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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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The density of functional BK channels at the basolateral pole increases with age. (A) Whole-cell recordings were made from embryonic and posthatch hair cells to confirm the onset of BK currents around E18/19. Example traces from three ages are shown. In these examples, currents were elicited by voltage steps from -80 mV to -20 or 0 mV. A fast-activating outward current was present at E18 under whole-cell recording conditions, but no such current could be recorded from earlier ages. (B) Single-channel current traces are shown for a cell-attached patch from a posthatch hair cell held at three different pipette potentials. Bath and pipette salines consisted of standard ECF and high potassium electrode solutions, respectively. Channel openings are downward. In this configuration, more negative pipette voltages correspond to increasing depolarization. The channel was voltage-sensitive, as evidenced by an increased open probability for more depolarized voltages. (C) Single-channel current amplitudes were estimated from all-points histograms using traces in (B). Linear regression to these data (dotted line) indicates a unitary conductance of 150 pS. Reversal was estimated to be about -60 mV, near the presumed resting potential of the cell. Therefore, all inclusion criteria were satisfied, establishing the identity of this channel as BK. (D) The percent of cell-attached patches (C.A.P.) exhibiting channel fluctuations attributed to BK are plotted for cells from embryonic and posthatch basilar papilla. The total number of attempts is shown in parentheses. The breakdown for the E17-E19 data set was: E17 (6%), E18 (7%), E19 (17%).
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Figure 5: The density of functional BK channels at the basolateral pole increases with age. (A) Whole-cell recordings were made from embryonic and posthatch hair cells to confirm the onset of BK currents around E18/19. Example traces from three ages are shown. In these examples, currents were elicited by voltage steps from -80 mV to -20 or 0 mV. A fast-activating outward current was present at E18 under whole-cell recording conditions, but no such current could be recorded from earlier ages. (B) Single-channel current traces are shown for a cell-attached patch from a posthatch hair cell held at three different pipette potentials. Bath and pipette salines consisted of standard ECF and high potassium electrode solutions, respectively. Channel openings are downward. In this configuration, more negative pipette voltages correspond to increasing depolarization. The channel was voltage-sensitive, as evidenced by an increased open probability for more depolarized voltages. (C) Single-channel current amplitudes were estimated from all-points histograms using traces in (B). Linear regression to these data (dotted line) indicates a unitary conductance of 150 pS. Reversal was estimated to be about -60 mV, near the presumed resting potential of the cell. Therefore, all inclusion criteria were satisfied, establishing the identity of this channel as BK. (D) The percent of cell-attached patches (C.A.P.) exhibiting channel fluctuations attributed to BK are plotted for cells from embryonic and posthatch basilar papilla. The total number of attempts is shown in parentheses. The breakdown for the E17-E19 data set was: E17 (6%), E18 (7%), E19 (17%).

Mentions: The magnitude of calcium-sensitive BK currents in the development of the chick basilar papilla has been quantified previously [11]. In that study, BK current substantially increased between E18 and E19, from little measurable calcium-sensitive current at the younger age to maximum levels only one day later. Since embryogenesis is highly dependent on flock age and health, incubation temperature, air flow around the egg, and humidity levels [46], we confirmed the time course of BK functional development using whole-cell voltage-clamp recordings. To isolate these currents, 15 mM 4-AP was included in the intracellular (electrode) solution to block slowly-activating delayed rectifier potassium channels [16]. The remaining fast-activating current was attributed to BK, and this was subsequently verified by removing extracellular calcium, leading to the elimination of the fast current (data not shown). Cells from animals at E18 and older exhibited this fast-activating, calcium-sensitive current, but cells from earlier ages showed no BK current, as reported previously (Figure 5A). The average steady-state currents measured during voltage steps to 0 mV were 652.1 ± 77.4 pA for posthatch (N = 5), 342.5 ± 69.2 pA for E18 (N = 4), and 27.7 ± 14.7 pA for E14 (N = 5) (mean ± SEM). All recordings were obtained from tall hair cells located 20% to 50% from the apical end of the papilla. These currents may be contaminated by small amounts of residual delayed rectifier channels, but the presence of a fast-activating current suggests that clustered channels at E18 are functional and sensitive to extracellular calcium.


