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Splice variants of Na(V)1.7 sodium channels have distinct β subunit-dependent biophysical properties.

Farmer C, Cox JJ, Fletcher EV, Woods CG, Wood JN, Schorge S - PLoS ONE (2012)

Bottom Line: In conditions where the intrinsic properties of the Na(V)1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit.The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version.Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.

View Article: PubMed Central - PubMed

Affiliation: UCL Institute of Neurology, Queen Square, London, United Kingdom.

ABSTRACT
Genes encoding the α subunits of neuronal sodium channels have evolutionarily conserved sites of alternative splicing but no functional differences have been attributed to the splice variants. Here, using Na(V)1.7 as an exemplar, we show that the sodium channel isoforms are functionally distinct when co-expressed with β subunits. The gene, SCN9A, encodes the α subunit of the Na(V)1.7 channel, and contains both sites of alternative splicing that are highly conserved. In conditions where the intrinsic properties of the Na(V)1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit. While the identity of exon 5 determined the degree to which β1 subunits altered voltage-dependence of activation (P = 0.027), the length of exon 11 regulated how far β1 subunits depolarised voltage-dependence of inactivation (P = 0.00012). The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version. Our data suggest that splicing can change the way that Na(V) channels interact with β subunits. Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.

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β1 subunits have different effects on current densities depending on splicing in SCN9A.(A) Un-normalised current density plots showing increased current due to expression of β1 subunits. Triangles  =  exon 5A, Squares  =  exon 5N. Grey  =  with β1 subunits. To assess the impact of the β subunits we held cells at physiological potentials (−80 mV) and stepped to a range of potentials (−80 to +70 mV). (B) Consequences of β1 subunit co-expression for steps to different potentials. The same data are shown in top and bottom panels, but in the top panel the greyed-out triangular area indicates the difference in current amplitude between variants that differ at exon 5, while in the bottom panel the shaded areas indicate the difference introduced by changing the length of exon 11. In contrast to the changes due to exon 5, which are much larger at −20 mV than at most other potentials, the changes imposed by exon 11 are of similar amplitude over the range of potentials tested. Exon 11 length (bottom panel) is associated with approximately a two fold increase of current density over all voltages. The identity of exon 5 induces a specific increase (∼2 fold) in the neighbourhood of −20 mV (top panel), but virtually no difference at more depolarised potentials. Cell numbers are as in Table 2.
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pone-0041750-g003: β1 subunits have different effects on current densities depending on splicing in SCN9A.(A) Un-normalised current density plots showing increased current due to expression of β1 subunits. Triangles  =  exon 5A, Squares  =  exon 5N. Grey  =  with β1 subunits. To assess the impact of the β subunits we held cells at physiological potentials (−80 mV) and stepped to a range of potentials (−80 to +70 mV). (B) Consequences of β1 subunit co-expression for steps to different potentials. The same data are shown in top and bottom panels, but in the top panel the greyed-out triangular area indicates the difference in current amplitude between variants that differ at exon 5, while in the bottom panel the shaded areas indicate the difference introduced by changing the length of exon 11. In contrast to the changes due to exon 5, which are much larger at −20 mV than at most other potentials, the changes imposed by exon 11 are of similar amplitude over the range of potentials tested. Exon 11 length (bottom panel) is associated with approximately a two fold increase of current density over all voltages. The identity of exon 5 induces a specific increase (∼2 fold) in the neighbourhood of −20 mV (top panel), but virtually no difference at more depolarised potentials. Cell numbers are as in Table 2.

Mentions: The shift in inactivation introduced by β1 subunits in the presence of exon 11 L introduced potentially important changes in channel availability near typical resting membrane potentials. For example, variant 5N11L changes from more than 60% inactivated at −80 mV in the absence of β1 subunits to less than 20% inactive when β1 is co-expressed, indicating that the presence of β1 alters the availability of nearly half the channels at physiological resting potentials. However a direct prediction of channel availability during voltage steps may be confounded by a changing voltage dependence of activation, such as that caused by splicing of exon 5, or by altered trafficking in the presence of β1 subunits. We therefore measured the current densities generated by the different splice variants over a range of potentials in the presence and absence of β1 subunits when cells were held near physiological resting potential (Figure 3).


Splice variants of Na(V)1.7 sodium channels have distinct β subunit-dependent biophysical properties.

