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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 appear at E18 during hair cell maturation. (A) Western blot analysis on membrane-bound protein from chick brain reveals a 120 kDa band, near the predicted weight of the minimal BK α variant (127 kDa; no inclusion of alternative exons). A fainter, lower-weight band is also apparent. No other spurious bands were observed in the blot. Both bands were absent when the primary antibody was preadsorbed with the immunogen. (B) Isolated hair cells from E10 to posthatch basilar papilla are shown labeled with antibodies to BK channels (Alomone). The apical surface and hair bundle are outlined to show orientation of the cell (dotted lines). Punctate clusters appear at E18. Bright label at E10 to E12 is inside the cell at the level of the nucleolus. (C) Several preparations were triple labeled for anti-BK (Alomone; red), hair bundles (phalloidin; green), and a nuclear stain (Hoechst; blue). Two examples for each age are shown (left two columns) along with no primary controls (far right column). Anti-BK clusters appear supranuclear at E18 and subnuclear from E20 onward. Scale bars = 10 μm.
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Figure 3: BK channel clusters appear at E18 during hair cell maturation. (A) Western blot analysis on membrane-bound protein from chick brain reveals a 120 kDa band, near the predicted weight of the minimal BK α variant (127 kDa; no inclusion of alternative exons). A fainter, lower-weight band is also apparent. No other spurious bands were observed in the blot. Both bands were absent when the primary antibody was preadsorbed with the immunogen. (B) Isolated hair cells from E10 to posthatch basilar papilla are shown labeled with antibodies to BK channels (Alomone). The apical surface and hair bundle are outlined to show orientation of the cell (dotted lines). Punctate clusters appear at E18. Bright label at E10 to E12 is inside the cell at the level of the nucleolus. (C) Several preparations were triple labeled for anti-BK (Alomone; red), hair bundles (phalloidin; green), and a nuclear stain (Hoechst; blue). Two examples for each age are shown (left two columns) along with no primary controls (far right column). Anti-BK clusters appear supranuclear at E18 and subnuclear from E20 onward. Scale bars = 10 μm.

Mentions: To specifically investigate the emergence of anti-BK label in hair cells, immunocytochemical procedures were conducted on mechanically isolated hair cell preparations. Similar single-cell preparations have been used to identify the expression of BK channels in mammalian inner hair cells [14] and voltage-gated calcium channels in bullfrog saccular hair cells [42]. For these experiments, several polyclonal BK α antibodies were used with similar results (Chemicon and Alomone anti-BK, see METHODS). Data presented in the remainder of this report reflect staining from Alomone APC-107 anti-BK. The BK α subunit is highly alternatively spliced, and most antibodies--including the Chemicon antibody used in Figure 2--target epitopes that cross splice boundaries or include sequence from alternative exons. In contrast, the Alomone APC-107 anti-BK targets an epitope common to all splice forms. Specificity for the chick BK α homologue was confirmed using Western blot (Figure 3A). In chick cochlear hair cells isolated from E10 and E12 animals, the brightest anti-BK label was intracellular (Figure 3B). Confocal stacks clarified that this label was intranuclear. While surprising, this pattern is similar to that in chick ciliary ganglion at the same embryonic ages [43]. Diffuse cytoplasmic staining at E14 to E16 gave way to punctate staining from E18 onward. Punctate clusters in posthatch hair cells were located in subnuclear regions and appeared to be associated with the plasma membrane. Notably, the appearance of BK clusters coincided with the appearance of calcium-sensitive BK currents at E18-E19. These data strongly suggest that translation and trafficking of BK subunits into clustered domains, likely near voltage-gated calcium sources, underlie the delayed and sudden acquisition of BK currents during hair cell maturation.


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 appear at E18 during hair cell maturation. (A) Western blot analysis on membrane-bound protein from chick brain reveals a 120 kDa band, near the predicted weight of the minimal BK α variant (127 kDa; no inclusion of alternative exons). A fainter, lower-weight band is also apparent. No other spurious bands were observed in the blot. Both bands were absent when the primary antibody was preadsorbed with the immunogen. (B) Isolated hair cells from E10 to posthatch basilar papilla are shown labeled with antibodies to BK channels (Alomone). The apical surface and hair bundle are outlined to show orientation of the cell (dotted lines). Punctate clusters appear at E18. Bright label at E10 to E12 is inside the cell at the level of the nucleolus. (C) Several preparations were triple labeled for anti-BK (Alomone; red), hair bundles (phalloidin; green), and a nuclear stain (Hoechst; blue). Two examples for each age are shown (left two columns) along with no primary controls (far right column). Anti-BK clusters appear supranuclear at E18 and subnuclear from E20 onward. Scale bars = 10 μm.
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Figure 3: BK channel clusters appear at E18 during hair cell maturation. (A) Western blot analysis on membrane-bound protein from chick brain reveals a 120 kDa band, near the predicted weight of the minimal BK α variant (127 kDa; no inclusion of alternative exons). A fainter, lower-weight band is also apparent. No other spurious bands were observed in the blot. Both bands were absent when the primary antibody was preadsorbed with the immunogen. (B) Isolated hair cells from E10 to posthatch basilar papilla are shown labeled with antibodies to BK channels (Alomone). The apical surface and hair bundle are outlined to show orientation of the cell (dotted lines). Punctate clusters appear at E18. Bright label at E10 to E12 is inside the cell at the level of the nucleolus. (C) Several preparations were triple labeled for anti-BK (Alomone; red), hair bundles (phalloidin; green), and a nuclear stain (Hoechst; blue). Two examples for each age are shown (left two columns) along with no primary controls (far right column). Anti-BK clusters appear supranuclear at E18 and subnuclear from E20 onward. Scale bars = 10 μm.
Mentions: To specifically investigate the emergence of anti-BK label in hair cells, immunocytochemical procedures were conducted on mechanically isolated hair cell preparations. Similar single-cell preparations have been used to identify the expression of BK channels in mammalian inner hair cells [14] and voltage-gated calcium channels in bullfrog saccular hair cells [42]. For these experiments, several polyclonal BK α antibodies were used with similar results (Chemicon and Alomone anti-BK, see METHODS). Data presented in the remainder of this report reflect staining from Alomone APC-107 anti-BK. The BK α subunit is highly alternatively spliced, and most antibodies--including the Chemicon antibody used in Figure 2--target epitopes that cross splice boundaries or include sequence from alternative exons. In contrast, the Alomone APC-107 anti-BK targets an epitope common to all splice forms. Specificity for the chick BK α homologue was confirmed using Western blot (Figure 3A). In chick cochlear hair cells isolated from E10 and E12 animals, the brightest anti-BK label was intracellular (Figure 3B). Confocal stacks clarified that this label was intranuclear. While surprising, this pattern is similar to that in chick ciliary ganglion at the same embryonic ages [43]. Diffuse cytoplasmic staining at E14 to E16 gave way to punctate staining from E18 onward. Punctate clusters in posthatch hair cells were located in subnuclear regions and appeared to be associated with the plasma membrane. Notably, the appearance of BK clusters coincided with the appearance of calcium-sensitive BK currents at E18-E19. These data strongly suggest that translation and trafficking of BK subunits into clustered domains, likely near voltage-gated calcium sources, underlie the delayed and sudden acquisition of BK currents during hair cell maturation.

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