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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 7. Ca2+ and CaM modulate interactions of SK2 and SK2-ARK with α-actinin-1. (A) Recombinant peptide binding assays showing effects of Ca2+ and CaM on binding of α-actinin-1 to SK2 and SK2-ARK. Purified recombinant CaM at the indicated concentrations was incubated with equal amounts of MBP-tagged SK2 or SK2-ARK C-terminus constructs and amylose beads in buffer containing 5mM BAPTA or 1mM CaCl2 prior to incubation with GST-tagged α-actinin-1. Immunoblots and graph (lower left) show that elevated Ca2+ (buffer containing 1mM CaCl2) increases the levels of GST-tagged α-actinin-1 bound to SK2 and SK2-ARK C-terminal peptides, compared with low Ca2+ (buffer containing 5mM BAPTA). Immunoblots and graph (lower right) show that CaM, in the presence of Ca2+, decreases the amount of α-actinin-1 bound to both isoformsbut only to SK2-ARK in the absence of Ca2+ (B) Immunoblots and graph show decreased co-precipitation of exogenous α-actinin-1 with SK2 and SK2-ARK from oocytes incubated with BAPTA-AM (10mM) to chelate intracellular Ca2+ prior to and during lysis. All graphs show normalized band densities of bound α-actinin-1-GST (A) or α-actinin-1 (B) normalized to MBP-tagged SK2 construct or SK2 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 ± SEM * 95% confidence interval was 59.15–98.29% of SK2 values with BAPTA. ** P < 0.0005 Students t test; † 99.99% confidence interval was 234.91–400.48% of SK2 values with BAPTA. *** 99.99% confidence interval was 55.21–90.01% of SK2-ARK values with 10 μM CaM; † 99.99% confidence intervals were 5.27–59.20% of SK2 values and 11.43–52.56% of SK2-ARK values with 10 μM CaM. n = 3 separate experiments (A and B).
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Figure 7: Figure 7. Ca2+ and CaM modulate interactions of SK2 and SK2-ARK with α-actinin-1. (A) Recombinant peptide binding assays showing effects of Ca2+ and CaM on binding of α-actinin-1 to SK2 and SK2-ARK. Purified recombinant CaM at the indicated concentrations was incubated with equal amounts of MBP-tagged SK2 or SK2-ARK C-terminus constructs and amylose beads in buffer containing 5mM BAPTA or 1mM CaCl2 prior to incubation with GST-tagged α-actinin-1. Immunoblots and graph (lower left) show that elevated Ca2+ (buffer containing 1mM CaCl2) increases the levels of GST-tagged α-actinin-1 bound to SK2 and SK2-ARK C-terminal peptides, compared with low Ca2+ (buffer containing 5mM BAPTA). Immunoblots and graph (lower right) show that CaM, in the presence of Ca2+, decreases the amount of α-actinin-1 bound to both isoformsbut only to SK2-ARK in the absence of Ca2+ (B) Immunoblots and graph show decreased co-precipitation of exogenous α-actinin-1 with SK2 and SK2-ARK from oocytes incubated with BAPTA-AM (10mM) to chelate intracellular Ca2+ prior to and during lysis. All graphs show normalized band densities of bound α-actinin-1-GST (A) or α-actinin-1 (B) normalized to MBP-tagged SK2 construct or SK2 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 ± SEM * 95% confidence interval was 59.15–98.29% of SK2 values with BAPTA. ** P < 0.0005 Students t test; † 99.99% confidence interval was 234.91–400.48% of SK2 values with BAPTA. *** 99.99% confidence interval was 55.21–90.01% of SK2-ARK values with 10 μM CaM; † 99.99% confidence intervals were 5.27–59.20% of SK2 values and 11.43–52.56% of SK2-ARK values with 10 μM CaM. n = 3 separate experiments (A and B).

