Figure 1. KCNE1 suppresses the intermediate-open state of KCNQ1.
(A–H) Fluorescence (green) and current (black) signals from Xenopus oocytes injected with cRNA encoding pseudo-WT (C214A/G219C/C331A) KCNQ1 alone (KCNQ1, A–D) or coinjected with cRNAs encoding pseudo-WT KCNQ1 and KCNE1 (KCNQ1+KCNE1, E–H). The cells were labeled with Alexa 488 C5-maleimide. (A and E) GV and FV relationships (solid) with the main and high voltage FV components plotted (dotted lines). (B and F) normalized fluorescence and current responses to a 60 mV pulse shown with fits (thin grey lines) to a single- or bi-exponential function. Averaged fast (C and G) and slow (D and H) tau values of fluorescence and current responses to various voltage pulses. (I) Intermediate- (E1-R2, top) and activated- (E1-R4, bottom) state homology models of KCNQ1 after 100 ns of MD simulation. Side view of one VSD (left) and bottom view of the pore (right). (J) Currents from the cells expressing E160R/R231E (E1R/R2E, top) or E160R/R237E (E1R/R4E, bottom) both alone (−KCNE1, middle) or with KCNE1 (+KCNE1, right).
Figure 1—figure supplement 1. Improved resolution of Fhigh.
(A and B) VCF data from cells expressing pseudo-WT
KCNQ1, labeled with Alexa 546 C5-maleimide (KCNQ1 [Alexa 546]).
(C and D) VCF data from pseudo-WT
KCNQ1+R67E/K69E/K70E KCNE1 labeled with Alexa 488 C5-maleimide
(KCNQ1+RKK/EEE [Alexa488]). (A and C)
Normalized current and fluorescence responses to a 60 mV pulse.
(B and D) GV and FV relationships (solid) with
the main and high voltage FV components plotted (dotted lines). Current
signals (black), fluorescence signals (color, red = Alexa 546, green
= Alexa 488). The RKK/EEE mutation in KCNE1 causes a leftward shift of
Fmain, relative to that of WT KCNE1, which increases the separation between
Fmain and Fhigh.
Figure 1—figure supplement 2. GV/FV relationships are maintained in channels the mutation R243Q in KCNQ1.
GV (solid black) and FV (solid green) relationships for R243Q/psWT KCNQ1
channels expressed alone (R243Q) (A) or with R67E/K69E/K70E
KCNE1 (R243Q+RKK/EEE) (B). Green dotted lines show the
main and high voltage FV components. The GV relationship of pseudo-WT
channels (black dashed lines) is significantly different from that of R243Q;
however, the relationship of the GV to different FV components are
preserved.
Figure 1—figure supplement 3. MD simulations predict that, unlike the resting-state, both the intermediate- and activated-states of the VSD stabilize pore opening through state-dependent protein and lipid interactions.
(A) Snapshots of one VSD (left, side view) or the pore domain
(right, bottom view) following 100 ns of MD simulations. In the resting,
intermediate or activated VSD, E160 (E1, red) forms a salt bridge with R228
(R1), R231 (R2) or R237 (R4), respectively. (B) Averaged (over
several trajectories) pore radius vs position along the axis normal to the
membrane (Z). (C) Snapshots of the PIP2
intrasubunit-binding site in the three states. In the resting/closed state,
PIP2 interacts with positive residues of S4 (cyan). When the
VSD is intermediate or activated, PIP2 shifts closer to S6
(yellow) and anchors its positive residues (K354 and K358). (D)
Probability of salt bridges formation between positive residues of S6 (K354
and K358) and PIP2. The lipid interacts with S6 only when the VSD
is intermediate or activated, not when it is resting. Error bars represent
SD. K354 and K358 interactions are not statistically different for the E1-R2
and E1-R4 states. Figure 1—figure
supplement 3B represents the averaged pore radius profiles along
the axis normal to the membrane (Z). In the activated/open and intermediate
states, the minimal radiuses of the pore at this level are 3.5 ± 0.4
Å and 2.8 ± 0.6 Å respectively. For comparison, in the Kv1.2
open state, the corresponding radius (pdb 3LUT [Chen et al., 2010]) is 4.2 Å, in the Kv1.2/2.1
paddle chimera open state (pdb 2 R9R [Long
et al., 2007]) it is 4.2 Å also, and in the NavMS open state
(pdb 3ZJZ [Loussouarn et al., 2003])
it is 2.3 Å. Therefore, the minimal pore radius at the intercellular
gate level in the models of the Kv7.1 activated and intermediate states
corresponds to the open pore. In the resting/closed state, this radius
decreases to 1.5 ± 0.5 Å. This is similar to the closed states of
KcsA (pdb 1K4C [Zhou et al.,
2001]), NavAB (pdb 4EKW [Payandeh
et al., 2012]) and NavAP (pdb 4DXW [Zhang et al., 2012]), where these values are 1.1, 1.2
and 0.9 Å respectively. The activated/open, intermediate and
resting/closed states of Kv7.1 differ by their properties as evidenced from
the reported experimental data. Taking advantage of our simulations, we
attempted to investigate whether the interactions between PIP2
and positive residues of the Kv7.1 intrasubunit binding site are different.
Indeed PIP2 interacts preferably with the VSD (S4) when the
channel is resting/closed or with the pore (S6) when the channel is
activated/open (Kasimova et al.,
2014) (Figure 1—figure
supplement 3C, top and bottom panels). In the intermediate state,
the lipid forms salt bridges with both S4 (R243) and S6 (K354 and K358)
simultaneously (Figure 1—figure
supplement 3C, middle panel). Its equilibrium position is also
between these in the activated/open and resting/closed states.
Interestingly, the probability of interaction between PIP2 and S6
(K354 and K358) is rather high (Figure
1—figure supplement 3D). The average values are slightly
higher for the intermediate than for the activated/open states: 40 and 26%
for K354, 68 and 42% for K358 respectively. However, this difference is
statistically insignificant due to the estimated error bars.
Figure 1—figure supplement 4. VSD mutations reveal that KCNE1 suppresses currents from intermediate-open states and increases those from activated-open states.
(A) Cartoons illustrating the mutational strategy used to
arrest the VSD near the intermediate- and activated-states. The E160R (E1R)
mutation was used to disrupt the native electrostatic interactions between
the S2 and S4 segments that stabilize the VSD as it undergoes activation.
E1R was paired with R231E (R2E) or R237E (R4E) to stabilize the putative
intermediate- and activated-states, respectively. (B) Currents
from oocytes expressing WT or mutant KCNQ1 subunits alone (−KCNE1,
left) or with KCNE1 (+KCNE1, right) in response various voltage test
pulses. (C and D) Averaged steady-state
current–voltage relationships for cells expressing WT or mutant KCNQ1
subunits alone (C, −KCNE1) or with KCNE1
(D, +KCNE1).
Figure 1—figure supplement 5. Surface membrane expression of E1R/R2E and E1R/R4E.
Biotinylation of intact oocytes allowed separation of membrane proteins from
the cell lysate using streptavidin beads. The membrane fraction and the cell
lysate were subjected to Western Blot using antibodies against KCNQ1 (Top)
or Gβ, a soluble protein not found in the membrane. (A)
E1R/R2E and E1R/R4E reached the cell membrane with similar efficiency.
(B) KCNE1 did not decrease the expression of E1R/R2E to the
membrane.