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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 4. Ca2+ gating of SK2 and SK2-ARK channels. (A) Mean normalized Ca2+ response curves for SK2 (solid line) and SK2-ARK (dashed line) currents recorded in inside-out patches of Xenopus oocyte membranes. SK2 and SK2-ARK currents during 50mV voltage steps were recorded in different Ca2+ concentrations. Normalized curves were fitted with the Hill equation. * P < 5 x 10−7, Student t test; n = 13 for both SK2 and SK2-ARK. (B) Recombinant peptide binding assay shows direct binding of CaM to MBP-tagged SK2 and SK2-ARK C-termini in the presence of 5mM BAPTA or 1mM CaCl2. Input, 1% of CaM used in pulldown. Histograms show band densities of co-precipitated CaM normalized to SK2-C-MBP or SK2-ARK-C-MBP in each lane (detected with anti-MBP antibody). In each experiment, normalized levels of CaM co-precipitated with SK2-ARK-MBP were calculated as a percentage of CaM co-precipitation with SK2-MBP (100%). Bars represent mean percentage ± SEM ** 95% confidence interval was 103.73–179.95% of SK2 values. n = 3 separate experiments.
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Figure 4: Figure 4. Ca2+ gating of SK2 and SK2-ARK channels. (A) Mean normalized Ca2+ response curves for SK2 (solid line) and SK2-ARK (dashed line) currents recorded in inside-out patches of Xenopus oocyte membranes. SK2 and SK2-ARK currents during 50mV voltage steps were recorded in different Ca2+ concentrations. Normalized curves were fitted with the Hill equation. * P < 5 x 10−7, Student t test; n = 13 for both SK2 and SK2-ARK. (B) Recombinant peptide binding assay shows direct binding of CaM to MBP-tagged SK2 and SK2-ARK C-termini in the presence of 5mM BAPTA or 1mM CaCl2. Input, 1% of CaM used in pulldown. Histograms show band densities of co-precipitated CaM normalized to SK2-C-MBP or SK2-ARK-C-MBP in each lane (detected with anti-MBP antibody). In each experiment, normalized levels of CaM co-precipitated with SK2-ARK-MBP were calculated as a percentage of CaM co-precipitation with SK2-MBP (100%). Bars represent mean percentage ± SEM ** 95% confidence interval was 103.73–179.95% of SK2 values. n = 3 separate experiments.

Mentions: We tested for differential properties between SK2 and SK2-ARK in order to gain insights into the functional significance of the increased developmental expression of SK2-ARK. We utilized heterologous expression in Xenopus laevis oocytes because: (1) no reagents are available to distinguish between the two SK2 isoforms, which differ by only 3 amino acids and (2) exogenous SK2 forms functional channels on the oocyte surface.4 Since the ARK splice insertion is located within the Ca2+-dependent CaM binding domain of the SK2 C-terminus, we tested for effects on CaM-mediated Ca2+ gating. We used patch-clamp recording to look for differences in Ca2+ sensitivity between channels composed of SK2-ARK vs. SK2 subunits. SK2 and SK2-ARK isoforms were separately expressed in oocytes, and Ca2+ responses were recorded from inside-out patches to generate Ca2+ dose-response curves. Ca2+-activated potassium currents recorded from SK2-expressing patches had an apparent KD of 0.48 ± 0.0067 μM and a Hill coefficient of 4.90 ± 0.40 (n = 13). SK2-ARK channels demonstrated a steeper, right-shifted Ca2+ response curve caused by a significantly reduced normalized response to 0.5 μM Ca2+, with an increased apparent KD of 0.61 ± 0.019 μM and a Hill coefficient of 10.14 ± 1.84 (Fig. 4A, n = 13). These results demonstrate that the ARK insertion alters the response of the assembled channels to Ca2+, which could suggest altered interactions with CaM.


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 4. Ca2+ gating of SK2 and SK2-ARK channels. (A) Mean normalized Ca2+ response curves for SK2 (solid line) and SK2-ARK (dashed line) currents recorded in inside-out patches of Xenopus oocyte membranes. SK2 and SK2-ARK currents during 50mV voltage steps were recorded in different Ca2+ concentrations. Normalized curves were fitted with the Hill equation. * P < 5 x 10−7, Student t test; n = 13 for both SK2 and SK2-ARK. (B) Recombinant peptide binding assay shows direct binding of CaM to MBP-tagged SK2 and SK2-ARK C-termini in the presence of 5mM BAPTA or 1mM CaCl2. Input, 1% of CaM used in pulldown. Histograms show band densities of co-precipitated CaM normalized to SK2-C-MBP or SK2-ARK-C-MBP in each lane (detected with anti-MBP antibody). In each experiment, normalized levels of CaM co-precipitated with SK2-ARK-MBP were calculated as a percentage of CaM co-precipitation with SK2-MBP (100%). Bars represent mean percentage ± SEM ** 95% confidence interval was 103.73–179.95% of SK2 values. n = 3 separate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Figure 4. Ca2+ gating of SK2 and SK2-ARK channels. (A) Mean normalized Ca2+ response curves for SK2 (solid line) and SK2-ARK (dashed line) currents recorded in inside-out patches of Xenopus oocyte membranes. SK2 and SK2-ARK currents during 50mV voltage steps were recorded in different Ca2+ concentrations. Normalized curves were fitted with the Hill equation. * P < 5 x 10−7, Student t test; n = 13 for both SK2 and SK2-ARK. (B) Recombinant peptide binding assay shows direct binding of CaM to MBP-tagged SK2 and SK2-ARK C-termini in the presence of 5mM BAPTA or 1mM CaCl2. Input, 1% of CaM used in pulldown. Histograms show band densities of co-precipitated CaM normalized to SK2-C-MBP or SK2-ARK-C-MBP in each lane (detected with anti-MBP antibody). In each experiment, normalized levels of CaM co-precipitated with SK2-ARK-MBP were calculated as a percentage of CaM co-precipitation with SK2-MBP (100%). Bars represent mean percentage ± SEM ** 95% confidence interval was 103.73–179.95% of SK2 values. n = 3 separate experiments.
Mentions: We tested for differential properties between SK2 and SK2-ARK in order to gain insights into the functional significance of the increased developmental expression of SK2-ARK. We utilized heterologous expression in Xenopus laevis oocytes because: (1) no reagents are available to distinguish between the two SK2 isoforms, which differ by only 3 amino acids and (2) exogenous SK2 forms functional channels on the oocyte surface.4 Since the ARK splice insertion is located within the Ca2+-dependent CaM binding domain of the SK2 C-terminus, we tested for effects on CaM-mediated Ca2+ gating. We used patch-clamp recording to look for differences in Ca2+ sensitivity between channels composed of SK2-ARK vs. SK2 subunits. SK2 and SK2-ARK isoforms were separately expressed in oocytes, and Ca2+ responses were recorded from inside-out patches to generate Ca2+ dose-response curves. Ca2+-activated potassium currents recorded from SK2-expressing patches had an apparent KD of 0.48 ± 0.0067 μM and a Hill coefficient of 4.90 ± 0.40 (n = 13). SK2-ARK channels demonstrated a steeper, right-shifted Ca2+ response curve caused by a significantly reduced normalized response to 0.5 μM Ca2+, with an increased apparent KD of 0.61 ± 0.019 μM and a Hill coefficient of 10.14 ± 1.84 (Fig. 4A, n = 13). These results demonstrate that the ARK insertion alters the response of the assembled channels to Ca2+, which could suggest altered interactions with CaM.

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