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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.

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Figure 1. α-actinin localizes to olivocochlear postsynaptic sites in sensory hair cells and interacts with SK2 channels. (A–C) Micrographs of fluorescent immunolabeling of E19 chicken hair cells show that SK2 (red, A) and the postsynaptic scaffold protein S-SCAM (red, B) are predominantly concentrated at olivocochlear postsynaptic sites, based on juxtaposition and partial overlap (yellow) with SV2 labeled presynaptic terminal vesicle clusters (green, A and B). α-actinin (red, C) is also enriched at postsynaptic sites, based on co-localization and strong overlap with S-SCAM (green, C). Lower panels: Profiles of pixel intensity peaks show partial overlap of SK2 with SV2 (A) and S-SCAM with SV2 (B), and almost complete overlap of α-actinin with S-SCAM (C). Pixel intensities were measured along a line drawn vertically across the postsynaptic and presynaptic membranes (white lines shown in middle panels). Scale bar = 5μM. (D) Co-precipitation of SK2 channels with α-actinin-1 from E20 chicken cochlea lysates, detected by immunoprecipitating (IP) SK2 and immunoblotting (IB) with an anti-α-actinin-1 antibody. Co-IP was seen in the absence and, to a lesser extent, presence of BAPTA (5mM) to chelate Ca2+. In contrast, α-actinin-1 did not co-precipitate with anti-HA antibody, as a negative control. Input, 6% of total lysate. (E) Direct binding of recombinant GST-tagged α-actinin-1 with the MBP-tagged SK2 C-terminus (SK2-C). There are no non-specific interactions between SK2 or α-actinin-1 with only GST or MBP, respectively (lanes 2,3). IB; GST antibody. Inputs, 0.5% of total peptide used in IP (lane 4, α-actinin-1-GST; lane 5, GST alone). There is a lower band in lane 4 that is likely a degradation product that does not bind to SK2. (A–E) n = 3 separate experiments.
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Figure 1: Figure 1. α-actinin localizes to olivocochlear postsynaptic sites in sensory hair cells and interacts with SK2 channels. (A–C) Micrographs of fluorescent immunolabeling of E19 chicken hair cells show that SK2 (red, A) and the postsynaptic scaffold protein S-SCAM (red, B) are predominantly concentrated at olivocochlear postsynaptic sites, based on juxtaposition and partial overlap (yellow) with SV2 labeled presynaptic terminal vesicle clusters (green, A and B). α-actinin (red, C) is also enriched at postsynaptic sites, based on co-localization and strong overlap with S-SCAM (green, C). Lower panels: Profiles of pixel intensity peaks show partial overlap of SK2 with SV2 (A) and S-SCAM with SV2 (B), and almost complete overlap of α-actinin with S-SCAM (C). Pixel intensities were measured along a line drawn vertically across the postsynaptic and presynaptic membranes (white lines shown in middle panels). Scale bar = 5μM. (D) Co-precipitation of SK2 channels with α-actinin-1 from E20 chicken cochlea lysates, detected by immunoprecipitating (IP) SK2 and immunoblotting (IB) with an anti-α-actinin-1 antibody. Co-IP was seen in the absence and, to a lesser extent, presence of BAPTA (5mM) to chelate Ca2+. In contrast, α-actinin-1 did not co-precipitate with anti-HA antibody, as a negative control. Input, 6% of total lysate. (E) Direct binding of recombinant GST-tagged α-actinin-1 with the MBP-tagged SK2 C-terminus (SK2-C). There are no non-specific interactions between SK2 or α-actinin-1 with only GST or MBP, respectively (lanes 2,3). IB; GST antibody. Inputs, 0.5% of total peptide used in IP (lane 4, α-actinin-1-GST; lane 5, GST alone). There is a lower band in lane 4 that is likely a degradation product that does not bind to SK2. (A–E) n = 3 separate experiments.

Mentions: To begin to define mechanisms of SK2 channel localization and functional coupling to its local Ca2+ source in cochlear sensory hair cells, we sought to identify proteins that interact with SK2. In cardiac muscle, SK2 channels interact with α-actinin-2, which is necessary for surface membrane expression.23,28 To determine whether α-actinins may interact with SK2 channels in a distinct cell type, inner ear hair cells, we first identified α-actinin isoforms expressed in these cells. We performed reverse-transcription (RT)-PCR with specific primers to amplify α-actinin from total RNA isolated from E19 chicken basilar papillae (equivalent of the mammalian cochlea). The PCR primers were designed against conserved regions flanking the EF-hand region of α-actinin, which differs in each isoform. Sequencing indicated that the α-actinin isoform expressed in the basilar papilla is not α-actinin-2, a muscle-specific, non-Ca2+-sensitive isoform, but a Ca2+-sensitive non-muscle isoform. We tested whether α-actinin localizes at olivocochlear synapses in sensory hair cells in vivo, using a pan-specific α-actinin monoclonal antibody. SK2 is concentrated at these postsynaptic sites, as indicated by the juxtaposition of SK2 immunolabeled surface clusters to the large calyx-type olivocochlear presynaptic terminal, marked by SV2 synaptic vesicle staining (Fig. 1A). We were unable to directly demonstrate co-localization of SK2 with α-actinin because of the poor match between optimal fixation conditions for their immunostaining. Instead, we showed that α-actinin, like SK2, is enriched postsynaptically, as indicated by co-localization with the synapse specific cell adhesion molecule (S-SCAM) (Fig. 1C; yellow = overlap of the red and green double labeling). S-SCAM is a scaffold protein that is enriched at nicotinic postsynaptic sites in neurons31 and in sensory hair cells (Fig. 1B). These results demonstrate that both α-actinin and SK2 are concentrated postsynaptically at the basal synaptic pole of inner ear hair cells.


