Limits...
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

Increased KCNE5 concentrations result in increased modulations of Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A) Current densities determined at 0 mV after co-expression of Kv2.1 with CFP (black) or KCNE5 (white) in a 1:0.5 (250 ng + 125 ng), 1:1 (250 ng + 250 ng), 1:4 (250 ng + 1 μg) and 1:8 (250 ng + 2 μg) cDNA transfection ratio. (B) Relative KCNE5-induced reduction of the Kv2.1 current density determined by dividing the Kv2.1 current density obtained upon co-expression with CFP by that upon co-expression with KCNE5 (black and white bars in panel A, respectively). The different transfection cDNA ratios are the same as in panel A. (C) Fast activation component (at 60 mV) of 0.5 μg Kv2.1 + 5 μg Kv6.4 (1:10 transfection ratio) in the absence or presence of 250 ng, 0.5 μg, 1 μg or 2 μg KCNE5 (1:10:0.5, 1:10:1, 1:10:2 and 1:10:4 cDNA transfection ratios, respectively). (D) Slope factor of the voltage-dependence of inactivation of Kv2.1/Kv6.4 in the absence or presence of KCNE5 transfected in different cDNA ratios. The used cDNA transfection ratios are the same as in panel C. In each panel, the numbers in italic represent the number of cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4525287&req=5

f5: Increased KCNE5 concentrations result in increased modulations of Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A) Current densities determined at 0 mV after co-expression of Kv2.1 with CFP (black) or KCNE5 (white) in a 1:0.5 (250 ng + 125 ng), 1:1 (250 ng + 250 ng), 1:4 (250 ng + 1 μg) and 1:8 (250 ng + 2 μg) cDNA transfection ratio. (B) Relative KCNE5-induced reduction of the Kv2.1 current density determined by dividing the Kv2.1 current density obtained upon co-expression with CFP by that upon co-expression with KCNE5 (black and white bars in panel A, respectively). The different transfection cDNA ratios are the same as in panel A. (C) Fast activation component (at 60 mV) of 0.5 μg Kv2.1 + 5 μg Kv6.4 (1:10 transfection ratio) in the absence or presence of 250 ng, 0.5 μg, 1 μg or 2 μg KCNE5 (1:10:0.5, 1:10:1, 1:10:2 and 1:10:4 cDNA transfection ratios, respectively). (D) Slope factor of the voltage-dependence of inactivation of Kv2.1/Kv6.4 in the absence or presence of KCNE5 transfected in different cDNA ratios. The used cDNA transfection ratios are the same as in panel C. In each panel, the numbers in italic represent the number of cells.

Mentions: Next, we determined the impact of different levels of KCNE5 expression on Kv2.1 and Kv2.1/Kv6.4 channels (Fig. 5). We co-transfected Kv2.1 or Kv2.1/Kv6.4 with KCNE5 in different ratios and determined the impact of KCNE5 focusing on those properties that we had seen to be mostly influenced by KCNE5 co-expression, i.e. Kv2.1 current density (Fig. 5A,B), and Kv2.1/Kv6.4 activation rate (Fig. 5C) and voltage-dependence of inactivation (Fig. 5D). Co-expression of Kv2.1 with KCNE5 in a 1:0.5, 1:1, 1:4 and 1:8 (Kv2.1:KCNE5) cDNA ratio reduced Kv2.1 current densities gradually. Although Kv2.1 current densities also decreased with increasing amounts of transfected peCFP vector alone (used to correct for effects of cDNA dilution and/or overload of the transcriptional/translational machinery), we observed a clear KCNE5-mediated decrease in Kv2.1 current density (Fig. 5A,B). These results demonstrated that at least a part of the Kv2.1 current reduction observed upon co-expression with KCNE5 is caused by KCNE5 and this KCNE5-induced current reduction depends on the KCNE5 expression level. Similarly, co-expression of Kv2.1/Kv6.4 channels (obtained by a 1:10 Kv2.1:Kv6.4 cDNA transfection ratio to minimize the presence of Kv2.1 homotetramers) with KCNE5 in 1:10:0.5, 1:10:1, 1:10:2 and 1:10:4 (Kv2.1:Kv6.4:KCNE5) cDNA ratios revealed that KCNE5 gradually accelerated the activation kinetics and steepened the voltage-dependence of inactivation (Fig. 5C,D).


