www.landesbioscience.com Channels 3
Channels 4:1, 3-11; January/February 2010; © 2010 Landes Bioscience
pKa and pKC partially rescue long QT type 1
phenotype by restoring channel-pIp2 interactions
Long QT syndrome (LQT) is the most common form of inher-
ited cardiac arrhythmia. It is estimated that one of 5,000 to
7,000 newborns have the disease and it may cause sudden death
in 3,000 to 4,000 children and young adults each year in the US.
The most common form of LQT syndrome (Romano-Ward syn-
drome) is a heterogeneous, autosomal dominant genetic disease
caused by mutations of cardiac ion channel genes. This syndrome
is associated with delayed ventricular repolarization and is mani-
fested by syncope and sudden death from ventricular arrhyth-
mias. LQT is identified by abnormal QT interval prolongation
on the electrocardiogram (ECG). The QT prolongation may
arise from either a decrease in repolarizing membrane currents or
an increase in depolarizing currents. QT prolongation produced
by delayed repolarization due to reductions of the slow repolariz-
ing cardiac K+ currents (IKs) is associated with the most common
form of LQT, LQT1.
Stress and exercise are known to precipitate arrhythmias associ-
ated with the LQT1 syndrome.1 Stimulation of the β1-adrenergic
receptor signaling cascade is one of the most important means
of increasing cardiac output in response to stress and exercise.
IKs is thought to be particularly important in the heart during
adrenergic stimulation.2,3 Activation of β1-adrenergic receptors is
coupled to activation of adenylyl cyclase which catalyzes the con-
version of ATP to cAMP and activates protein kinase A (PKA).
β-Adrenergic stimulation increases IKs via channel activation by
direct phosphylation by PKA.4,5 β-Blockers are commonly used
to treat these patients although some patients are refractory to the
treatment.1 Normally, β-adrenergic stimulation of repolarizing
cardiac K+ currents suppresses β-adrenergic-induced premature
beats and afterdepolarizations which are believed to induced, at
least in part, by a concomitant increase in Ca2+ currents.6-11
G-proteins of the Gq/G11 family (GqPCRs) are also known
to mediate positive inotropism in human ventricular myo-
cardium.12 GqPCRs, when activated, stimulate phosphatidyl
*Correspondence to: Coeli M.B. Lopes; Email: email@example.com
Submitted: 04/29/09; Revised: 10/03/09; Accepted: 10/05/09
Previously published online: www.landesbioscience.com/journals/channels/article/10227
Long-QT syndrome causes torsade de pointes arrhythmia, ventricular fibrillation, and sudden death. The most commonly
inherited form of long-QT syndrome, LQT1, is due to mutations on the potassium channel gene KCNQ1, which forms one
of the main repolarizing cardiac K+ channels, IKs. IKs has been shown to be regulated by both β-adrenergic receptors, via
protein kinase a (pKa), and by Gq protein coupled receptors (GqpCR), via protein kinase C (pKC) and phosphatidylinosi-
tol 4,5-bisphosphate (pIp2). These regulatory pathways were shown to crosstalk, with pKa phosphorylation increasing
the apparent affinity of IKs to PIP2. here we study the effects of LQT1 mutations in putative pIp2-KCNQ1 interaction sites
on regulation of IKs by pKa and GqpCR. The effect of the LQT1 mutations on IKs regulation was tested for mutations
in conserved, positively charged amino acids, located in four distinct cytoplamic domains of the KCNQ1 subunit: R174C
(s2-s3), R243C (s4-s5), R366Q (proximal c-terminus) and R555C (distal c-terminus). Mutations in the c-terminus of IKs
(both proximal and distal) enhanced channel sensitivity to changes in membrane pIp2 levels, consistent with a decrease in
apparent channel-pIp2 affinity. These mutant channels were more sensitive to inhibition caused by receptor mediated PIP2-
depletion and more sensitive to stimulation of pIp2 production, by overexpression of phosphatidylinositol-4-phosphate-
5-kinase (pI5-kinase). In addition, c-terminus mutants showed a potentiated regulation by pKa. On the other hand, for
the two cytoplasmic-loop mutations, an impaired activation by pKa was observed. The effects of the mutations on pKC
stimulation of the channel paralleled the effects on pKa stimulation, suggesting that both regulatory inputs are similarly
affected by the mutations. We tested whether pKC-mediated activation of IKs, similarly to the pKa-mediated activation,
can regulate the channel response to pIp2. after pKC activation, channel was less sensitive to changes in membrane pIp2
levels, consistent with an increase in apparent channel-pIp2 affinity. PKC-activated channel was less sensitive to inhibition
caused by block of synthesis of pIp2 by the lipid kinase inhibitor wortmannin and less sensitive to stimulation of pIp2 pro-
duction. Our data indicates that stimulation by pKa and pKC can partially rescue LQT1 mutant channels with weakened
response to pIp2 by strengthening channel interactions with pIp2.