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 1. α-actinin localizes to olivocochlear postsynaptic sites in sensory hair cells and interacts with SK2 channels. (A–C) Micrographs of fluorescent immunolabeling of E19 chicken hair cells show that SK2 (red, A) and the postsynaptic scaffold protein S-SCAM (red, B) are predominantly concentrated at olivocochlear postsynaptic sites, based on juxtaposition and partial overlap (yellow) with SV2 labeled presynaptic terminal vesicle clusters (green, A and B). α-actinin (red, C) is also enriched at postsynaptic sites, based on co-localization and strong overlap with S-SCAM (green, C). Lower panels: Profiles of pixel intensity peaks show partial overlap of SK2 with SV2 (A) and S-SCAM with SV2 (B), and almost complete overlap of α-actinin with S-SCAM (C). Pixel intensities were measured along a line drawn vertically across the postsynaptic and presynaptic membranes (white lines shown in middle panels). Scale bar = 5μM. (D) Co-precipitation of SK2 channels with α-actinin-1 from E20 chicken cochlea lysates, detected by immunoprecipitating (IP) SK2 and immunoblotting (IB) with an anti-α-actinin-1 antibody. Co-IP was seen in the absence and, to a lesser extent, presence of BAPTA (5mM) to chelate Ca2+. In contrast, α-actinin-1 did not co-precipitate with anti-HA antibody, as a negative control. Input, 6% of total lysate. (E) Direct binding of recombinant GST-tagged α-actinin-1 with the MBP-tagged SK2 C-terminus (SK2-C). There are no non-specific interactions between SK2 or α-actinin-1 with only GST or MBP, respectively (lanes 2,3). IB; GST antibody. Inputs, 0.5% of total peptide used in IP (lane 4, α-actinin-1-GST; lane 5, GST alone). There is a lower band in lane 4 that is likely a degradation product that does not bind to SK2. (A–E) n = 3 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Figure 1. α-actinin localizes to olivocochlear postsynaptic sites in sensory hair cells and interacts with SK2 channels. (A–C) Micrographs of fluorescent immunolabeling of E19 chicken hair cells show that SK2 (red, A) and the postsynaptic scaffold protein S-SCAM (red, B) are predominantly concentrated at olivocochlear postsynaptic sites, based on juxtaposition and partial overlap (yellow) with SV2 labeled presynaptic terminal vesicle clusters (green, A and B). α-actinin (red, C) is also enriched at postsynaptic sites, based on co-localization and strong overlap with S-SCAM (green, C). Lower panels: Profiles of pixel intensity peaks show partial overlap of SK2 with SV2 (A) and S-SCAM with SV2 (B), and almost complete overlap of α-actinin with S-SCAM (C). Pixel intensities were measured along a line drawn vertically across the postsynaptic and presynaptic membranes (white lines shown in middle panels). Scale bar = 5μM. (D) Co-precipitation of SK2 channels with α-actinin-1 from E20 chicken cochlea lysates, detected by immunoprecipitating (IP) SK2 and immunoblotting (IB) with an anti-α-actinin-1 antibody. Co-IP was seen in the absence and, to a lesser extent, presence of BAPTA (5mM) to chelate Ca2+. In contrast, α-actinin-1 did not co-precipitate with anti-HA antibody, as a negative control. Input, 6% of total lysate. (E) Direct binding of recombinant GST-tagged α-actinin-1 with the MBP-tagged SK2 C-terminus (SK2-C). There are no non-specific interactions between SK2 or α-actinin-1 with only GST or MBP, respectively (lanes 2,3). IB; GST antibody. Inputs, 0.5% of total peptide used in IP (lane 4, α-actinin-1-GST; lane 5, GST alone). There is a lower band in lane 4 that is likely a degradation product that does not bind to SK2. (A–E) n = 3 separate experiments.
Mentions: To begin to define mechanisms of SK2 channel localization and functional coupling to its local Ca2+ source in cochlear sensory hair cells, we sought to identify proteins that interact with SK2. In cardiac muscle, SK2 channels interact with α-actinin-2, which is necessary for surface membrane expression.23,28 To determine whether α-actinins may interact with SK2 channels in a distinct cell type, inner ear hair cells, we first identified α-actinin isoforms expressed in these cells. We performed reverse-transcription (RT)-PCR with specific primers to amplify α-actinin from total RNA isolated from E19 chicken basilar papillae (equivalent of the mammalian cochlea). The PCR primers were designed against conserved regions flanking the EF-hand region of α-actinin, which differs in each isoform. Sequencing indicated that the α-actinin isoform expressed in the basilar papilla is not α-actinin-2, a muscle-specific, non-Ca2+-sensitive isoform, but a Ca2+-sensitive non-muscle isoform. We tested whether α-actinin localizes at olivocochlear synapses in sensory hair cells in vivo, using a pan-specific α-actinin monoclonal antibody. SK2 is concentrated at these postsynaptic sites, as indicated by the juxtaposition of SK2 immunolabeled surface clusters to the large calyx-type olivocochlear presynaptic terminal, marked by SV2 synaptic vesicle staining (Fig. 1A). We were unable to directly demonstrate co-localization of SK2 with α-actinin because of the poor match between optimal fixation conditions for their immunostaining. Instead, we showed that α-actinin, like SK2, is enriched postsynaptically, as indicated by co-localization with the synapse specific cell adhesion molecule (S-SCAM) (Fig. 1C; yellow = overlap of the red and green double labeling). S-SCAM is a scaffold protein that is enriched at nicotinic postsynaptic sites in neurons31 and in sensory hair cells (Fig. 1B). These results demonstrate that both α-actinin and SK2 are concentrated postsynaptically at the basal synaptic pole of inner ear hair cells.

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