Limits...
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.

Show MeSH

Related in: MedlinePlus

Figure 3. Increases in developmental expression of SK2-ARK splice variant in chicken cochlear hair cells in vivo. (A) Sequence alignments of the C-terminus of chicken (ch) splice variants, SK2 and SK2-ARK (24), mouse SK2 (GenBank accession number P58390) and SK2-ARK, trout SK2 (NP_001117783) and mouse SK1 (Q9EQR3). Numbers indicate amino acids of chicken SK2. Source: http://multalin.toulouse.inra.fr/multalin/. (B) ARK insertion (lower sequence) creates a unique restriction endonuclease site (Hpy188I) that is not present in chicken SK2 (upper sequence) that lacks the insert. (C) Quantification of relative abundance of SK2-ARK mRNA, compared with SK2, during chicken cochlear development, ranging from embryonic day (E)12–14 to E20. Identification of cDNA clones of SK2-ARK (lanes 1,3) and SK2 (lanes 2,4) by using Hpy188I restriction digestion and size separation of the products by agarose gel electrophoresis. The cDNAs were generated by RT-PCR amplification from cochleae total RNA with primers to conserved sequences that flank the ARK insertion site and subcloning (~150 clones analyzed in total per age). Bottom: quantification of the relative abundance of SK2-ARK transcript. n = 3 separate RT-PCR experiments with independent RNA extractions, 50–60 clones per age per experiment. (D) Graph of developmental increases in the relative abundance of SK2-ARK transcripts, detected by q-PCR, in the mammalian hippocampus (HC) and cortex (Ctx) from postnatal day 8 to 3 mo of age (adult). n = 6 animals per age, 3 separate q-PCR experiments with independent RNA extractions. Bars and data points in (C and D) represent mean ± SEM; * P < 0.01 Student t test compared with levels at E12–14.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048344&req=5

Figure 3: Figure 3. Increases in developmental expression of SK2-ARK splice variant in chicken cochlear hair cells in vivo. (A) Sequence alignments of the C-terminus of chicken (ch) splice variants, SK2 and SK2-ARK (24), mouse SK2 (GenBank accession number P58390) and SK2-ARK, trout SK2 (NP_001117783) and mouse SK1 (Q9EQR3). Numbers indicate amino acids of chicken SK2. Source: http://multalin.toulouse.inra.fr/multalin/. (B) ARK insertion (lower sequence) creates a unique restriction endonuclease site (Hpy188I) that is not present in chicken SK2 (upper sequence) that lacks the insert. (C) Quantification of relative abundance of SK2-ARK mRNA, compared with SK2, during chicken cochlear development, ranging from embryonic day (E)12–14 to E20. Identification of cDNA clones of SK2-ARK (lanes 1,3) and SK2 (lanes 2,4) by using Hpy188I restriction digestion and size separation of the products by agarose gel electrophoresis. The cDNAs were generated by RT-PCR amplification from cochleae total RNA with primers to conserved sequences that flank the ARK insertion site and subcloning (~150 clones analyzed in total per age). Bottom: quantification of the relative abundance of SK2-ARK transcript. n = 3 separate RT-PCR experiments with independent RNA extractions, 50–60 clones per age per experiment. (D) Graph of developmental increases in the relative abundance of SK2-ARK transcripts, detected by q-PCR, in the mammalian hippocampus (HC) and cortex (Ctx) from postnatal day 8 to 3 mo of age (adult). n = 6 animals per age, 3 separate q-PCR experiments with independent RNA extractions. Bars and data points in (C and D) represent mean ± SEM; * P < 0.01 Student t test compared with levels at E12–14.

Mentions: Previous studies of posthatch chicken short (outer) hair cells identified an SK2 splice variant, containing a 3-residue “ARK” insertion within the C terminus (Fig. 3A), referred to here as SK2-ARK.30 We and others have also detected the ARK splice insertion in mammalian SK2.32 The same insertion is constitutively present in trout SK2, and a similar AQK sequence is found in the same region in mammalian SK1 (Fig. 3A).30 Because the ARK insertion localizes to the domain that binds CaM, a protein that regulates SK2 surface expression and Ca2+-gating, we speculated that SK2 isoforms may exhibit differences in protein interactions and functional properties.


