First published online October 17, 2008
Journal of Experimental Biology 211, 3442-3453 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.022608
Non-linear intramolecular interactions and voltage sensitivity of a KV1 family potassium channel from Polyorchis penicillatus (Eschscholtz 1829)
Tara L. Klassen1,
Megan L. O'Mara2,
Megan Redstone2,
Andrew N. Spencer1,3 and
Warren J. Gallin1,*
1 Department of Biological Sciences, University of Alberta, Edmonton, Alberta,
Canada T6G 2E9
2 Department of Biological Sciences, University of Calgary, Calgary, Alberta,
Canada T2N 1N4
3 Malaspina University College, 900 Fifth Street, Nanaimo, British Columbia,
Canada V9R 5S5
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Fig. 1. The jShak1 channel lacks one acidic residue in S2 and one basic triplet
motif in S4, compared with Drosophila Shaker and RatKV1.2.
(A)A schematic of a single 6TM alpha subunit of the Drosophila Shaker
voltage-gated potassium channel. The S5-pore, S5-S6 loop and S6 domain are
shown in grey and the S1-S4 transmembrane helices are white. The conserved
acidic residues in S2 and S3 are labelled as are the characteristic basic
residues in S4. (B)Alignments of the transmembrane helices S2, S3 and S4 in
selected Drosophila, rat and hydrozoan KV channels. The
jellyfish (Polyorchis penicillatus) channel jShak1 lacks one (N227)
of the two acidic residues in S2 but contains the stabilizing acidic residue
(D260) in S3. The S4 voltage sensor of jShak1 lacks one basic motif found in
other KV1 channels but has the same helical length. The highly
conserved acidic residues (E283 and E293) in S2 and the conserved acidic
residue (D316) in S3 and the basic residues in the S4 voltage sensor of
Drosophila Shaker and other Shaker-type channels are highlighted in
grey. (C)Alignment of the S4 helix of jShak1 with the S4 helices of three
other channels, indicating the sites of insertion of the S4 mutants used in
this study on either side of position K294 (highlighted in black) in
jShak1.
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Fig. 3. Single mutations in the S2 helix and triplet insertions in the S4 helix
modify steady-state activation properties. (A)The steady-state activation
curve for jShak1 S2 mutants revealed that the N227E (inverted triangles)
channels were activated at far more hyperpolarized potentials than wild-type
channels (filled circles) and N227D mutant channels (open circles). The solid
curve represents the fit to a Boltzmann function giving the voltage of half
activation (V50) and slope parameter (b) for each
channel (error bars indicate s.e.m.). Fit parameters are summarized in
Table 1. (B)The steady-state
activation curve for jShak1 S4 insertion mutants revealed that insertion of
the QIF motif (inverted triangles) to the N-terminal side of K294 shifted the
activation curve in the hyperpolarized direction, the RIF insertion (open
circles) shifted the activation curve in the depolarized direction, and the
IFR insertion (open triangles) had no significant effect compared with
wild-type channels (filled circles). The solid curve represents the fit to a
Boltzmann function. Voltage of half activation (V50) and
Boltzmann slope factors (b) for each channel are summarized in
Table 1 (error bars indicate
s.e.m.).
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Fig. 4. The effects of combining S2 point mutations and S4 insertional mutations on
steady-state activation properties. In all panels the wild-type jShak1 channel
is indicated by the red circles, the S2 single mutants are indicated by
triangles, the S4 insertion mutants are indicated by squares and the double
mutants are indicated by diamonds. (A,C,E) The effect of combining the N227D
mutation with the S4 triplet insertions in the double mutant channels. (B,D,F)
The effect of combining the N227E mutation with the S4 triplet insertions in
the double mutant channels. The solid curve represents the fit to a Boltzmann
function. Voltage of half activation (V50) and Boltzmann
slope factors (b) for each channel are summarized in
Table 1 (error bars indicate
s.e.m.). (A)The effect of the N227D mutation in the S2 helix in combination
with the QIF insertion mutation in S4 is similar to its effect on the
wild-type channel. There appears to be a slight shift in slope factor and
V50, but the effect is not statistically significant. (B)The
steady-state activation curve for the QIF-E double mutant (pink diamonds), the
single QIF mutant (green squares), and the N227E point mutant (black
triangles) were all shifted leftward approximately the same amount relative to
the wild-type channel (red circles), but had different slopes, although the
differences were not statistically significant. (C)The steady-state activation
curves for the IFR-D double mutant (turquoise diamonds), the single mutant
N227D (orange triangles), IFR insertion alone (green squares) were not
significantly different from the wild-type channel (red circles). (D)The
steady-state activation curve for the IFR-E double mutant (pink diamonds) was
shifted in a hyperpolarized direction relative to the IFR mutant alone (green
squares) and the wild-type channel (red circles) and had a similar
V50 of activation to that of the N227E single mutant (blue
triangles), but with a larger slope factor, b. (E)The steady-state
activation curve for the RIF-D double mutant (turquoise diamonds) was shifted
in a depolarized direction relative to the single N227D mutant (orange
triangles), to the same extent that the RIF mutant (green squares) was shifted
relative to the wild-type channel (red circles). (F)The steady-state
activation curve for the RIF-E double mutant (pink diamonds) was shifted in a
depolarized direction relative to the wild-type channel (red circles), which
was in the opposite direction to the shift for the N227E single mutant (black
triangles). The RIF-E double mutant is also shifted in the hyperpolarizing
direction relative to the RIF mutant alone (green squares).
