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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 8. Ca2+ differentially modulates interactions of SK2 and SK2-ARK with α9/10-nAChRs. Immunoblots show that incubation with BAPTA-AM (10mM) to chelate intracellular Ca2+ leads to increased co-precipitation of HA-tagged α9/10-nAChRs with SK2, but not with SK2-ARK, from oocytes. Graph shows normalized band densities of co-precipitated α9/10-nAChRs relative to precipitated SK2 in each lane. In each experiment, normalized nAChR levels co-precipitated with SK2-ARK were calculated as a percentage of co-precipitation with SK2 (100%). Bars represent mean ± SEM * 99.99% confidence interval was 109.34–290.84% of control SK2 values. n = 4 separate experiments.
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Figure 8: Figure 8. Ca2+ differentially modulates interactions of SK2 and SK2-ARK with α9/10-nAChRs. Immunoblots show that incubation with BAPTA-AM (10mM) to chelate intracellular Ca2+ leads to increased co-precipitation of HA-tagged α9/10-nAChRs with SK2, but not with SK2-ARK, from oocytes. Graph shows normalized band densities of co-precipitated α9/10-nAChRs relative to precipitated SK2 in each lane. In each experiment, normalized nAChR levels co-precipitated with SK2-ARK were calculated as a percentage of co-precipitation with SK2 (100%). Bars represent mean ± SEM * 99.99% confidence interval was 109.34–290.84% of control SK2 values. n = 4 separate experiments.

Mentions: Interestingly, SK2 and SK2-ARK showed different effects of Ca2+ chelation on interactions with α9/10-nAChRs. Co-precipitation of α9/10-nAChRs with SK2 from oocytes increased 2-fold after treating with BAPTA-AM (Fig. 8; n = 4), suggesting that Ca2+ influx may reduce linkage of SK2 to α9/10-nAChRs. In contrast, co-precipitation of α9/10-nAChRs with SK2-ARK was not significantly altered in response to BAPTA-AM treatment, suggesting that Ca2+ does not inhibit the association of α9/10-nAChRs with SK2-ARK. These findings suggest that the isoform composition of SK2 channels may provide a novel mechanism for modulating protein interactions at the SK2 postsynaptic complex in response to synaptic activity-induced increases in local Ca2+ and Ca2+-CaM binding.


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 8. Ca2+ differentially modulates interactions of SK2 and SK2-ARK with α9/10-nAChRs. Immunoblots show that incubation with BAPTA-AM (10mM) to chelate intracellular Ca2+ leads to increased co-precipitation of HA-tagged α9/10-nAChRs with SK2, but not with SK2-ARK, from oocytes. Graph shows normalized band densities of co-precipitated α9/10-nAChRs relative to precipitated SK2 in each lane. In each experiment, normalized nAChR levels co-precipitated with SK2-ARK were calculated as a percentage of co-precipitation with SK2 (100%). Bars represent mean ± SEM * 99.99% confidence interval was 109.34–290.84% of control SK2 values. n = 4 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 8: Figure 8. Ca2+ differentially modulates interactions of SK2 and SK2-ARK with α9/10-nAChRs. Immunoblots show that incubation with BAPTA-AM (10mM) to chelate intracellular Ca2+ leads to increased co-precipitation of HA-tagged α9/10-nAChRs with SK2, but not with SK2-ARK, from oocytes. Graph shows normalized band densities of co-precipitated α9/10-nAChRs relative to precipitated SK2 in each lane. In each experiment, normalized nAChR levels co-precipitated with SK2-ARK were calculated as a percentage of co-precipitation with SK2 (100%). Bars represent mean ± SEM * 99.99% confidence interval was 109.34–290.84% of control SK2 values. n = 4 separate experiments.
Mentions: Interestingly, SK2 and SK2-ARK showed different effects of Ca2+ chelation on interactions with α9/10-nAChRs. Co-precipitation of α9/10-nAChRs with SK2 from oocytes increased 2-fold after treating with BAPTA-AM (Fig. 8; n = 4), suggesting that Ca2+ influx may reduce linkage of SK2 to α9/10-nAChRs. In contrast, co-precipitation of α9/10-nAChRs with SK2-ARK was not significantly altered in response to BAPTA-AM treatment, suggesting that Ca2+ does not inhibit the association of α9/10-nAChRs with SK2-ARK. These findings suggest that the isoform composition of SK2 channels may provide a novel mechanism for modulating protein interactions at the SK2 postsynaptic complex in response to synaptic activity-induced increases in local Ca2+ and Ca2+-CaM binding.

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