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)

Increased KCNE5 concentrations result in increased modulations of Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A) Current densities determined at 0 mV after co-expression of Kv2.1 with CFP (black) or KCNE5 (white) in a 1:0.5 (250 ng + 125 ng), 1:1 (250 ng + 250 ng), 1:4 (250 ng + 1 μg) and 1:8 (250 ng + 2 μg) cDNA transfection ratio. (B) Relative KCNE5-induced reduction of the Kv2.1 current density determined by dividing the Kv2.1 current density obtained upon co-expression with CFP by that upon co-expression with KCNE5 (black and white bars in panel A, respectively). The different transfection cDNA ratios are the same as in panel A. (C) Fast activation component (at 60 mV) of 0.5 μg Kv2.1 + 5 μg Kv6.4 (1:10 transfection ratio) in the absence or presence of 250 ng, 0.5 μg, 1 μg or 2 μg KCNE5 (1:10:0.5, 1:10:1, 1:10:2 and 1:10:4 cDNA transfection ratios, respectively). (D) Slope factor of the voltage-dependence of inactivation of Kv2.1/Kv6.4 in the absence or presence of KCNE5 transfected in different cDNA ratios. The used cDNA transfection ratios are the same as in panel C. In each panel, the numbers in italic represent the number of cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525287&req=5

f5: Increased KCNE5 concentrations result in increased modulations of Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A) Current densities determined at 0 mV after co-expression of Kv2.1 with CFP (black) or KCNE5 (white) in a 1:0.5 (250 ng + 125 ng), 1:1 (250 ng + 250 ng), 1:4 (250 ng + 1 μg) and 1:8 (250 ng + 2 μg) cDNA transfection ratio. (B) Relative KCNE5-induced reduction of the Kv2.1 current density determined by dividing the Kv2.1 current density obtained upon co-expression with CFP by that upon co-expression with KCNE5 (black and white bars in panel A, respectively). The different transfection cDNA ratios are the same as in panel A. (C) Fast activation component (at 60 mV) of 0.5 μg Kv2.1 + 5 μg Kv6.4 (1:10 transfection ratio) in the absence or presence of 250 ng, 0.5 μg, 1 μg or 2 μg KCNE5 (1:10:0.5, 1:10:1, 1:10:2 and 1:10:4 cDNA transfection ratios, respectively). (D) Slope factor of the voltage-dependence of inactivation of Kv2.1/Kv6.4 in the absence or presence of KCNE5 transfected in different cDNA ratios. The used cDNA transfection ratios are the same as in panel C. In each panel, the numbers in italic represent the number of cells.
Mentions: Next, we determined the impact of different levels of KCNE5 expression on Kv2.1 and Kv2.1/Kv6.4 channels (Fig. 5). We co-transfected Kv2.1 or Kv2.1/Kv6.4 with KCNE5 in different ratios and determined the impact of KCNE5 focusing on those properties that we had seen to be mostly influenced by KCNE5 co-expression, i.e. Kv2.1 current density (Fig. 5A,B), and Kv2.1/Kv6.4 activation rate (Fig. 5C) and voltage-dependence of inactivation (Fig. 5D). Co-expression of Kv2.1 with KCNE5 in a 1:0.5, 1:1, 1:4 and 1:8 (Kv2.1:KCNE5) cDNA ratio reduced Kv2.1 current densities gradually. Although Kv2.1 current densities also decreased with increasing amounts of transfected peCFP vector alone (used to correct for effects of cDNA dilution and/or overload of the transcriptional/translational machinery), we observed a clear KCNE5-mediated decrease in Kv2.1 current density (Fig. 5A,B). These results demonstrated that at least a part of the Kv2.1 current reduction observed upon co-expression with KCNE5 is caused by KCNE5 and this KCNE5-induced current reduction depends on the KCNE5 expression level. Similarly, co-expression of Kv2.1/Kv6.4 channels (obtained by a 1:10 Kv2.1:Kv6.4 cDNA transfection ratio to minimize the presence of Kv2.1 homotetramers) with KCNE5 in 1:10:0.5, 1:10:1, 1:10:2 and 1:10:4 (Kv2.1:Kv6.4:KCNE5) cDNA ratios revealed that KCNE5 gradually accelerated the activation kinetics and steepened the voltage-dependence of inactivation (Fig. 5C,D).

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