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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 accelerates the recovery of Kv2.1/Kv6.4 closed-state inactivation.(A) Representative current recordings in the absence (left) and presence (right) of KCNE5 obtained from the pulse protocol shown on top to investigate the recovery of Kv2.1/Kv6.4 closed-state inactivation. (B) Recovery of Kv2.1/Kv6.4 closed-state inactivation in the absence (filled symbols) or presence (open symbols) of KCNE5 obtained by plotting the normalized P2/P1 current amplitudes as function of the pulse duration at the −90 mV voltage step. Single exponential functions were fitted to the Kv2.1/Kv6.4 recovery in absence or presence of KCNE5 (solid and dotted line, respectively). Inset represents a close-up of the initial phase of the recovery of closed-state inactivation. (C) Recovery time constants of the closed-state inactivation of Kv2.1/Kv6.4 in the absence (black) or presence of KCNE5 (white). KCNE5 accelerated the recovery of Kv2.1/Kv6.4 closed-state inactivation significantly (*p < 0.05); n=number of cells.
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f4: KCNE5 accelerates the recovery of Kv2.1/Kv6.4 closed-state inactivation.(A) Representative current recordings in the absence (left) and presence (right) of KCNE5 obtained from the pulse protocol shown on top to investigate the recovery of Kv2.1/Kv6.4 closed-state inactivation. (B) Recovery of Kv2.1/Kv6.4 closed-state inactivation in the absence (filled symbols) or presence (open symbols) of KCNE5 obtained by plotting the normalized P2/P1 current amplitudes as function of the pulse duration at the −90 mV voltage step. Single exponential functions were fitted to the Kv2.1/Kv6.4 recovery in absence or presence of KCNE5 (solid and dotted line, respectively). Inset represents a close-up of the initial phase of the recovery of closed-state inactivation. (C) Recovery time constants of the closed-state inactivation of Kv2.1/Kv6.4 in the absence (black) or presence of KCNE5 (white). KCNE5 accelerated the recovery of Kv2.1/Kv6.4 closed-state inactivation significantly (*p < 0.05); n=number of cells.

Mentions: Since Kv6.4 subunits shift the voltage-dependence of inactivation of Kv2.1/Kv6.4 heterotetramers ~40 mV into hyperpolarized direction compared to that of Kv2.1 homotetramers14, we investigated the effect of KCNE5 on Kv2.1 and Kv2.1/Kv6.4 inactivation properties (Fig. 3). Figure 3A shows typical current recordings to determine the voltage-dependence of inactivation of Kv2.1 and Kv2.1/Kv6.4 channels in the absence and presence of KCNE5. Co-expression of KCNE5 with Kv2.1 did not alter the voltage-dependence of Kv2.1 channels (Fig. 3B, Table 1). However, when co-expressed with Kv2.1 and Kv6.4 subunits, KCNE5 significantly altered the voltage-dependency of Kv2.1/Kv6.4 inactivation, rendering the inactivation curve steeper without altering the midpoint significantly (Fig. 3B, Table 1). The slope of the inactivation curve depends on the ratio of initiation and recovery of the Kv6.4-induced closed-state inactivation. Therefore, we determined both the initiation and recovery rate constant of Kv2.1/Kv6.4 closed-state inactivation in the absence and presence of KCNE5 (Fig. 4). The recovery rate was determined using the protocol illustrated in Fig. 4A: an initial control pulse to +60 mV (P1) was used to record the initial current amplitude, followed by a 1 s step to −130 mV to recover all channels from inactivation, then a 10 s pulse to −60 mV to induce a certain degree of closed-state inactivation, a pulse of variable duration to −90 mV allowing the channels to recover from inactivation and a second test pulse to +60 mV (P2). The fraction P2/P1 represents the degree of channels that have recovered from their closed-inactivated state and was plotted as function of time spent at the recovery pulse to −90 mV (Fig. 4B). Kv2.1/Kv6.4 channels recovered with a time constant of 2.9 ± 0.5 s (n = 8) and KCNE5 significantly modulated this process, increasing the rate of recovery to 1.2 ± 0.2 s (Fig. 4C). In addition, the rate of initiation of Kv2.1/Kv6.4 closed-state inactivation and the extent of inactivation was determined in the absence and presence of KCNE5 but no significant differences could be observed (data not shown).