alessandra Matavel, emiliano Medei and Coeli M.B. Lopes*
Cardiovascular Research Institute; Department of Medicine; University of Rochester; Rochester, NY USA
Key words: cardiac arrhythmia, channelopathies, ion channel modulation by GPCRs, K+ channels, modulation of channels,
potassium channels, MinK, PI(4,5)P2, KvLQT1, potassium, arrhythmias
4 Channels Volume 4 Issue 1
depletion,14 we investigated the consequences of altered channel-
PIP2 sensitivity on PKA and PKC regulation.
Two of the four mutants tested (R366Q, in the proximal
c-terminus, and R555C, in the distal c-terminus) show an
enhanced sensitivity to changes in membrane PIP2 levels, consis-
tent with channels that have a weaker apparent affinity to PIP2.
Function of these c-terminal mutations could be partially rescued
by PKA and PKC activation. In contrast, the c-loop mutations
tested (R174C and R243C) disrupted the transduction of regula-
tory inputs that led to channel activation. Our data shows that
normal GqPCR regulation is disrupted by LQT1 mutations and
that changes in IKs regulation by GPCRs may contribute to the
pathophysiology of long-QT syndrome.
LQT1 related mutations affect IKs regulation by PIP2. The
inhibitory phase of the IKs channel regulation by GqPCRs has
been shown to be due to PIP2 depletion of the channel.13,14 We
studied the effect of four naturally occurring LQT1 mutations on
PIP2-mediated inhibition of IKs. The LQT1 mutations studied
here were neutralizing mutations in positively charged cytoplas-
mic residues highly conserved among the KCNQ channel fam-
ily. Because PIP2 has been shown to activate all KCNQ channel
family members, these four residues are putative PIP2 interaction
sites. To assess the effect of the LQT1 mutations on the chan-
nel, we expressed either the wild-type or the mutant KCNQ1
subunit together with KCNE1 at a ratio 1:1 (KCNQ1:KCNE1)
in Xenopus oocytes. To determine the voltage dependence of
IKs, we constructed isochronal (t = 6 s) activation curves. We
measured the IKs tail current at -40 mV after depolarization
to a series of voltage steps from -60 to +80 mV. A Boltzmann
fit of this data was used to determine the V1/2 and the maximal
conductance (Gmax) of activation. IKs and LQT1 mutant chan-
nels did not reach a steady level even after long depolarizations
at room temperature. Experiments using depolarizing pulses of
varying length (18 s and 2.7 s) showed that V1/2 of activation
inositol-specific phospholipase C (PLC). The substrate for PLC
is phosphotidylinositol 4,5-biphosphate (PIP2), so that agonist
stimulation of the receptor causes reduction of PIP2 in the plasma
membrane. Hydrolysis of PIP2 generates inositol 1,4,5-triphos-
phate (IP3) and diacylglycerol (DAG). DAG activates protein
kinase C (PKC). We have recently shown that stimulation of a
number of GqPCRs regulates the IKs channel in a biphasic man-
ner. The channel inhibition phase is due to PIP2 depletion from
the channel and the activation phase is mediated by PKC.13 In
addition, PKA modulates PIP2 mediated inhibition of IKs both
in heterologous and in the native system, suggesting that the
GqPCR and β-adrenergic IKs regulatory pathways crosstalk.14
In proteins shown to interact with PIP2, including Kir chan-
nels, several basic residues were shown to form the PIP2 binding
site.15-18 Several positively charged residues present in cytoplas-
mic portions of the KCNQ1 channel may come together to
form a binding-site for PIP2. Mutations that decrease channel-
PIP2 interactions also increase the channel sensitivity to PIP2-
depletion.14,19-23 Approximately 250 LQT1 mutations have been
identified to date (http://www.fsm.it/cardmoc/). About 75% of
these mutations are missense mutations, which have been asso-
ciated with the greatest cardiac risk.24 Twenty percent of the