Farmer C, Cox JJ, Fletcher EV, Woods CG, Wood JN, Schorge S - PLoS ONE (2012)

β1 subunits have different effects on current densities depending on splicing in SCN9A.(A) Un-normalised current density plots showing increased current due to expression of β1 subunits. Triangles  =  exon 5A, Squares  =  exon 5N. Grey  =  with β1 subunits. To assess the impact of the β subunits we held cells at physiological potentials (−80 mV) and stepped to a range of potentials (−80 to +70 mV). (B) Consequences of β1 subunit co-expression for steps to different potentials. The same data are shown in top and bottom panels, but in the top panel the greyed-out triangular area indicates the difference in current amplitude between variants that differ at exon 5, while in the bottom panel the shaded areas indicate the difference introduced by changing the length of exon 11. In contrast to the changes due to exon 5, which are much larger at −20 mV than at most other potentials, the changes imposed by exon 11 are of similar amplitude over the range of potentials tested. Exon 11 length (bottom panel) is associated with approximately a two fold increase of current density over all voltages. The identity of exon 5 induces a specific increase (∼2 fold) in the neighbourhood of −20 mV (top panel), but virtually no difference at more depolarised potentials. Cell numbers are as in Table 2.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3404004&req=5

pone-0041750-g003: β1 subunits have different effects on current densities depending on splicing in SCN9A.(A) Un-normalised current density plots showing increased current due to expression of β1 subunits. Triangles  =  exon 5A, Squares  =  exon 5N. Grey  =  with β1 subunits. To assess the impact of the β subunits we held cells at physiological potentials (−80 mV) and stepped to a range of potentials (−80 to +70 mV). (B) Consequences of β1 subunit co-expression for steps to different potentials. The same data are shown in top and bottom panels, but in the top panel the greyed-out triangular area indicates the difference in current amplitude between variants that differ at exon 5, while in the bottom panel the shaded areas indicate the difference introduced by changing the length of exon 11. In contrast to the changes due to exon 5, which are much larger at −20 mV than at most other potentials, the changes imposed by exon 11 are of similar amplitude over the range of potentials tested. Exon 11 length (bottom panel) is associated with approximately a two fold increase of current density over all voltages. The identity of exon 5 induces a specific increase (∼2 fold) in the neighbourhood of −20 mV (top panel), but virtually no difference at more depolarised potentials. Cell numbers are as in Table 2.
Mentions: The shift in inactivation introduced by β1 subunits in the presence of exon 11 L introduced potentially important changes in channel availability near typical resting membrane potentials. For example, variant 5N11L changes from more than 60% inactivated at −80 mV in the absence of β1 subunits to less than 20% inactive when β1 is co-expressed, indicating that the presence of β1 alters the availability of nearly half the channels at physiological resting potentials. However a direct prediction of channel availability during voltage steps may be confounded by a changing voltage dependence of activation, such as that caused by splicing of exon 5, or by altered trafficking in the presence of β1 subunits. We therefore measured the current densities generated by the different splice variants over a range of potentials in the presence and absence of β1 subunits when cells were held near physiological resting potential (Figure 3).

Bottom Line: In conditions where the intrinsic properties of the Na(V)1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit.The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version.Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.

View Article: PubMed Central - PubMed

Affiliation: UCL Institute of Neurology, Queen Square, London, United Kingdom.

ABSTRACT
Genes encoding the α subunits of neuronal sodium channels have evolutionarily conserved sites of alternative splicing but no functional differences have been attributed to the splice variants. Here, using Na(V)1.7 as an exemplar, we show that the sodium channel isoforms are functionally distinct when co-expressed with β subunits. The gene, SCN9A, encodes the α subunit of the Na(V)1.7 channel, and contains both sites of alternative splicing that are highly conserved. In conditions where the intrinsic properties of the Na(V)1.7 splice variants were similar when expressed alone, co-expression of β1 subunits had different effects on channel availability that were determined by splicing at either site in the α subunit. While the identity of exon 5 determined the degree to which β1 subunits altered voltage-dependence of activation (P = 0.027), the length of exon 11 regulated how far β1 subunits depolarised voltage-dependence of inactivation (P = 0.00012). The results could have a significant impact on channel availability, for example with the long version of exon 11, the co-expression of β1 subunits could lead to nearly twice as large an increase in channel availability compared to channels containing the short version. Our data suggest that splicing can change the way that Na(V) channels interact with β subunits. Because splicing is conserved, its unexpected role in regulating the functional impact of β subunits may apply to multiple voltage-gated sodium channels, and the full repertoire of β subunit function may depend on splicing in α subunits.

Show MeSH