Tong M, Duncan RK - BMC Dev. Biol. (2009)

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Illinois at Chicago, USA. yli71@uic.edu

ABSTRACT

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.

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BK channel clusters are associated with the hair cell membrane. (A) Isolated hair cells were imaged using confocal microscopy to determine whether anti-BK clusters were associated with the membrane or found intracellularly. Two exemplar hair cells are shown, both oriented with hair bundles toward the top of the image. Between 13 and 17 optical sections were taken at increments of 0.5 μm. Projections of full Z-stack are shown alongside orthogonal cuts through the reconstructed stack. Cuts were made at the level of each anti-BK puncta and positioned to the right of each hair cell example. In all cases, puncta were present along the outer edge of the cut-view, indicating overlap with the hair cell plasma membrane. (B) Hair cells and auditory neurons were acutely isolated onto the same slide in some preparations and stained for glutamate receptor 2 (GluR2) in order to detect residual afferent terminals on the dissociated hair cells. Afferent auditory neurons were identified according to cell size and the presence of bipolar axonal projections. Under epifluorescence and using constant exposure conditions, anti-GluR2 label was found on the cell soma of auditory neurons but was absent from hair cells. (C) Anti-BK puncta were distant from synaptic ribbons in isolated hair cells. Isolated hair cells were co-labeled with anti-BK (red) and anti-ctbp2/RIBEYE (green), a marker for presynaptic ribbons. An exemplar tall hair cell is shown using epifluorescence imaging. Debris that might be associated with afferent terminals is absent from the matching differential interference contrast image (left). The image shown is representative of over 5 tall hair cells imaged in this co-labeling experiment. Scale bars in all panels = 10 μm.
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Figure 4: BK channel clusters are associated with the hair cell membrane. (A) Isolated hair cells were imaged using confocal microscopy to determine whether anti-BK clusters were associated with the membrane or found intracellularly. Two exemplar hair cells are shown, both oriented with hair bundles toward the top of the image. Between 13 and 17 optical sections were taken at increments of 0.5 μm. Projections of full Z-stack are shown alongside orthogonal cuts through the reconstructed stack. Cuts were made at the level of each anti-BK puncta and positioned to the right of each hair cell example. In all cases, puncta were present along the outer edge of the cut-view, indicating overlap with the hair cell plasma membrane. (B) Hair cells and auditory neurons were acutely isolated onto the same slide in some preparations and stained for glutamate receptor 2 (GluR2) in order to detect residual afferent terminals on the dissociated hair cells. Afferent auditory neurons were identified according to cell size and the presence of bipolar axonal projections. Under epifluorescence and using constant exposure conditions, anti-GluR2 label was found on the cell soma of auditory neurons but was absent from hair cells. (C) Anti-BK puncta were distant from synaptic ribbons in isolated hair cells. Isolated hair cells were co-labeled with anti-BK (red) and anti-ctbp2/RIBEYE (green), a marker for presynaptic ribbons. An exemplar tall hair cell is shown using epifluorescence imaging. Debris that might be associated with afferent terminals is absent from the matching differential interference contrast image (left). The image shown is representative of over 5 tall hair cells imaged in this co-labeling experiment. Scale bars in all panels = 10 μm.

Mentions: Localization of anti-BK puncta to the surface of the hair cell membrane was supported by confocal microscopy (Figure 4A). Cuts through reconstructed Z-stacks showed that puncta were always positioned along the membrane border rather than internally (see also confocal movies in Additional files 1 and 2). Based on the neuronal labeling in Figure 2, it was possible that BK puncta reflected residual post-synaptic terminals rather than pre-synaptic clusters in the hair cell membrane. To address this possibility, we labeled isolated hair cells with pre- and post-synaptic markers to identify prevalence of post-synaptic terminals in our preparations and to determine the relationship between BK puncta and the synaptic active zone. Anti-GluR2 staining was negative on dissociated cells (Figure 4B), suggesting that terminals were adequately removed during enzymatic treatment and mechanical dissociation. Co-label experiments with antibodies to BK channels and the synaptic ribbon protein RIBEYE (anti-ctbp2) revealed a dislocalization of BK clusters and active zones (Figure 4C). Staining for the ribbon synapse can be considered a proxy for the location of synaptic active zones, even though the postsynaptic membrane covers a larger surface area than that marked by the ribbon itself [44,45]. For BK plaques to be associated with terminals, the postsynaptic footprint would have to be large enough to encapsulate both BK and ribbon puncta. Such a large amount of extraneous membrane would be visible under DIC optics. However, images in Figures 3 and 4 were taken from hair cells exhibiting a smooth surface. These data support an association between BK plaques and the hair cell membrane, and they challenge the widely held view that BK channels and voltage-gated calcium channels are clustered at presynaptic active zones in non-mammalian hair cells [18,19]. This observation requires further study, but it is possible that channel density at active zones is below our detection limits.


Developmental expression of BK channels in chick cochlear hair cells.

