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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 expression of BK transcripts during development of the chick auditory epithelium. (A) Cycle-by-cycle amplification data are shown for S16 using serial dilutions of total RNA from chick basilar papilla. Threshold (dotted line) was manually drawn at a point during the log-linear phase of amplification. Cycle threshold was determined by identifying the threshold crossing-point for each amplification curve. (B) Total input RNA was serially diluted starting from a concentrated sample of chick basilar papilla. Diluted RNA was reverse transcribed and analyzed by qPCR. Efficiency data were fit by linear least-squares regression to Ct = M*log2(dilution)+B. The slope (M) for each curve was between -1.0 and -1.1, indicating equal efficiency and 2-fold amplification per cycle. (C) The expression of BK transcripts, assessed by the non-variant-specific probe αX, gradually increased during late-stage maturation but decreased again in the first week after hatching. The time course for acquiring calcium-sensitive BK currents is illustrated (black dots) based on data from Fuchs and Sokolwski (1990). (D) The proportion of STREX splice variants among the total population of BK transcripts steadily increased during development, whereas the proportion of β1 decreased at the onset of hearing.
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Figure 1: The expression of BK transcripts during development of the chick auditory epithelium. (A) Cycle-by-cycle amplification data are shown for S16 using serial dilutions of total RNA from chick basilar papilla. Threshold (dotted line) was manually drawn at a point during the log-linear phase of amplification. Cycle threshold was determined by identifying the threshold crossing-point for each amplification curve. (B) Total input RNA was serially diluted starting from a concentrated sample of chick basilar papilla. Diluted RNA was reverse transcribed and analyzed by qPCR. Efficiency data were fit by linear least-squares regression to Ct = M*log2(dilution)+B. The slope (M) for each curve was between -1.0 and -1.1, indicating equal efficiency and 2-fold amplification per cycle. (C) The expression of BK transcripts, assessed by the non-variant-specific probe αX, gradually increased during late-stage maturation but decreased again in the first week after hatching. The time course for acquiring calcium-sensitive BK currents is illustrated (black dots) based on data from Fuchs and Sokolwski (1990). (D) The proportion of STREX splice variants among the total population of BK transcripts steadily increased during development, whereas the proportion of β1 decreased at the onset of hearing.

Mentions: Real-time PCR was used to quantify the relative abundance of BK transcripts during late-stage cochlear development in the chick to test whether an upregulation of these transcripts near E19 could explain the acquisition of currents at this time point. TaqMan probes were developed for the pore-forming BK α subunit and the housekeeping gene S16 (Table 1). An example of the cycle-by-cycle QPCR fluorescence data is shown in Figure 1A for the S16 probe using serially diluted template. Reactions were run in triplicate and the data proved highly reproducible even for low-copy templates amplifying at later cycles. Efficiency curves were generated using serial dilutions of the total RNA samples (Figure 1B). This approach allowed us to test for amplification efficiency and for differential effects of reverse transcription depending on the amount of starting material. Amplification efficiency is determined by least-squares fits to the dilution curves using a log2 scale. A slope of -1.0 indicates an amplification efficiency of 100%. The slopes of the efficiency curves for αX and S16 were similar and near unity, validating the use of these probes for relative gene expression studies. QPCR products visualized on agarose gels were restricted to a single, specific amplicon of expected size (data not shown). Amplification curves for dissection saline and no-RT controls fell below threshold lines (undetected) or exhibited Ct values far greater than the experimental samples (i.e. a difference of greater than 7 Cts or a reduction of more than 128-fold in starting material).


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 expression of BK transcripts during development of the chick auditory epithelium. (A) Cycle-by-cycle amplification data are shown for S16 using serial dilutions of total RNA from chick basilar papilla. Threshold (dotted line) was manually drawn at a point during the log-linear phase of amplification. Cycle threshold was determined by identifying the threshold crossing-point for each amplification curve. (B) Total input RNA was serially diluted starting from a concentrated sample of chick basilar papilla. Diluted RNA was reverse transcribed and analyzed by qPCR. Efficiency data were fit by linear least-squares regression to Ct = M*log2(dilution)+B. The slope (M) for each curve was between -1.0 and -1.1, indicating equal efficiency and 2-fold amplification per cycle. (C) The expression of BK transcripts, assessed by the non-variant-specific probe αX, gradually increased during late-stage maturation but decreased again in the first week after hatching. The time course for acquiring calcium-sensitive BK currents is illustrated (black dots) based on data from Fuchs and Sokolwski (1990). (D) The proportion of STREX splice variants among the total population of BK transcripts steadily increased during development, whereas the proportion of β1 decreased at the onset of hearing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC2803478&req=5

Figure 1: The expression of BK transcripts during development of the chick auditory epithelium. (A) Cycle-by-cycle amplification data are shown for S16 using serial dilutions of total RNA from chick basilar papilla. Threshold (dotted line) was manually drawn at a point during the log-linear phase of amplification. Cycle threshold was determined by identifying the threshold crossing-point for each amplification curve. (B) Total input RNA was serially diluted starting from a concentrated sample of chick basilar papilla. Diluted RNA was reverse transcribed and analyzed by qPCR. Efficiency data were fit by linear least-squares regression to Ct = M*log2(dilution)+B. The slope (M) for each curve was between -1.0 and -1.1, indicating equal efficiency and 2-fold amplification per cycle. (C) The expression of BK transcripts, assessed by the non-variant-specific probe αX, gradually increased during late-stage maturation but decreased again in the first week after hatching. The time course for acquiring calcium-sensitive BK currents is illustrated (black dots) based on data from Fuchs and Sokolwski (1990). (D) The proportion of STREX splice variants among the total population of BK transcripts steadily increased during development, whereas the proportion of β1 decreased at the onset of hearing.
Mentions: Real-time PCR was used to quantify the relative abundance of BK transcripts during late-stage cochlear development in the chick to test whether an upregulation of these transcripts near E19 could explain the acquisition of currents at this time point. TaqMan probes were developed for the pore-forming BK α subunit and the housekeeping gene S16 (Table 1). An example of the cycle-by-cycle QPCR fluorescence data is shown in Figure 1A for the S16 probe using serially diluted template. Reactions were run in triplicate and the data proved highly reproducible even for low-copy templates amplifying at later cycles. Efficiency curves were generated using serial dilutions of the total RNA samples (Figure 1B). This approach allowed us to test for amplification efficiency and for differential effects of reverse transcription depending on the amount of starting material. Amplification efficiency is determined by least-squares fits to the dilution curves using a log2 scale. A slope of -1.0 indicates an amplification efficiency of 100%. The slopes of the efficiency curves for αX and S16 were similar and near unity, validating the use of these probes for relative gene expression studies. QPCR products visualized on agarose gels were restricted to a single, specific amplicon of expected size (data not shown). Amplification curves for dissection saline and no-RT controls fell below threshold lines (undetected) or exhibited Ct values far greater than the experimental samples (i.e. a difference of greater than 7 Cts or a reduction of more than 128-fold in starting material).

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