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Auxiliary KCNE subunits modulate both homotetrameric Kv2.1 and heterotetrameric Kv2.1/Kv6.4 channels.

David JP, Stas JI, Schmitt N, Bocksteins E - Sci Rep (2015)

Bottom Line: Co-expression of KCNE5 with Kv2.1 and Kv6.4 did not alter the Kv2.1/Kv6.4 current density but modulated the biophysical properties significantly; KCNE5 accelerated the activation, slowed the deactivation and steepened the slope of the voltage-dependence of the Kv2.1/Kv6.4 inactivation by accelerating recovery of the closed-state inactivation.In contrast, KCNE5 reduced the current density ~2-fold without affecting the biophysical properties of Kv2.1 homotetramers.These results suggest that a triple complex consisting of Kv2.1, Kv6.4 and KCNE5 subunits can be formed.

View Article: PubMed Central - PubMed

Affiliation: Danish National Research Foundation Centre for Cardiac Arrhythmia and Department for Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
The diversity of the voltage-gated K(+) (Kv) channel subfamily Kv2 is increased by interactions with auxiliary β-subunits and by assembly with members of the modulatory so-called silent Kv subfamilies (Kv5-Kv6 and Kv8-Kv9). However, it has not yet been investigated whether these two types of modulating subunits can associate within and modify a single channel complex simultaneously. Here, we demonstrate that the transmembrane β-subunit KCNE5 modifies the Kv2.1/Kv6.4 current extensively, whereas KCNE2 and KCNE4 only exert minor effects. Co-expression of KCNE5 with Kv2.1 and Kv6.4 did not alter the Kv2.1/Kv6.4 current density but modulated the biophysical properties significantly; KCNE5 accelerated the activation, slowed the deactivation and steepened the slope of the voltage-dependence of the Kv2.1/Kv6.4 inactivation by accelerating recovery of the closed-state inactivation. In contrast, KCNE5 reduced the current density ~2-fold without affecting the biophysical properties of Kv2.1 homotetramers. Co-localization of Kv2.1, Kv6.4 and KCNE5 was demonstrated with immunocytochemistry and formation of Kv2.1/Kv6.4/KCNE5 and Kv2.1/KCNE5 complexes was confirmed by Fluorescence Resonance Energy Transfer experiments performed in HEK293 cells. These results suggest that a triple complex consisting of Kv2.1, Kv6.4 and KCNE5 subunits can be formed. In vivo, formation of such tripartite Kv2.1/Kv6.4/KCNE5 channel complexes might contribute to tissue-specific fine-tuning of excitability.

No MeSH data available.


Related in: MedlinePlus

KCNE5 associates with both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.Average FRET efficiencies determined after co-expression of CFP- and YFP- labeled subunits. CFP-Kv6.4 + YFP-Kv2.1 and CFP + YFP combinations represent the positive and negative control, respectively. Note the increased FRET efficiency after co-expression of CFP-Kv6.4 with YFP-KCNE5 and untagged Kv2.1 compared to the CFP-Kv6.4 + YFP-KCNE5 co-expression; n = number of cells; *p < 0.05.
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f7: KCNE5 associates with both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.Average FRET efficiencies determined after co-expression of CFP- and YFP- labeled subunits. CFP-Kv6.4 + YFP-Kv2.1 and CFP + YFP combinations represent the positive and negative control, respectively. Note the increased FRET efficiency after co-expression of CFP-Kv6.4 with YFP-KCNE5 and untagged Kv2.1 compared to the CFP-Kv6.4 + YFP-KCNE5 co-expression; n = number of cells; *p < 0.05.