Li Y, Atkin GM, Morales MM, Liu LQ, Tong M, Duncan RK - BMC Dev. Biol. (2009)

BK channel clusters are associated with the hair cell membrane. (A) Isolated hair cells were imaged using confocal microscopy to determine whether anti-BK clusters were associated with the membrane or found intracellularly. Two exemplar hair cells are shown, both oriented with hair bundles toward the top of the image. Between 13 and 17 optical sections were taken at increments of 0.5 μm. Projections of full Z-stack are shown alongside orthogonal cuts through the reconstructed stack. Cuts were made at the level of each anti-BK puncta and positioned to the right of each hair cell example. In all cases, puncta were present along the outer edge of the cut-view, indicating overlap with the hair cell plasma membrane. (B) Hair cells and auditory neurons were acutely isolated onto the same slide in some preparations and stained for glutamate receptor 2 (GluR2) in order to detect residual afferent terminals on the dissociated hair cells. Afferent auditory neurons were identified according to cell size and the presence of bipolar axonal projections. Under epifluorescence and using constant exposure conditions, anti-GluR2 label was found on the cell soma of auditory neurons but was absent from hair cells. (C) Anti-BK puncta were distant from synaptic ribbons in isolated hair cells. Isolated hair cells were co-labeled with anti-BK (red) and anti-ctbp2/RIBEYE (green), a marker for presynaptic ribbons. An exemplar tall hair cell is shown using epifluorescence imaging. Debris that might be associated with afferent terminals is absent from the matching differential interference contrast image (left). The image shown is representative of over 5 tall hair cells imaged in this co-labeling experiment. Scale bars in all panels = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803478&req=5

Figure 4: BK channel clusters are associated with the hair cell membrane. (A) Isolated hair cells were imaged using confocal microscopy to determine whether anti-BK clusters were associated with the membrane or found intracellularly. Two exemplar hair cells are shown, both oriented with hair bundles toward the top of the image. Between 13 and 17 optical sections were taken at increments of 0.5 μm. Projections of full Z-stack are shown alongside orthogonal cuts through the reconstructed stack. Cuts were made at the level of each anti-BK puncta and positioned to the right of each hair cell example. In all cases, puncta were present along the outer edge of the cut-view, indicating overlap with the hair cell plasma membrane. (B) Hair cells and auditory neurons were acutely isolated onto the same slide in some preparations and stained for glutamate receptor 2 (GluR2) in order to detect residual afferent terminals on the dissociated hair cells. Afferent auditory neurons were identified according to cell size and the presence of bipolar axonal projections. Under epifluorescence and using constant exposure conditions, anti-GluR2 label was found on the cell soma of auditory neurons but was absent from hair cells. (C) Anti-BK puncta were distant from synaptic ribbons in isolated hair cells. Isolated hair cells were co-labeled with anti-BK (red) and anti-ctbp2/RIBEYE (green), a marker for presynaptic ribbons. An exemplar tall hair cell is shown using epifluorescence imaging. Debris that might be associated with afferent terminals is absent from the matching differential interference contrast image (left). The image shown is representative of over 5 tall hair cells imaged in this co-labeling experiment. Scale bars in all panels = 10 μm.
Mentions: Localization of anti-BK puncta to the surface of the hair cell membrane was supported by confocal microscopy (Figure 4A). Cuts through reconstructed Z-stacks showed that puncta were always positioned along the membrane border rather than internally (see also confocal movies in Additional files 1 and 2). Based on the neuronal labeling in Figure 2, it was possible that BK puncta reflected residual post-synaptic terminals rather than pre-synaptic clusters in the hair cell membrane. To address this possibility, we labeled isolated hair cells with pre- and post-synaptic markers to identify prevalence of post-synaptic terminals in our preparations and to determine the relationship between BK puncta and the synaptic active zone. Anti-GluR2 staining was negative on dissociated cells (Figure 4B), suggesting that terminals were adequately removed during enzymatic treatment and mechanical dissociation. Co-label experiments with antibodies to BK channels and the synaptic ribbon protein RIBEYE (anti-ctbp2) revealed a dislocalization of BK clusters and active zones (Figure 4C). Staining for the ribbon synapse can be considered a proxy for the location of synaptic active zones, even though the postsynaptic membrane covers a larger surface area than that marked by the ribbon itself [44,45]. For BK plaques to be associated with terminals, the postsynaptic footprint would have to be large enough to encapsulate both BK and ribbon puncta. Such a large amount of extraneous membrane would be visible under DIC optics. However, images in Figures 3 and 4 were taken from hair cells exhibiting a smooth surface. These data support an association between BK plaques and the hair cell membrane, and they challenge the widely held view that BK channels and voltage-gated calcium channels are clustered at presynaptic active zones in non-mammalian hair cells [18,19]. This observation requires further study, but it is possible that channel density at active zones is below our detection limits.

Bottom Line: 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).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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Illinois at Chicago, USA. yli71@uic.edu

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus