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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 co-localizes with both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A–C) Horizontal confocal images of HEK293 cells singly expressing KCNE5, HA-Kv6.4, and Kv2.1, respectively. Both KCNE5 and Kv2.1 were found in clusters at the plasma membrane, whereas HA-Kv6.4 was retained intracellularly. Phalloidin (Pha) that labels the submembranous actin cytoskeleton was used to visualize plasma membrane localization. (D) Co-expressing KCNE5 and HA-Kv6.4 did not result in co-localization of the subunits. Hence, KCNE5 was unable to rescue HA-Kv6.4 to the cell surface. (E–F) Co-expressing Kv2.1 and KCNE5 or HA-Kv6.4 resulted in partial overlap of the subunits in small clusters at the cell membrane. Thus, Kv2.1 is capable of redistributing the HA-Kv6.4 subunit to the cell surface and potentially form complexes with both KCNE5 and HA-Kv6.4, respectively. (G) Co-localization of all three subunits was detected when they were transiently expressed in the same cells, which indicates the possibility of a triple complex formation. Merged images are shown in the right column of each row.
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f6: KCNE5 co-localizes with both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A–C) Horizontal confocal images of HEK293 cells singly expressing KCNE5, HA-Kv6.4, and Kv2.1, respectively. Both KCNE5 and Kv2.1 were found in clusters at the plasma membrane, whereas HA-Kv6.4 was retained intracellularly. Phalloidin (Pha) that labels the submembranous actin cytoskeleton was used to visualize plasma membrane localization. (D) Co-expressing KCNE5 and HA-Kv6.4 did not result in co-localization of the subunits. Hence, KCNE5 was unable to rescue HA-Kv6.4 to the cell surface. (E–F) Co-expressing Kv2.1 and KCNE5 or HA-Kv6.4 resulted in partial overlap of the subunits in small clusters at the cell membrane. Thus, Kv2.1 is capable of redistributing the HA-Kv6.4 subunit to the cell surface and potentially form complexes with both KCNE5 and HA-Kv6.4, respectively. (G) Co-localization of all three subunits was detected when they were transiently expressed in the same cells, which indicates the possibility of a triple complex formation. Merged images are shown in the right column of each row.

Mentions: Since the modulation of Kv2.1 and Kv2.1/Kv6.4 channels by KCNE5 subunits has previously not been demonstrated and co-expression of KCNE5 modified Kv2.1/Kv6.4 currents most extensively (compared to the other KCNE subunits), we assessed whether KCNE5 associates with Kv2.1 and Kv6.4 subunits into Kv2.1/KCNE5 and Kv2.1/Kv6.4/KCNE5 channel complexes in HEK293 cells (Fig. 6). We performed immunocytochemical experiments and found Kv2.1 subunits in small clusters at the cell surface when expressed alone in line with previous studies (Fig. 6C)18. In addition, Kv6.4 subunits were retained intracellularly (Fig. 6B) which corresponds to the previously demonstrated ER retention of Kv6.4 subunits when expressed alone14. As described earlier, this ER retention is relieved upon co-expression with Kv2.1 subunits resulting in Kv2.1/Kv6.4 heterotetramers at the plasma membrane1419 which we also observed in our immunocytochemical experiments (Fig. 6F). To examine whether KCNE5 interacts with Kv2.1 and Kv6.4 in (tripartite) channels, we first deduced the KCNE5 localization pattern when expressed alone in HEK293 cells using a custom antibody directed against KCNE5 (Suppl. Material and Methods, Suppl. Fig. S3). We found that KCNE5 was located mainly in small vesicles or clusters which appeared to be at the plasma membrane (Fig. 6A). Performing surface stainings using a N-terminally HA-tagged KCNE5 construct, we found that KCNE5 was able to traffic to the cell membrane on its own and was located in small clusters (Suppl. Fig. S4, arrows) as seen for Kv2.1 (Fig. 6C). From the whole cell stainings it was noticeable that KCNE5 was also located in some vesicles beneath the membrane. To determine the origin of these vesicles/clusters we employed different compartmental markers (Suppl. Fig. S5). These vesicles were not part of the ER, Golgi Apparatus, late endosomes/prelysosomes or early endosomes (Suppl. Fig. S5A-C, E, respectively), yet some were found in vesicles positive for the transferrin receptor, a marker for recycling endosomes. (Suppl. Fig. S5D). Co-expression of KCNE5 and Kv2.1 revealed that the channel subunits located in the same clusters (Fig. 6E). In contrast to Kv2.1, KCNE5 was incapable of rescuing Kv6.4 from the ER (Fig. 6D), yet when all three subunits were co-expressed in the same cell they co-localized at the cell surface (Fig. 6G). To ensure that this observed co-localization was originating from the formed Kv2.1/Kv6.4/KCNE5 channel complexes (rather than from non-specific interactions caused by the presence of the HA tag), we confirmed electrophysiologically that the channel properties were not affected by the HA tag (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 co-localizes with both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A–C) Horizontal confocal images of HEK293 cells singly expressing KCNE5, HA-Kv6.4, and Kv2.1, respectively. Both KCNE5 and Kv2.1 were found in clusters at the plasma membrane, whereas HA-Kv6.4 was retained intracellularly. Phalloidin (Pha) that labels the submembranous actin cytoskeleton was used to visualize plasma membrane localization. (D) Co-expressing KCNE5 and HA-Kv6.4 did not result in co-localization of the subunits. Hence, KCNE5 was unable to rescue HA-Kv6.4 to the cell surface. (E–F) Co-expressing Kv2.1 and KCNE5 or HA-Kv6.4 resulted in partial overlap of the subunits in small clusters at the cell membrane. Thus, Kv2.1 is capable of redistributing the HA-Kv6.4 subunit to the cell surface and potentially form complexes with both KCNE5 and HA-Kv6.4, respectively. (G) Co-localization of all three subunits was detected when they were transiently expressed in the same cells, which indicates the possibility of a triple complex formation. Merged images are shown in the right column of each row.
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f6: KCNE5 co-localizes with both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A–C) Horizontal confocal images of HEK293 cells singly expressing KCNE5, HA-Kv6.4, and Kv2.1, respectively. Both KCNE5 and Kv2.1 were found in clusters at the plasma membrane, whereas HA-Kv6.4 was retained intracellularly. Phalloidin (Pha) that labels the submembranous actin cytoskeleton was used to visualize plasma membrane localization. (D) Co-expressing KCNE5 and HA-Kv6.4 did not result in co-localization of the subunits. Hence, KCNE5 was unable to rescue HA-Kv6.4 to the cell surface. (E–F) Co-expressing Kv2.1 and KCNE5 or HA-Kv6.4 resulted in partial overlap of the subunits in small clusters at the cell membrane. Thus, Kv2.1 is capable of redistributing the HA-Kv6.4 subunit to the cell surface and potentially form complexes with both KCNE5 and HA-Kv6.4, respectively. (G) Co-localization of all three subunits was detected when they were transiently expressed in the same cells, which indicates the possibility of a triple complex formation. Merged images are shown in the right column of each row.
Mentions: Since the modulation of Kv2.1 and Kv2.1/Kv6.4 channels by KCNE5 subunits has previously not been demonstrated and co-expression of KCNE5 modified Kv2.1/Kv6.4 currents most extensively (compared to the other KCNE subunits), we assessed whether KCNE5 associates with Kv2.1 and Kv6.4 subunits into Kv2.1/KCNE5 and Kv2.1/Kv6.4/KCNE5 channel complexes in HEK293 cells (Fig. 6). We performed immunocytochemical experiments and found Kv2.1 subunits in small clusters at the cell surface when expressed alone in line with previous studies (Fig. 6C)18. In addition, Kv6.4 subunits were retained intracellularly (Fig. 6B) which corresponds to the previously demonstrated ER retention of Kv6.4 subunits when expressed alone14. As described earlier, this ER retention is relieved upon co-expression with Kv2.1 subunits resulting in Kv2.1/Kv6.4 heterotetramers at the plasma membrane1419 which we also observed in our immunocytochemical experiments (Fig. 6F). To examine whether KCNE5 interacts with Kv2.1 and Kv6.4 in (tripartite) channels, we first deduced the KCNE5 localization pattern when expressed alone in HEK293 cells using a custom antibody directed against KCNE5 (Suppl. Material and Methods, Suppl. Fig. S3). We found that KCNE5 was located mainly in small vesicles or clusters which appeared to be at the plasma membrane (Fig. 6A). Performing surface stainings using a N-terminally HA-tagged KCNE5 construct, we found that KCNE5 was able to traffic to the cell membrane on its own and was located in small clusters (Suppl. Fig. S4, arrows) as seen for Kv2.1 (Fig. 6C). From the whole cell stainings it was noticeable that KCNE5 was also located in some vesicles beneath the membrane. To determine the origin of these vesicles/clusters we employed different compartmental markers (Suppl. Fig. S5). These vesicles were not part of the ER, Golgi Apparatus, late endosomes/prelysosomes or early endosomes (Suppl. Fig. S5A-C, E, respectively), yet some were found in vesicles positive for the transferrin receptor, a marker for recycling endosomes. (Suppl. Fig. S5D). Co-expression of KCNE5 and Kv2.1 revealed that the channel subunits located in the same clusters (Fig. 6E). In contrast to Kv2.1, KCNE5 was incapable of rescuing Kv6.4 from the ER (Fig. 6D), yet when all three subunits were co-expressed in the same cell they co-localized at the cell surface (Fig. 6G). To ensure that this observed co-localization was originating from the formed Kv2.1/Kv6.4/KCNE5 channel complexes (rather than from non-specific interactions caused by the presence of the HA tag), we confirmed electrophysiologically that the channel properties were not affected by the HA tag (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