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Title
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Conserved negative charges in the N-terminal tetramerization domain mediate efficient assembly of Kv2.1 and Kv2.1/Kv6.4 channels
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Author
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Abstract
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| Voltage-gated potassium (Kv) channels are transmembrane tetramers of individual α-subunits. Eight different Shaker-related Kv subfamilies have been identified in which the tetramerization domain T1, located on the intracellular N terminus, facilitates and controls the assembly of both homo- and heterotetrameric channels. Only the Kv2 α-subunits are able to form heterotetramers with members of the silent Kv subfamilies (Kv5, Kv6, Kv8, and Kv9). The T1 domain contains two subdomains, A and B box, which presumably determine subfamily specificity by preventing incompatible subunits to assemble. In contrast, little is known about the involvement of the A/B linker sequence. Both Kv2 and silent Kv subfamilies contain a fully conserved and negatively charged sequence (CDD) in this linker that is lacking in the other subfamilies. Neutralizing these aspartates in Kv2.1 by mutating them to alanines did not affect the gating properties, but reduced the current density moderately. However, charge reversal arginine substitutions strongly reduced the current density of these homotetrameric mutant Kv2.1 channels and immunocytochemistry confirmed the reduced expression at the plasma membrane. Förster resonance energy transfer measurements using confocal microscopy showed that the latter was not due to impaired trafficking, but to a failure to assemble the tetramer. This was further confirmed with co-immunoprecipitation experiments. The corresponding arginine substitution in Kv6.4 prevented its heterotetrameric interaction with Kv2.1. These results indicate that these aspartates (especially the first one) in the A/B box linker of the T1 domain are required for efficient assembly of both homotetrameric Kv2.1 and heterotetrameric Kv2.1/silent Kv6.4 channels. |
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Language
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English
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Source (journal)
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Journal of biological chemistry. - Baltimore, Md
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Publication
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Baltimore, Md
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2009
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ISSN
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0021-9258
[print]
1083-351X
[online]
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DOI
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10.1074/JBC.M109.039479
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Volume/pages
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284
:6
(2009)
, p. 31625-31634
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ISI
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000271572700017
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Full text (Publisher's DOI)
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Full text (publisher's version - intranet only)
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