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 3. Increases in developmental expression of SK2-ARK splice variant in chicken cochlear hair cells in vivo. (A) Sequence alignments of the C-terminus of chicken (ch) splice variants, SK2 and SK2-ARK (24), mouse SK2 (GenBank accession number P58390) and SK2-ARK, trout SK2 (NP_001117783) and mouse SK1 (Q9EQR3). Numbers indicate amino acids of chicken SK2. Source: http://multalin.toulouse.inra.fr/multalin/. (B) ARK insertion (lower sequence) creates a unique restriction endonuclease site (Hpy188I) that is not present in chicken SK2 (upper sequence) that lacks the insert. (C) Quantification of relative abundance of SK2-ARK mRNA, compared with SK2, during chicken cochlear development, ranging from embryonic day (E)12–14 to E20. Identification of cDNA clones of SK2-ARK (lanes 1,3) and SK2 (lanes 2,4) by using Hpy188I restriction digestion and size separation of the products by agarose gel electrophoresis. The cDNAs were generated by RT-PCR amplification from cochleae total RNA with primers to conserved sequences that flank the ARK insertion site and subcloning (~150 clones analyzed in total per age). Bottom: quantification of the relative abundance of SK2-ARK transcript. n = 3 separate RT-PCR experiments with independent RNA extractions, 50–60 clones per age per experiment. (D) Graph of developmental increases in the relative abundance of SK2-ARK transcripts, detected by q-PCR, in the mammalian hippocampus (HC) and cortex (Ctx) from postnatal day 8 to 3 mo of age (adult). n = 6 animals per age, 3 separate q-PCR experiments with independent RNA extractions. Bars and data points in (C and D) represent mean ± SEM; * P < 0.01 Student t test compared with levels at E12–14.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Figure 3. Increases in developmental expression of SK2-ARK splice variant in chicken cochlear hair cells in vivo. (A) Sequence alignments of the C-terminus of chicken (ch) splice variants, SK2 and SK2-ARK (24), mouse SK2 (GenBank accession number P58390) and SK2-ARK, trout SK2 (NP_001117783) and mouse SK1 (Q9EQR3). Numbers indicate amino acids of chicken SK2. Source: http://multalin.toulouse.inra.fr/multalin/. (B) ARK insertion (lower sequence) creates a unique restriction endonuclease site (Hpy188I) that is not present in chicken SK2 (upper sequence) that lacks the insert. (C) Quantification of relative abundance of SK2-ARK mRNA, compared with SK2, during chicken cochlear development, ranging from embryonic day (E)12–14 to E20. Identification of cDNA clones of SK2-ARK (lanes 1,3) and SK2 (lanes 2,4) by using Hpy188I restriction digestion and size separation of the products by agarose gel electrophoresis. The cDNAs were generated by RT-PCR amplification from cochleae total RNA with primers to conserved sequences that flank the ARK insertion site and subcloning (~150 clones analyzed in total per age). Bottom: quantification of the relative abundance of SK2-ARK transcript. n = 3 separate RT-PCR experiments with independent RNA extractions, 50–60 clones per age per experiment. (D) Graph of developmental increases in the relative abundance of SK2-ARK transcripts, detected by q-PCR, in the mammalian hippocampus (HC) and cortex (Ctx) from postnatal day 8 to 3 mo of age (adult). n = 6 animals per age, 3 separate q-PCR experiments with independent RNA extractions. Bars and data points in (C and D) represent mean ± SEM; * P < 0.01 Student t test compared with levels at E12–14.
Mentions: Previous studies of posthatch chicken short (outer) hair cells identified an SK2 splice variant, containing a 3-residue “ARK” insertion within the C terminus (Fig. 3A), referred to here as SK2-ARK.30 We and others have also detected the ARK splice insertion in mammalian SK2.32 The same insertion is constitutively present in trout SK2, and a similar AQK sequence is found in the same region in mammalian SK1 (Fig. 3A).30 Because the ARK insertion localizes to the domain that binds CaM, a protein that regulates SK2 surface expression and Ca2+-gating, we speculated that SK2 isoforms may exhibit differences in protein interactions and functional properties.

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