Limits...
Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels.

Scholl ES, Pirone A, Cox DH, Duncan RK, Jacob MH - Channels (Austin) (2014)

Bottom Line: SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3' terminus, modulates these interactions.Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca(2+) influx.Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience; Tufts University Sackler School of Graduate Biomedical Sciences; Boston, MA USA.

ABSTRACT
Small conductance Ca(2+)-sensitive potassium (SK2) channels are voltage-independent, Ca(2+)-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca(2+) permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3' terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca(2+) and Ca(2+)-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca(2+) influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.

Show MeSH

Related in: MedlinePlus

Figure 5. ARK alternative splicing alters interactions of SK2 with α9/10-nAChRs and α-actinin-1. Co-immunoprecipitation of α -actinin-1 (A; see also Fig. 2A) and HA-tagged α9/10-nAChRs (C) with SK2 and SK2-ARK exogenously expressed in Xenopus oocytes. (A and C) Negative controls show no α-actinin-1 or α9/10-nAChR co-precipitation with an unrelated antibody (ctl lanes) or (C) with SK2 antibody from oocytes not transfected with exogeneous SK2. Input in (A and C), 6% of total membrane fraction (M) and lysate (tot). (B) Direct interactions of GST-tagged α -actinin-1 with MBP-tagged SK2 or SK2-ARK C-termini. IP: MBP antibody to pull down SK2, IB: GST antibody. Negative controls show little or no nonspecific interactions with MBP or GST alone (lanes 2,4,5). Input, 0.5% of GST-α-actinin-1 and GST used in pulldown. The lower band in lane 6 is likely a degradation product. (A–C) Graphs show normalized band densities of co-precipitated proteins relative to precipitated SK2 or MBP-tagged SK2 or SK2-ARK peptide in each lane. In each experiment, normalized protein levels co-precipitated with SK2-ARK were calculated as a percentage of co-precipitation with SK2 (100%). Bars represent mean percentage ± SEM * 95% confidence interval was 103.83–241.27% of SK2 values. ** 99.99% confidence interval was 70.00–92.50% of SK2 values. n = 4 separate experiments (A) and 3 separate experiments (B and C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048344&req=5

Figure 5: Figure 5. ARK alternative splicing alters interactions of SK2 with α9/10-nAChRs and α-actinin-1. Co-immunoprecipitation of α -actinin-1 (A; see also Fig. 2A) and HA-tagged α9/10-nAChRs (C) with SK2 and SK2-ARK exogenously expressed in Xenopus oocytes. (A and C) Negative controls show no α-actinin-1 or α9/10-nAChR co-precipitation with an unrelated antibody (ctl lanes) or (C) with SK2 antibody from oocytes not transfected with exogeneous SK2. Input in (A and C), 6% of total membrane fraction (M) and lysate (tot). (B) Direct interactions of GST-tagged α -actinin-1 with MBP-tagged SK2 or SK2-ARK C-termini. IP: MBP antibody to pull down SK2, IB: GST antibody. Negative controls show little or no nonspecific interactions with MBP or GST alone (lanes 2,4,5). Input, 0.5% of GST-α-actinin-1 and GST used in pulldown. The lower band in lane 6 is likely a degradation product. (A–C) Graphs show normalized band densities of co-precipitated proteins relative to precipitated SK2 or MBP-tagged SK2 or SK2-ARK peptide in each lane. In each experiment, normalized protein levels co-precipitated with SK2-ARK were calculated as a percentage of co-precipitation with SK2 (100%). Bars represent mean percentage ± SEM * 95% confidence interval was 103.83–241.27% of SK2 values. ** 99.99% confidence interval was 70.00–92.50% of SK2 values. n = 4 separate experiments (A) and 3 separate experiments (B and C).