Mentions: To address whether KCNE5 subunits physically associate with Kv2.1 and Kv6.4 within Kv2.1/KCNE5 and Kv2.1/Kv6.4/KCNE5 channel complexes we determined the Fluorescence Resonance Energy Transfer (FRET) efficiency between N-terminally CFP-labeled Kv2.1 and Kv6.4 and C-terminally YFP-labeled KCNE5 (Fig. 7). The FRET efficiency of CFP-Kv6.4 + YFP-Kv2.1 (positive control) and CFP + YFP (negative control) combinations yielded FRET efficiencies of 15.7 ± 1.1% and 1.8 ± 1.0%, respectively. Upon co-expression of KCNE5-YFP with CFP-Kv2.1, we obtained a FRET efficiency of 9.7 ± 0.9% which is lower than the FRET efficiency of the positive control but significantly higher than the FRET efficiency of the negative CFP+YFP combination. This indicated that KCNE5 physically associates with Kv2.1 subunits. Co-expression of KCNE5-YFP with CFP-Kv6.4 yielded a FRET efficiency of 2.4 ± 1.0%. This FRET efficiency was similar to the negative control indicating that KCNE5 does not associate with Kv6.4 subunits alone. These results suggest that KCNE5 subunits modulate the Kv2.1/Kv6.4 heterotetramers via association with Kv2.1 subunits. To investigate this further, we performed FRET experiments co-expressing unlabeled Kv2.1 subunits; an increased FRET efficiency between CFP-Kv6.4 and KCNE5-YFP subunits upon co-expression with unlabeled Kv2.1 would indicate that Kv6.4 and KCNE5 are in each other’s proximity caused by an association with the Kv2.1 subunits into a tripartite complex. Indeed, co-expression of CFP-Kv6.4 with KCNE5-YFP and unlabeled Kv2.1 yielded a FRET efficiency of 6.0 ± 0.9% which was significantly higher than the FRET efficiency obtained with the CFP-Kv6.4 + KCNE5-YFP combination. In addition, this FRET efficiency was similar to the FRET efficiency between CFP-Kv2.1 and KCNE5-YFP upon co-expression with unlabeled Kv6.4 subunits (5.9 ± 1.7%). To ensure that these observed FRET efficiencies were originating from the formed (tripartite) channel complexes (rather than from non-specific interactions caused by the presence of the different tags), we confirmed that the present tags do not affect the biophysical properties of the channels (Suppl. Fig. S6).


Auxiliary KCNE subunits modulate both homotetrameric Kv2.1 and heterotetrameric Kv2.1/Kv6.4 channels.

David JP, Stas JI, Schmitt N, Bocksteins E - Sci Rep (2015)

KCNE5 associates with both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.Average FRET efficiencies determined after co-expression of CFP- and YFP- labeled subunits. CFP-Kv6.4 + YFP-Kv2.1 and CFP + YFP combinations represent the positive and negative control, respectively. Note the increased FRET efficiency after co-expression of CFP-Kv6.4 with YFP-KCNE5 and untagged Kv2.1 compared to the CFP-Kv6.4 + YFP-KCNE5 co-expression; n = number of cells; *p < 0.05.
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Related In: Results  -  Collection