Mentions: We tested first for effects of Ca2+ alone on the binding of α-actinin-1 to SK2 and SK2-ARK using recombinant peptide binding assays. Ca2+ strongly promoted α-actinin-1 binding; we found a 3.2-fold increase in binding of α-actinin-1 to SK2 and a 6.8-fold increase in binding to SK2-ARK in the presence of 1mM CaCl2, compared with 5mM BAPTA (Fig. 7A; compare lanes 1 and 3, 5 and 7 with no CaM; n = 3). Similarly, α-actinin-1 co-precipitation with SK2 was reduced by loading oocytes with BAPTA-AM to chelate intracellular Ca2+ prior to SK2 immunoprecipitation (Fig. 7B; 78.72 ± 9.98% compared with untreated controls; n = 3). These data suggest that increased intracellular Ca2+ levels likely strengthen α-actinin-1 interactions with both SK2 and SK2-ARK subunits. Such changes may occur upon olivocochlear synaptic activation in sensory 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 7. Ca2+ and CaM modulate interactions of SK2 and SK2-ARK with α-actinin-1. (A) Recombinant peptide binding assays showing effects of Ca2+ and CaM on binding of α-actinin-1 to SK2 and SK2-ARK. Purified recombinant CaM at the indicated concentrations was incubated with equal amounts of MBP-tagged SK2 or SK2-ARK C-terminus constructs and amylose beads in buffer containing 5mM BAPTA or 1mM CaCl2 prior to incubation with GST-tagged α-actinin-1. Immunoblots and graph (lower left) show that elevated Ca2+ (buffer containing 1mM CaCl2) increases the levels of GST-tagged α-actinin-1 bound to SK2 and SK2-ARK C-terminal peptides, compared with low Ca2+ (buffer containing 5mM BAPTA). Immunoblots and graph (lower right) show that CaM, in the presence of Ca2+, decreases the amount of α-actinin-1 bound to both isoformsbut only to SK2-ARK in the absence of Ca2+ (B) Immunoblots and graph show decreased co-precipitation of exogenous α-actinin-1 with SK2 and SK2-ARK from oocytes incubated with BAPTA-AM (10mM) to chelate intracellular Ca2+ prior to and during lysis. All graphs show normalized band densities of bound α-actinin-1-GST (A) or α-actinin-1 (B) normalized to MBP-tagged SK2 construct or SK2 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 ± SEM * 95% confidence interval was 59.15–98.29% of SK2 values with BAPTA. ** P < 0.0005 Students t test; † 99.99% confidence interval was 234.91–400.48% of SK2 values with BAPTA. *** 99.99% confidence interval was 55.21–90.01% of SK2-ARK values with 10 μM CaM; † 99.99% confidence intervals were 5.27–59.20% of SK2 values and 11.43–52.56% of SK2-ARK values with 10 μM CaM. n = 3 separate experiments (A and B).
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Figure 7: Figure 7. Ca2+ and CaM modulate interactions of SK2 and SK2-ARK with α-actinin-1. (A) Recombinant peptide binding assays showing effects of Ca2+ and CaM on binding of α-actinin-1 to SK2 and SK2-ARK. Purified recombinant CaM at the indicated concentrations was incubated with equal amounts of MBP-tagged SK2 or SK2-ARK C-terminus constructs and amylose beads in buffer containing 5mM BAPTA or 1mM CaCl2 prior to incubation with GST-tagged α-actinin-1. Immunoblots and graph (lower left) show that elevated Ca2+ (buffer containing 1mM CaCl2) increases the levels of GST-tagged α-actinin-1 bound to SK2 and SK2-ARK C-terminal peptides, compared with low Ca2+ (buffer containing 5mM BAPTA). Immunoblots and graph (lower right) show that CaM, in the presence of Ca2+, decreases the amount of α-actinin-1 bound to both isoformsbut only to SK2-ARK in the absence of Ca2+ (B) Immunoblots and graph show decreased co-precipitation of exogenous α-actinin-1 with SK2 and SK2-ARK from oocytes incubated with BAPTA-AM (10mM) to chelate intracellular Ca2+ prior to and during lysis. All graphs show normalized band densities of bound α-actinin-1-GST (A) or α-actinin-1 (B) normalized to MBP-tagged SK2 construct or SK2 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 ± SEM * 95% confidence interval was 59.15–98.29% of SK2 values with BAPTA. ** P < 0.0005 Students t test; † 99.99% confidence interval was 234.91–400.48% of SK2 values with BAPTA. *** 99.99% confidence interval was 55.21–90.01% of SK2-ARK values with 10 μM CaM; † 99.99% confidence intervals were 5.27–59.20% of SK2 values and 11.43–52.56% of SK2-ARK values with 10 μM CaM. n = 3 separate experiments (A and B).
Mentions: We tested first for effects of Ca2+ alone on the binding of α-actinin-1 to SK2 and SK2-ARK using recombinant peptide binding assays. Ca2+ strongly promoted α-actinin-1 binding; we found a 3.2-fold increase in binding of α-actinin-1 to SK2 and a 6.8-fold increase in binding to SK2-ARK in the presence of 1mM CaCl2, compared with 5mM BAPTA (Fig. 7A; compare lanes 1 and 3, 5 and 7 with no CaM; n = 3). Similarly, α-actinin-1 co-precipitation with SK2 was reduced by loading oocytes with BAPTA-AM to chelate intracellular Ca2+ prior to SK2 immunoprecipitation (Fig. 7B; 78.72 ± 9.98% compared with untreated controls; n = 3). These data suggest that increased intracellular Ca2+ levels likely strengthen α-actinin-1 interactions with both SK2 and SK2-ARK subunits. Such changes may occur upon olivocochlear synaptic activation in sensory 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