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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

KCNE4 modulates both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A) Voltage-dependence of activation and inactivation of Kv2.1 and Kv2.1/Kv6.4 in the absence or presence of KCNE4. The activation curve was determined by plotting the normalized tail currents at −35 mV as a function of the prepulse potential and the inactivation curve was determined by plotting the normalized current amplitude at +60 mV as a function of the 5-s prepulse potential. Solid lines represent the Boltzmann fit. KCNE4 (open symbols) did not modulate Kv2.1 (circles) or Kv2.1/Kv6.4 (triangles) voltage-dependence of activation and inactivation. (B) Activation and deactivation kinetics of Kv2.1 and Kv2.1/Kv6.4 in the absence or presence of KCNE4 derived from a single or double exponential fit of the raw current recordings. KCNE4 did not affect Kv2.1 activation or deactivation kinetics significantly whereas it slightly fastened Kv2.1/Kv6.4 activation kinetics at higher potentials. (C) Current densities obtained at 0 mV after co-expression of 250 ng Kv2.1 + 1 μg CFP or 1 μg KCNE4 (1st and 2nd combination, respectively) and 0.5 μg Kv2.1 and 5 μg Kv6.4 + 1 μg CFP or 1 μg KCNE4 (3rd and 4th combination, respectively). Co-expression of KCNE4 reduced Kv2.1 current density significantly (*p < 0.05) while co-expression of KCNE4 with Kv2.1/Kv6.4 had no significant effect.
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f1: KCNE4 modulates both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A) Voltage-dependence of activation and inactivation of Kv2.1 and Kv2.1/Kv6.4 in the absence or presence of KCNE4. The activation curve was determined by plotting the normalized tail currents at −35 mV as a function of the prepulse potential and the inactivation curve was determined by plotting the normalized current amplitude at +60 mV as a function of the 5-s prepulse potential. Solid lines represent the Boltzmann fit. KCNE4 (open symbols) did not modulate Kv2.1 (circles) or Kv2.1/Kv6.4 (triangles) voltage-dependence of activation and inactivation. (B) Activation and deactivation kinetics of Kv2.1 and Kv2.1/Kv6.4 in the absence or presence of KCNE4 derived from a single or double exponential fit of the raw current recordings. KCNE4 did not affect Kv2.1 activation or deactivation kinetics significantly whereas it slightly fastened Kv2.1/Kv6.4 activation kinetics at higher potentials. (C) Current densities obtained at 0 mV after co-expression of 250 ng Kv2.1 + 1 μg CFP or 1 μg KCNE4 (1st and 2nd combination, respectively) and 0.5 μg Kv2.1 and 5 μg Kv6.4 + 1 μg CFP or 1 μg KCNE4 (3rd and 4th combination, respectively). Co-expression of KCNE4 reduced Kv2.1 current density significantly (*p < 0.05) while co-expression of KCNE4 with Kv2.1/Kv6.4 had no significant effect.

Mentions: Co-expression of KCNE4 did not affect the biophysical properties of Kv2.1 homotetramers (Fig. 1A,B, Table 1) but did reduce the Kv2.1 current density ~10-fold (508 ± 114 pA/pF, n = 21 and 46 ± 18, n = 5 in the absence and presence of KCNE4, respectively, Fig. 1C). Compared to Kv2.1 homotetramers, Kv2.1/Kv6.4 heterotetramers display a significantly reduced current density17 yet co-expression of KCNE4 did not reduce the Kv2.1/Kv6.4 current density further. In contrast, current density was slightly, yet not significantly increased (77 ± 27 pA/pF, n = 12 vs. with KCNE4 91 ± 20 pA/pF, n = 7, Fig. 1C). In addition, co-expression of KCNE4 modulated the biophysical Kv2.1/Kv6.4 properties slightly. KCNE4 accelerated the fast component of Kv2.1/Kv6.4 activation significantly at higher potentials (p < 0.05) but did not affect the slow Kv2.1/Kv6.4 activation component, the deactivation kinetics, or the voltage-dependencies of activation and inactivation (Fig. 1A,B, Table 1).