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Show All Figures
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f7: KCNE5 associates with both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.Average FRET efficiencies determined after co-expression of CFP- and YFP- labeled subunits. CFP-Kv6.4 + YFP-Kv2.1 and CFP + YFP combinations represent the positive and negative control, respectively. Note the increased FRET efficiency after co-expression of CFP-Kv6.4 with YFP-KCNE5 and untagged Kv2.1 compared to the CFP-Kv6.4 + YFP-KCNE5 co-expression; n = number of cells; *p < 0.05.
Mentions: To address whether KCNE5 subunits physically associate with Kv2.1 and Kv6.4 within Kv2.1/KCNE5 and Kv2.1/Kv6.4/KCNE5 channel complexes we determined the Fluorescence Resonance Energy Transfer (FRET) efficiency between N-terminally CFP-labeled Kv2.1 and Kv6.4 and C-terminally YFP-labeled KCNE5 (Fig. 7). The FRET efficiency of CFP-Kv6.4 + YFP-Kv2.1 (positive control) and CFP + YFP (negative control) combinations yielded FRET efficiencies of 15.7 ± 1.1% and 1.8 ± 1.0%, respectively. Upon co-expression of KCNE5-YFP with CFP-Kv2.1, we obtained a FRET efficiency of 9.7 ± 0.9% which is lower than the FRET efficiency of the positive control but significantly higher than the FRET efficiency of the negative CFP+YFP combination. This indicated that KCNE5 physically associates with Kv2.1 subunits. Co-expression of KCNE5-YFP with CFP-Kv6.4 yielded a FRET efficiency of 2.4 ± 1.0%. This FRET efficiency was similar to the negative control indicating that KCNE5 does not associate with Kv6.4 subunits alone. These results suggest that KCNE5 subunits modulate the Kv2.1/Kv6.4 heterotetramers via association with Kv2.1 subunits. To investigate this further, we performed FRET experiments co-expressing unlabeled Kv2.1 subunits; an increased FRET efficiency between CFP-Kv6.4 and KCNE5-YFP subunits upon co-expression with unlabeled Kv2.1 would indicate that Kv6.4 and KCNE5 are in each other’s proximity caused by an association with the Kv2.1 subunits into a tripartite complex. Indeed, co-expression of CFP-Kv6.4 with KCNE5-YFP and unlabeled Kv2.1 yielded a FRET efficiency of 6.0 ± 0.9% which was significantly higher than the FRET efficiency obtained with the CFP-Kv6.4 + KCNE5-YFP combination. In addition, this FRET efficiency was similar to the FRET efficiency between CFP-Kv2.1 and KCNE5-YFP upon co-expression with unlabeled Kv6.4 subunits (5.9 ± 1.7%). To ensure that these observed FRET efficiencies were originating from the formed (tripartite) channel complexes (rather than from non-specific interactions caused by the presence of the different tags), we confirmed that the present tags do not affect the biophysical properties of the channels (Suppl. Fig. S6).

Bottom Line: Co-expression of KCNE5 with Kv2.1 and Kv6.4 did not alter the Kv2.1/Kv6.4 current density but modulated the biophysical properties significantly; KCNE5 accelerated the activation, slowed the deactivation and steepened the slope of the voltage-dependence of the Kv2.1/Kv6.4 inactivation by accelerating recovery of the closed-state inactivation.In contrast, KCNE5 reduced the current density ~2-fold without affecting the biophysical properties of Kv2.1 homotetramers.These results suggest that a triple complex consisting of Kv2.1, Kv6.4 and KCNE5 subunits can be formed.

View Article: PubMed Central - PubMed

Affiliation: Danish National Research Foundation Centre for Cardiac Arrhythmia and Department for Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.

ABSTRACT
The diversity of the voltage-gated K(+) (Kv) channel subfamily Kv2 is increased by interactions with auxiliary β-subunits and by assembly with members of the modulatory so-called silent Kv subfamilies (Kv5-Kv6 and Kv8-Kv9). However, it has not yet been investigated whether these two types of modulating subunits can associate within and modify a single channel complex simultaneously. Here, we demonstrate that the transmembrane β-subunit KCNE5 modifies the Kv2.1/Kv6.4 current extensively, whereas KCNE2 and KCNE4 only exert minor effects. Co-expression of KCNE5 with Kv2.1 and Kv6.4 did not alter the Kv2.1/Kv6.4 current density but modulated the biophysical properties significantly; KCNE5 accelerated the activation, slowed the deactivation and steepened the slope of the voltage-dependence of the Kv2.1/Kv6.4 inactivation by accelerating recovery of the closed-state inactivation. In contrast, KCNE5 reduced the current density ~2-fold without affecting the biophysical properties of Kv2.1 homotetramers. Co-localization of Kv2.1, Kv6.4 and KCNE5 was demonstrated with immunocytochemistry and formation of Kv2.1/Kv6.4/KCNE5 and Kv2.1/KCNE5 complexes was confirmed by Fluorescence Resonance Energy Transfer experiments performed in HEK293 cells. These results suggest that a triple complex consisting of Kv2.1, Kv6.4 and KCNE5 subunits can be formed. In vivo, formation of such tripartite Kv2.1/Kv6.4/KCNE5 channel complexes might contribute to tissue-specific fine-tuning of excitability.

No MeSH data available.


Related in: MedlinePlus