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)

The density of functional BK channels at the basolateral pole increases with age. (A) Whole-cell recordings were made from embryonic and posthatch hair cells to confirm the onset of BK currents around E18/19. Example traces from three ages are shown. In these examples, currents were elicited by voltage steps from -80 mV to -20 or 0 mV. A fast-activating outward current was present at E18 under whole-cell recording conditions, but no such current could be recorded from earlier ages. (B) Single-channel current traces are shown for a cell-attached patch from a posthatch hair cell held at three different pipette potentials. Bath and pipette salines consisted of standard ECF and high potassium electrode solutions, respectively. Channel openings are downward. In this configuration, more negative pipette voltages correspond to increasing depolarization. The channel was voltage-sensitive, as evidenced by an increased open probability for more depolarized voltages. (C) Single-channel current amplitudes were estimated from all-points histograms using traces in (B). Linear regression to these data (dotted line) indicates a unitary conductance of 150 pS. Reversal was estimated to be about -60 mV, near the presumed resting potential of the cell. Therefore, all inclusion criteria were satisfied, establishing the identity of this channel as BK. (D) The percent of cell-attached patches (C.A.P.) exhibiting channel fluctuations attributed to BK are plotted for cells from embryonic and posthatch basilar papilla. The total number of attempts is shown in parentheses. The breakdown for the E17-E19 data set was: E17 (6%), E18 (7%), E19 (17%).
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Figure 5: The density of functional BK channels at the basolateral pole increases with age. (A) Whole-cell recordings were made from embryonic and posthatch hair cells to confirm the onset of BK currents around E18/19. Example traces from three ages are shown. In these examples, currents were elicited by voltage steps from -80 mV to -20 or 0 mV. A fast-activating outward current was present at E18 under whole-cell recording conditions, but no such current could be recorded from earlier ages. (B) Single-channel current traces are shown for a cell-attached patch from a posthatch hair cell held at three different pipette potentials. Bath and pipette salines consisted of standard ECF and high potassium electrode solutions, respectively. Channel openings are downward. In this configuration, more negative pipette voltages correspond to increasing depolarization. The channel was voltage-sensitive, as evidenced by an increased open probability for more depolarized voltages. (C) Single-channel current amplitudes were estimated from all-points histograms using traces in (B). Linear regression to these data (dotted line) indicates a unitary conductance of 150 pS. Reversal was estimated to be about -60 mV, near the presumed resting potential of the cell. Therefore, all inclusion criteria were satisfied, establishing the identity of this channel as BK. (D) The percent of cell-attached patches (C.A.P.) exhibiting channel fluctuations attributed to BK are plotted for cells from embryonic and posthatch basilar papilla. The total number of attempts is shown in parentheses. The breakdown for the E17-E19 data set was: E17 (6%), E18 (7%), E19 (17%).
Mentions: The magnitude of calcium-sensitive BK currents in the development of the chick basilar papilla has been quantified previously [11]. In that study, BK current substantially increased between E18 and E19, from little measurable calcium-sensitive current at the younger age to maximum levels only one day later. Since embryogenesis is highly dependent on flock age and health, incubation temperature, air flow around the egg, and humidity levels [46], we confirmed the time course of BK functional development using whole-cell voltage-clamp recordings. To isolate these currents, 15 mM 4-AP was included in the intracellular (electrode) solution to block slowly-activating delayed rectifier potassium channels [16]. The remaining fast-activating current was attributed to BK, and this was subsequently verified by removing extracellular calcium, leading to the elimination of the fast current (data not shown). Cells from animals at E18 and older exhibited this fast-activating, calcium-sensitive current, but cells from earlier ages showed no BK current, as reported previously (Figure 5A). The average steady-state currents measured during voltage steps to 0 mV were 652.1 ± 77.4 pA for posthatch (N = 5), 342.5 ± 69.2 pA for E18 (N = 4), and 27.7 ± 14.7 pA for E14 (N = 5) (mean ± SEM). All recordings were obtained from tall hair cells located 20% to 50% from the apical end of the papilla. These currents may be contaminated by small amounts of residual delayed rectifier channels, but the presence of a fast-activating current suggests that clustered channels at E18 are functional and sensitive to extracellular calcium.

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