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 accelerates the recovery of Kv2.1/Kv6.4 closed-state inactivation.(A) Representative current recordings in the absence (left) and presence (right) of KCNE5 obtained from the pulse protocol shown on top to investigate the recovery of Kv2.1/Kv6.4 closed-state inactivation. (B) Recovery of Kv2.1/Kv6.4 closed-state inactivation in the absence (filled symbols) or presence (open symbols) of KCNE5 obtained by plotting the normalized P2/P1 current amplitudes as function of the pulse duration at the −90 mV voltage step. Single exponential functions were fitted to the Kv2.1/Kv6.4 recovery in absence or presence of KCNE5 (solid and dotted line, respectively). Inset represents a close-up of the initial phase of the recovery of closed-state inactivation. (C) Recovery time constants of the closed-state inactivation of Kv2.1/Kv6.4 in the absence (black) or presence of KCNE5 (white). KCNE5 accelerated the recovery of Kv2.1/Kv6.4 closed-state inactivation significantly (*p < 0.05); n=number of cells.
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Related In: Results  -  Collection

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f4: KCNE5 accelerates the recovery of Kv2.1/Kv6.4 closed-state inactivation.(A) Representative current recordings in the absence (left) and presence (right) of KCNE5 obtained from the pulse protocol shown on top to investigate the recovery of Kv2.1/Kv6.4 closed-state inactivation. (B) Recovery of Kv2.1/Kv6.4 closed-state inactivation in the absence (filled symbols) or presence (open symbols) of KCNE5 obtained by plotting the normalized P2/P1 current amplitudes as function of the pulse duration at the −90 mV voltage step. Single exponential functions were fitted to the Kv2.1/Kv6.4 recovery in absence or presence of KCNE5 (solid and dotted line, respectively). Inset represents a close-up of the initial phase of the recovery of closed-state inactivation. (C) Recovery time constants of the closed-state inactivation of Kv2.1/Kv6.4 in the absence (black) or presence of KCNE5 (white). KCNE5 accelerated the recovery of Kv2.1/Kv6.4 closed-state inactivation significantly (*p < 0.05); n=number of cells.
Mentions: Since Kv6.4 subunits shift the voltage-dependence of inactivation of Kv2.1/Kv6.4 heterotetramers ~40 mV into hyperpolarized direction compared to that of Kv2.1 homotetramers14, we investigated the effect of KCNE5 on Kv2.1 and Kv2.1/Kv6.4 inactivation properties (Fig. 3). Figure 3A shows typical current recordings to determine the voltage-dependence of inactivation of Kv2.1 and Kv2.1/Kv6.4 channels in the absence and presence of KCNE5. Co-expression of KCNE5 with Kv2.1 did not alter the voltage-dependence of Kv2.1 channels (Fig. 3B, Table 1). However, when co-expressed with Kv2.1 and Kv6.4 subunits, KCNE5 significantly altered the voltage-dependency of Kv2.1/Kv6.4 inactivation, rendering the inactivation curve steeper without altering the midpoint significantly (Fig. 3B, Table 1). The slope of the inactivation curve depends on the ratio of initiation and recovery of the Kv6.4-induced closed-state inactivation. Therefore, we determined both the initiation and recovery rate constant of Kv2.1/Kv6.4 closed-state inactivation in the absence and presence of KCNE5 (Fig. 4). The recovery rate was determined using the protocol illustrated in Fig. 4A: an initial control pulse to +60 mV (P1) was used to record the initial current amplitude, followed by a 1 s step to −130 mV to recover all channels from inactivation, then a 10 s pulse to −60 mV to induce a certain degree of closed-state inactivation, a pulse of variable duration to −90 mV allowing the channels to recover from inactivation and a second test pulse to +60 mV (P2). The fraction P2/P1 represents the degree of channels that have recovered from their closed-inactivated state and was plotted as function of time spent at the recovery pulse to −90 mV (Fig. 4B). Kv2.1/Kv6.4 channels recovered with a time constant of 2.9 ± 0.5 s (n = 8) and KCNE5 significantly modulated this process, increasing the rate of recovery to 1.2 ± 0.2 s (Fig. 4C). In addition, the rate of initiation of Kv2.1/Kv6.4 closed-state inactivation and the extent of inactivation was determined in the absence and presence of KCNE5 but no significant differences could be observed (data not shown).

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