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Fig. 5. Homology models of the structural constraints in jShak1 based on the
crystal structure of RatKV1.2. (A)Overlay of S3 and S4 showing the
extremely short three amino acid S3-S4 linker which anchors the C-terminal end
of S3 closely to the N-terminal end of S4 in jShak1 during gating transitions.
The homology model of jShak1 (blue) is placed over the model of
RatKV1.2 (gold). The basic residues in S4 are illustrated as
side-chain sticks, and transmembrane helices are labelled. (B)Overlay of the
highly conserved S4-S5 linker (L4-5) that is conserved in both
RatKV1.2 (gold) and jShak1 (blue). The L4-5 linker couples the
translocation of the voltage sensor to the opening of activation gate. This
conserved mechanism suggests that length insertions in the short S4 of
jShak1 should move residues extracellularly rather than modify the
amphipathic packing of L4-5.
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Fig. 6. The S2 and S4 of jShak1 lack favourable electrostatic interactions in the
open state that are present in most other KV1 channels. (A)A
structural overlay of S2 transmembrane domains of RatKV1.2 (gold)
and jShak1 (blue) showing the homology of positions of E226
(RatKV1.2) to N227 (jShak1) and E236 (RatKV1.2) to E237
(jShak1), respectively. The short non-charged side-chain of N227 prevents
favourable electrostatic interactions with S4 residues in the open
conformation that can be recovered with mutations to glutamate (N227E). (B)A
structural overlay of S4 showing relative locations of the S4 basic residue
side chains in RatKV1.2 (gold) and the shortened S4 of jShak1
(blue). The periodicity and side-chain location of the arginine residues
between the channels is preserved at the C-terminal end of the helix.
Specifically, positions K306/R309 in RatKV1.2 overlay K274/R377 in
RatKV1.2. (C)Homology model of the interactions between S2 and S4
residues in the open conformation of the channel illustrating the favourable
salt bridge interactions between E226 and R303. The N227 residue in jShak1
(blue) does not form a salt bridge with R291, whereas E226 shows a strong
interaction with R303 in RatKV1.2 (gold).
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Fig. 7. Schematic of interactions occurring between the S2 and S4 helices in the
open and closed states in the jShak1 and QIF channels. (A)jShak1 in closed
state (left) is stabilized by charge-pair interactions between positively
charged R291 and K294 on the S4 helix and negatively charged E237 on S2 and
D260 on S3. In the transition to the open state the interactions between R291
and the two acidic residues are broken and R291 comes into proximity to N227,
which is uncharged. In the N227E mutant the open state is stabilized by the
R291–E227 charge interaction, shifting the equilibrium toward the open
state. (B)In the QIF mutation, where glutamine occupies the position normally
taken by R291, there are no charge interactions between the glutamine residue,
E237 and D260 to stabilize the closed state, so the equilibrium is shifted
towards the open state. Replacement of N227 with acidic residues does not
increase stabilization of the open state because the glutamine does not
interact strongly with the acidic residues.
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© The Company of Biologists Ltd 2008
23 N-truncated jShak1 channels lack N-type inactivation but exhibit
slow C-type inactivation. (A)The full-length wild-type jShak1 channel
expressed fast opening kinetics and fast N-type inactivation in
Xenopus oocytes. Outwardly directed current traces were evoked by 500
ms step depolarizations from a holding potential of –90 mV to a range of
potentials from –90 to +90 mV in 10 mV increments followed by a return
to –90 mV. (B)The