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)

KCNE4 modulates both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A) Voltage-dependence of activation and inactivation of Kv2.1 and Kv2.1/Kv6.4 in the absence or presence of KCNE4. The activation curve was determined by plotting the normalized tail currents at −35 mV as a function of the prepulse potential and the inactivation curve was determined by plotting the normalized current amplitude at +60 mV as a function of the 5-s prepulse potential. Solid lines represent the Boltzmann fit. KCNE4 (open symbols) did not modulate Kv2.1 (circles) or Kv2.1/Kv6.4 (triangles) voltage-dependence of activation and inactivation. (B) Activation and deactivation kinetics of Kv2.1 and Kv2.1/Kv6.4 in the absence or presence of KCNE4 derived from a single or double exponential fit of the raw current recordings. KCNE4 did not affect Kv2.1 activation or deactivation kinetics significantly whereas it slightly fastened Kv2.1/Kv6.4 activation kinetics at higher potentials. (C) Current densities obtained at 0 mV after co-expression of 250 ng Kv2.1 + 1 μg CFP or 1 μg KCNE4 (1st and 2nd combination, respectively) and 0.5 μg Kv2.1 and 5 μg Kv6.4 + 1 μg CFP or 1 μg KCNE4 (3rd and 4th combination, respectively). Co-expression of KCNE4 reduced Kv2.1 current density significantly (*p < 0.05) while co-expression of KCNE4 with Kv2.1/Kv6.4 had no significant effect.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4525287&req=5

f1: KCNE4 modulates both Kv2.1 homotetramers and Kv2.1/Kv6.4 heterotetramers.(A) Voltage-dependence of activation and inactivation of Kv2.1 and Kv2.1/Kv6.4 in the absence or presence of KCNE4. The activation curve was determined by plotting the normalized tail currents at −35 mV as a function of the prepulse potential and the inactivation curve was determined by plotting the normalized current amplitude at +60 mV as a function of the 5-s prepulse potential. Solid lines represent the Boltzmann fit. KCNE4 (open symbols) did not modulate Kv2.1 (circles) or Kv2.1/Kv6.4 (triangles) voltage-dependence of activation and inactivation. (B) Activation and deactivation kinetics of Kv2.1 and Kv2.1/Kv6.4 in the absence or presence of KCNE4 derived from a single or double exponential fit of the raw current recordings. KCNE4 did not affect Kv2.1 activation or deactivation kinetics significantly whereas it slightly fastened Kv2.1/Kv6.4 activation kinetics at higher potentials. (C) Current densities obtained at 0 mV after co-expression of 250 ng Kv2.1 + 1 μg CFP or 1 μg KCNE4 (1st and 2nd combination, respectively) and 0.5 μg Kv2.1 and 5 μg Kv6.4 + 1 μg CFP or 1 μg KCNE4 (3rd and 4th combination, respectively). Co-expression of KCNE4 reduced Kv2.1 current density significantly (*p < 0.05) while co-expression of KCNE4 with Kv2.1/Kv6.4 had no significant effect.
Mentions: Co-expression of KCNE4 did not affect the biophysical properties of Kv2.1 homotetramers (Fig. 1A,B, Table 1) but did reduce the Kv2.1 current density ~10-fold (508 ± 114 pA/pF, n = 21 and 46 ± 18, n = 5 in the absence and presence of KCNE4, respectively, Fig. 1C). Compared to Kv2.1 homotetramers, Kv2.1/Kv6.4 heterotetramers display a significantly reduced current density17 yet co-expression of KCNE4 did not reduce the Kv2.1/Kv6.4 current density further. In contrast, current density was slightly, yet not significantly increased (77 ± 27 pA/pF, n = 12 vs. with KCNE4 91 ± 20 pA/pF, n = 7, Fig. 1C). In addition, co-expression of KCNE4 modulated the biophysical Kv2.1/Kv6.4 properties slightly. KCNE4 accelerated the fast component of Kv2.1/Kv6.4 activation significantly at higher potentials (p < 0.05) but did not affect the slow Kv2.1/Kv6.4 activation component, the deactivation kinetics, or the voltage-dependencies of activation and inactivation (Fig. 1A,B, Table 1).

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