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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 6. Surface membrane levels of SK2, SK2-ARK, and α9/10-nAChRs. (A) Histogram of SK2 and SK2-ARK surface levels, normalized biotinylated band densities at time 0 (precipitation immediately following biotinylation). (B) Histogram of α9/10-nAChRs surface levels expressed alone, or co-expressed with SK2, or SK2-ARK, all with α-actinin-1, normalized band densities at time 0. (C) Graph of the remaining surface levels of biotinylated SK2 or SK2-ARK at indicated time points relative to levels at time 0, indicating their relative stability in the surface membrane. Graphs indicate band densities of precipitated SK2 or nAChRs normalized to input membrane expression. In each experiment, normalized levels of biotinylated SK2-ARK (A) or nAChRs co-expressed with SK2-ARK or no SK2 (B) were calculated as a percentage of biotinylated SK2 or nAChRs co-expressed with SK2 (100%). In each experiment (C), remaining levels of biotinylated SK2 or SK2-ARK were calculated as a percentage of biotinylated levels at time 0. Bars represent mean ± SEM * 99.5% confidence interval was 9.64–93.78% of co-expression with SK2. ** 99.99% confidence interval was -4.14–78.45% of co-expression with SK2. *** 99.99% confidence interval was -2.47–61.41% of SK2 values. n = 5 separate experiments (A) and 3–4 separate experiments (B and C).
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Figure 6: Figure 6. Surface membrane levels of SK2, SK2-ARK, and α9/10-nAChRs. (A) Histogram of SK2 and SK2-ARK surface levels, normalized biotinylated band densities at time 0 (precipitation immediately following biotinylation). (B) Histogram of α9/10-nAChRs surface levels expressed alone, or co-expressed with SK2, or SK2-ARK, all with α-actinin-1, normalized band densities at time 0. (C) Graph of the remaining surface levels of biotinylated SK2 or SK2-ARK at indicated time points relative to levels at time 0, indicating their relative stability in the surface membrane. Graphs indicate band densities of precipitated SK2 or nAChRs normalized to input membrane expression. In each experiment, normalized levels of biotinylated SK2-ARK (A) or nAChRs co-expressed with SK2-ARK or no SK2 (B) were calculated as a percentage of biotinylated SK2 or nAChRs co-expressed with SK2 (100%). In each experiment (C), remaining levels of biotinylated SK2 or SK2-ARK were calculated as a percentage of biotinylated levels at time 0. Bars represent mean ± SEM * 99.5% confidence interval was 9.64–93.78% of co-expression with SK2. ** 99.99% confidence interval was -4.14–78.45% of co-expression with SK2. *** 99.99% confidence interval was -2.47–61.41% of SK2 values. n = 5 separate experiments (A) and 3–4 separate experiments (B and C).

Mentions: We therefore tested for differences in surface membrane levels of α9/10-nAChRs co-expressed with either SK2-ARK or SK2 in oocytes. We used standard cell surface biotinylation assays with a membrane-impermeant biotinylation reagent. For each protein of interest, the levels on the surface (biotinylated) were normalized to their total expression levels. The two SK2 isoforms displayed similar total expression levels; however their surface levels differed. SK2-ARK surface levels were dramatically reduced, 29.47 ± 8.21% compared with SK2 levels (Fig. 6A; n = 5).


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 6. Surface membrane levels of SK2, SK2-ARK, and α9/10-nAChRs. (A) Histogram of SK2 and SK2-ARK surface levels, normalized biotinylated band densities at time 0 (precipitation immediately following biotinylation). (B) Histogram of α9/10-nAChRs surface levels expressed alone, or co-expressed with SK2, or SK2-ARK, all with α-actinin-1, normalized band densities at time 0. (C) Graph of the remaining surface levels of biotinylated SK2 or SK2-ARK at indicated time points relative to levels at time 0, indicating their relative stability in the surface membrane. Graphs indicate band densities of precipitated SK2 or nAChRs normalized to input membrane expression. In each experiment, normalized levels of biotinylated SK2-ARK (A) or nAChRs co-expressed with SK2-ARK or no SK2 (B) were calculated as a percentage of biotinylated SK2 or nAChRs co-expressed with SK2 (100%). In each experiment (C), remaining levels of biotinylated SK2 or SK2-ARK were calculated as a percentage of biotinylated levels at time 0. Bars represent mean ± SEM * 99.5% confidence interval was 9.64–93.78% of co-expression with SK2. ** 99.99% confidence interval was -4.14–78.45% of co-expression with SK2. *** 99.99% confidence interval was -2.47–61.41% of SK2 values. n = 5 separate experiments (A) and 3–4 separate experiments (B and C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Figure 6. Surface membrane levels of SK2, SK2-ARK, and α9/10-nAChRs. (A) Histogram of SK2 and SK2-ARK surface levels, normalized biotinylated band densities at time 0 (precipitation immediately following biotinylation). (B) Histogram of α9/10-nAChRs surface levels expressed alone, or co-expressed with SK2, or SK2-ARK, all with α-actinin-1, normalized band densities at time 0. (C) Graph of the remaining surface levels of biotinylated SK2 or SK2-ARK at indicated time points relative to levels at time 0, indicating their relative stability in the surface membrane. Graphs indicate band densities of precipitated SK2 or nAChRs normalized to input membrane expression. In each experiment, normalized levels of biotinylated SK2-ARK (A) or nAChRs co-expressed with SK2-ARK or no SK2 (B) were calculated as a percentage of biotinylated SK2 or nAChRs co-expressed with SK2 (100%). In each experiment (C), remaining levels of biotinylated SK2 or SK2-ARK were calculated as a percentage of biotinylated levels at time 0. Bars represent mean ± SEM * 99.5% confidence interval was 9.64–93.78% of co-expression with SK2. ** 99.99% confidence interval was -4.14–78.45% of co-expression with SK2. *** 99.99% confidence interval was -2.47–61.41% of SK2 values. n = 5 separate experiments (A) and 3–4 separate experiments (B and C).
Mentions: We therefore tested for differences in surface membrane levels of α9/10-nAChRs co-expressed with either SK2-ARK or SK2 in oocytes. We used standard cell surface biotinylation assays with a membrane-impermeant biotinylation reagent. For each protein of interest, the levels on the surface (biotinylated) were normalized to their total expression levels. The two SK2 isoforms displayed similar total expression levels; however their surface levels differed. SK2-ARK surface levels were dramatically reduced, 29.47 ± 8.21% compared with SK2 levels (Fig. 6A; n = 5).

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