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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 9. Summary model of the effects of SK2 alternative splicing and Ca2+ signaling on the α9/10-nAChR-SK2 channel postsynaptic complex. The model indicates that SK2 channels and α9/10-nAChRs physically interact within a multi-molecular complex that includes the actin-binding protein α-actinin-1 and that Ca2+ modulates the interactions. SK2-ARK, compared with SK2, channels exhibit reduced binding to α-actinin-1, and increased association with α9/10-nAChRs. However, SK2-ARK surface membrane levels are lower than those of SK2. Interactions between α-actinin-1 and SK2 (A) and SK2-ARK (B) are similarly modulated by Ca2+ (enhanced, arrow) and Ca2+-CaM (reduced). Ca2+ decreases SK2 interactions with α9/10-nAChRs, possibly serving as a mechanism to regulate efferent inhibition. In comparison, Ca2+ does not alter or modestly enhances the SK2-ARK::α9/10-nAChR interactions (dotted arrow), suggesting that they are less prone to inhibition by Ca2+. Additional differences, such as Ca2+ sensitivity (see text), suggest that developmental increases in SK2-ARK relative levels may modulate synaptic activity in cochlear hair cells and neurons.
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Figure 9: Figure 9. Summary model of the effects of SK2 alternative splicing and Ca2+ signaling on the α9/10-nAChR-SK2 channel postsynaptic complex. The model indicates that SK2 channels and α9/10-nAChRs physically interact within a multi-molecular complex that includes the actin-binding protein α-actinin-1 and that Ca2+ modulates the interactions. SK2-ARK, compared with SK2, channels exhibit reduced binding to α-actinin-1, and increased association with α9/10-nAChRs. However, SK2-ARK surface membrane levels are lower than those of SK2. Interactions between α-actinin-1 and SK2 (A) and SK2-ARK (B) are similarly modulated by Ca2+ (enhanced, arrow) and Ca2+-CaM (reduced). Ca2+ decreases SK2 interactions with α9/10-nAChRs, possibly serving as a mechanism to regulate efferent inhibition. In comparison, Ca2+ does not alter or modestly enhances the SK2-ARK::α9/10-nAChR interactions (dotted arrow), suggesting that they are less prone to inhibition by Ca2+. Additional differences, such as Ca2+ sensitivity (see text), suggest that developmental increases in SK2-ARK relative levels may modulate synaptic activity in cochlear hair cells and neurons.

Mentions: Our findings provide novel insights into molecular interactions of SK2 channels and the differential properties of SK2 alternative splice variants. We show that SK2 co-precipitates with α9/10-nAChRs, using heterologous expression. This is the first demonstration, to our knowledge, of a physical association between these 2 key functional channels of olivocochlear postsynaptic sites. Further, we show direct binding of SK2 to the actin-binding protein α-actinin-1 in vitro and in vivo. We also find developmentally regulated expression of SK2 splice variants, in vivo, in the avian cochlea and the mammalian cortex and hippocampus, suggesting widespread significance of SK2 molecular heterogeneity. The SK2-ARK isoform has been identified previously in the avian cochlea,30 but changes induced by the insert have not been defined prior to our study. We demonstrate that the two isoforms differ in surface membrane levels, interactions with α9/10-nAChRs, Ca2+ sensitivity, and modulation of their molecular interactions by Ca2+ and calmodulin (Fig. 9). Based on these differences, we speculate that the 2 SK2 isoforms provide an unanticipated complexity that may serve to differentially modulate responses to synaptic activity in sensory hair cells and neurons.


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 9. Summary model of the effects of SK2 alternative splicing and Ca2+ signaling on the α9/10-nAChR-SK2 channel postsynaptic complex. The model indicates that SK2 channels and α9/10-nAChRs physically interact within a multi-molecular complex that includes the actin-binding protein α-actinin-1 and that Ca2+ modulates the interactions. SK2-ARK, compared with SK2, channels exhibit reduced binding to α-actinin-1, and increased association with α9/10-nAChRs. However, SK2-ARK surface membrane levels are lower than those of SK2. Interactions between α-actinin-1 and SK2 (A) and SK2-ARK (B) are similarly modulated by Ca2+ (enhanced, arrow) and Ca2+-CaM (reduced). Ca2+ decreases SK2 interactions with α9/10-nAChRs, possibly serving as a mechanism to regulate efferent inhibition. In comparison, Ca2+ does not alter or modestly enhances the SK2-ARK::α9/10-nAChR interactions (dotted arrow), suggesting that they are less prone to inhibition by Ca2+. Additional differences, such as Ca2+ sensitivity (see text), suggest that developmental increases in SK2-ARK relative levels may modulate synaptic activity in cochlear hair cells and neurons.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 9: Figure 9. Summary model of the effects of SK2 alternative splicing and Ca2+ signaling on the α9/10-nAChR-SK2 channel postsynaptic complex. The model indicates that SK2 channels and α9/10-nAChRs physically interact within a multi-molecular complex that includes the actin-binding protein α-actinin-1 and that Ca2+ modulates the interactions. SK2-ARK, compared with SK2, channels exhibit reduced binding to α-actinin-1, and increased association with α9/10-nAChRs. However, SK2-ARK surface membrane levels are lower than those of SK2. Interactions between α-actinin-1 and SK2 (A) and SK2-ARK (B) are similarly modulated by Ca2+ (enhanced, arrow) and Ca2+-CaM (reduced). Ca2+ decreases SK2 interactions with α9/10-nAChRs, possibly serving as a mechanism to regulate efferent inhibition. In comparison, Ca2+ does not alter or modestly enhances the SK2-ARK::α9/10-nAChR interactions (dotted arrow), suggesting that they are less prone to inhibition by Ca2+. Additional differences, such as Ca2+ sensitivity (see text), suggest that developmental increases in SK2-ARK relative levels may modulate synaptic activity in cochlear hair cells and neurons.
Mentions: Our findings provide novel insights into molecular interactions of SK2 channels and the differential properties of SK2 alternative splice variants. We show that SK2 co-precipitates with α9/10-nAChRs, using heterologous expression. This is the first demonstration, to our knowledge, of a physical association between these 2 key functional channels of olivocochlear postsynaptic sites. Further, we show direct binding of SK2 to the actin-binding protein α-actinin-1 in vitro and in vivo. We also find developmentally regulated expression of SK2 splice variants, in vivo, in the avian cochlea and the mammalian cortex and hippocampus, suggesting widespread significance of SK2 molecular heterogeneity. The SK2-ARK isoform has been identified previously in the avian cochlea,30 but changes induced by the insert have not been defined prior to our study. We demonstrate that the two isoforms differ in surface membrane levels, interactions with α9/10-nAChRs, Ca2+ sensitivity, and modulation of their molecular interactions by Ca2+ and calmodulin (Fig. 9). Based on these differences, we speculate that the 2 SK2 isoforms provide an unanticipated complexity that may serve to differentially modulate responses to synaptic activity in sensory hair cells and neurons.

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