Mentions: Proper function of olivocochlear synapses on hair cells requires close physical proximity (co-localization) and functional coupling of SK2 channels with α9/10-nAChRs,1,6 but a physical association has not been demonstrated to date. To test for their interaction, we utilized heterologous expression in Xenopus laevis oocytes, rather than the native proteins in hair cells, because of the lack of reliable antibodies that recognize α9- and α10-nAChR subunits. We epitope tagged the chicken α10-nAChR subunit C-terminus end with hemagglutinin (HA). Oocytes were microinjected with cRNA encoding α9, α10-HA, SK2, and α-actinin-1. SK2 channels were immunoprecipitated from membrane fractions isolated from oocytes three days after injection, the time determined experimentally to provide optimal expression levels. As a positive control, exogenously expressed SK2 co-precipitated with α-actinin-1 from oocyte membrane fractions (Fig. 2A), consistent with the co-precipitation of these endogenous proteins from cochlear lysates (Fig. 1D). Importantly, SK2 channels co-precipitated with HA-tagged α9/10-nAChRs (Fig. 2B). The interaction is specific, as SK2 did not co-precipitate with other membrane proteins, such as the endogenous sodium potassium ATPase (Fig. 2B). As an additional negative control, SK2 antibody did not pull down HA-tagged α9/10-nAChRs from oocytes not co-expressing exogenous SK2 (see Fig. 5A). This is the first demonstration, to our knowledge, of a physical association between SK2 and α9/10-nAChRs.


Alternative splice isoforms of small conductance calcium-activated SK2 channels differ in molecular interactions and surface levels.

Scholl ES, Pirone A, Cox DH, Duncan RK, Jacob MH - Channels (Austin) (2014)

Figure 5. ARK alternative splicing alters interactions of SK2 with α9/10-nAChRs and α-actinin-1. Co-immunoprecipitation of α -actinin-1 (A; see also Fig. 2A) and HA-tagged α9/10-nAChRs (C) with SK2 and SK2-ARK exogenously expressed in Xenopus oocytes. (A and C) Negative controls show no α-actinin-1 or α9/10-nAChR co-precipitation with an unrelated antibody (ctl lanes) or (C) with SK2 antibody from oocytes not transfected with exogeneous SK2. Input in (A and C), 6% of total membrane fraction (M) and lysate (tot). (B) Direct interactions of GST-tagged α -actinin-1 with MBP-tagged SK2 or SK2-ARK C-termini. IP: MBP antibody to pull down SK2, IB: GST antibody. Negative controls show little or no nonspecific interactions with MBP or GST alone (lanes 2,4,5). Input, 0.5% of GST-α-actinin-1 and GST used in pulldown. The lower band in lane 6 is likely a degradation product. (A–C) Graphs show normalized band densities of co-precipitated proteins relative to precipitated SK2 or MBP-tagged SK2 or SK2-ARK peptide in each lane. In each experiment, normalized protein levels co-precipitated with SK2-ARK were calculated as a percentage of co-precipitation with SK2 (100%). Bars represent mean percentage ± SEM * 95% confidence interval was 103.83–241.27% of SK2 values. ** 99.99% confidence interval was 70.00–92.50% of SK2 values. n = 4 separate experiments (A) and 3 separate experiments (B and C).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048344&req=5

Figure 5: Figure 5. ARK alternative splicing alters interactions of SK2 with α9/10-nAChRs and α-actinin-1. Co-immunoprecipitation of α -actinin-1 (A; see also Fig. 2A) and HA-tagged α9/10-nAChRs (C) with SK2 and SK2-ARK exogenously expressed in Xenopus oocytes. (A and C) Negative controls show no α-actinin-1 or α9/10-nAChR co-precipitation with an unrelated antibody (ctl lanes) or (C) with SK2 antibody from oocytes not transfected with exogeneous SK2. Input in (A and C), 6% of total membrane fraction (M) and lysate (tot). (B) Direct interactions of GST-tagged α -actinin-1 with MBP-tagged SK2 or SK2-ARK C-termini. IP: MBP antibody to pull down SK2, IB: GST antibody. Negative controls show little or no nonspecific interactions with MBP or GST alone (lanes 2,4,5). Input, 0.5% of GST-α-actinin-1 and GST used in pulldown. The lower band in lane 6 is likely a degradation product. (A–C) Graphs show normalized band densities of co-precipitated proteins relative to precipitated SK2 or MBP-tagged SK2 or SK2-ARK peptide in each lane. In each experiment, normalized protein levels co-precipitated with SK2-ARK were calculated as a percentage of co-precipitation with SK2 (100%). Bars represent mean percentage ± SEM * 95% confidence interval was 103.83–241.27% of SK2 values. ** 99.99% confidence interval was 70.00–92.50% of SK2 values. n = 4 separate experiments (A) and 3 separate experiments (B and C).
Mentions: Proper function of olivocochlear synapses on hair cells requires close physical proximity (co-localization) and functional coupling of SK2 channels with α9/10-nAChRs,1,6 but a physical association has not been demonstrated to date. To test for their interaction, we utilized heterologous expression in Xenopus laevis oocytes, rather than the native proteins in hair cells, because of the lack of reliable antibodies that recognize α9- and α10-nAChR subunits. We epitope tagged the chicken α10-nAChR subunit C-terminus end with hemagglutinin (HA). Oocytes were microinjected with cRNA encoding α9, α10-HA, SK2, and α-actinin-1. SK2 channels were immunoprecipitated from membrane fractions isolated from oocytes three days after injection, the time determined experimentally to provide optimal expression levels. As a positive control, exogenously expressed SK2 co-precipitated with α-actinin-1 from oocyte membrane fractions (Fig. 2A), consistent with the co-precipitation of these endogenous proteins from cochlear lysates (Fig. 1D). Importantly, SK2 channels co-precipitated with HA-tagged α9/10-nAChRs (Fig. 2B). The interaction is specific, as SK2 did not co-precipitate with other membrane proteins, such as the endogenous sodium potassium ATPase (Fig. 2B). As an additional negative control, SK2 antibody did not pull down HA-tagged α9/10-nAChRs from oocytes not co-expressing exogenous SK2 (see Fig. 5A). This is the first demonstration, to our knowledge, of a physical association between SK2 and α9/10-nAChRs.

Bottom Line: SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3' terminus, modulates these interactions.Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca(2+) influx.Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuroscience; Tufts University Sackler School of Graduate Biomedical Sciences; Boston, MA USA.

ABSTRACT
Small conductance Ca(2+)-sensitive potassium (SK2) channels are voltage-independent, Ca(2+)-activated ion channels that conduct potassium cations and thereby modulate the intrinsic excitability and synaptic transmission of neurons and sensory hair cells. In the cochlea, SK2 channels are functionally coupled to the highly Ca(2+) permeant α9/10-nicotinic acetylcholine receptors (nAChRs) at olivocochlear postsynaptic sites. SK2 activation leads to outer hair cell hyperpolarization and frequency-selective suppression of afferent sound transmission. These inhibitory responses are essential for normal regulation of sound sensitivity, frequency selectivity, and suppression of background noise. However, little is known about the molecular interactions of these key functional channels. Here we show that SK2 channels co-precipitate with α9/10-nAChRs and with the actin-binding protein α-actinin-1. SK2 alternative splicing, resulting in a 3 amino acid insertion in the intracellular 3' terminus, modulates these interactions. Further, relative abundance of the SK2 splice variants changes during developmental stages of synapse maturation in both the avian cochlea and the mammalian forebrain. Using heterologous cell expression to separately study the 2 distinct isoforms, we show that the variants differ in protein interactions and surface expression levels, and that Ca(2+) and Ca(2+)-bound calmodulin differentially regulate their protein interactions. Our findings suggest that the SK2 isoforms may be distinctly modulated by activity-induced Ca(2+) influx. Alternative splicing of SK2 may serve as a novel mechanism to differentially regulate the maturation and function of olivocochlear and neuronal synapses.

Show MeSH
Related in: MedlinePlus