missense mutations are associated with positively charged cyto-
plasmic residues and at least another 30% are in close proximity
to these putative PIP2 interaction sites, suggesting a decrease in
interactions of the channel with PIP2 may be a common mecha-
nism underlying decrease in function observed in LQT1. Most
of these charged residues are highly conserved in other KCNQ
channel families, also known to be sensitive to PIP2. Here we
studied whether four mutations associated with LQT1, present
in positively charged amino acids, conserved among the KCNQ
family, in distinct cytoplasmic domains of the channel (S2-S3,
S4-S5, proximal c-terminus and distal c-terminus), altered chan-
nel regulation by PIP2. Because the strength of channel-PIP2 inter-
actions was shown to determine the sensitivity of Kir channels to
regulation by various modulators23 and because PKA phosphory-
lation was shown to modulate IKs sensitivity to membrane PIP2
Figure 1 (See opposite page). pIp2 regulation is modified by LQT1 mutants. (A) Typical IKs response in oocytes expressing either KCNQ1 or
mutant subunit together with KCNe1 subunits. Currents were measured to depolarized steps from -60 to +80 mV from a -80 mV holding potential
followed by a -40 mV step. (B) Left: summary data for current measured after 4 s depolarization to +40 mV for wild-type and mutant channels. Cur-
rent was normalized to wild-type current for each batch of oocytes (WT: 1.00 ± 0.05, n = 30; R174C: 0.47 ± 0.21, n = 14; R243C: 0.12 ± 0.06, n =
22; R366Q: 0.22 ± 0.12, n = 23; R555C: 0.08 ± 0.06, n = 24). Middle: V1/2 for current activation for wild-type and mutant channels. V1/2 was calculated
from a Boltzmann fit of the tail current data. Tail currents were measured at -40 mV after 6 seconds depolarizing pulses from -60 mV to +80 mV from
-80 mV holding potential (in mV: WT: 17 ± 2, n = 25; R174C: 34 ± 2, n = 11; R243C: 84 ± 5, n = 6; R366Q: 46 ± 2, n = 14; R555C: 52 ± 2, n = 16).
Right: Normalized Gmax for current activation calculated from Boltzmann fit for WT and mutant channels in mV: WT: 1.00 ± 0.04, n = 28; R174C:
0.52 ± 0.06, n = 9; R243C: 0.56 ± 0.19, n = 6; R366Q: 48 ± 0.06, n = 16; R555C: 0.20 ± 0.04, n = 18. (C–F) effect of BK2 receptor stimulation on the
KCNQ1(R174C)/KCNe1, KCNQ1(R243C)/KCNe1, KCNQ1(R366Q)/KCNe1 and KCNQ1(R555C)/KCNe1 mutant channel currents when compared to
WT regulation. Depolarizing steps to +40 mV from -80 mV. Left: typical current response to bradykinin 1 μM before (a) and during the inhibition (b) in-
duced by agonist stimulation. Right: time course of the effects of agonist stimulation measured at the end of the depolarizing pulse. (G) summary data.
The inhibition phase of the regulation was disrupted for three of the mutants tested. Regulation for each mutant was compared to wild-type regulation
measured in same oocyte batches. Inhibition for the R174C mutant channel was lower than wild-type (R174C: 39 ± 4%, n = 8; WT: 55 ± 5%, n = 9);
for R243C it was the same as wild-type channels (R243C: 48 ± 3%, n = 7; WT: 46 ± 7%, n = 8); for R366Q (R366Q: 72 ± 3%, n = 9; WT: 52 ± 6%, n =
9) and R555C (R555C: 48 ± 5%, n = 8, WT: 24 ± 2%, n = 8) inhibition were stronger than wild-type channels. (h) effect of expression of pI5kinase on
wild-type and mutant currents measured at +40 mV after 4 s depolarization from -80 mV. Currents were normalized to the current without pI5kinase
expressed. Current in cells expressing pI5kinase was higher than in cells without pI5 kinase being expressed for WT for R366Q (3.5 ± 0.3, n = 18) and
R555C (2.0 ± 0.1, n = 8) mutant channels and lower for R174C (0.9 ± 0.1, n = 20) when compared to wild-type. For R243C (1.7 ± 0.1, n = 27) activation
was not significantly different than wild-type (1.5 ± 0.1, n = 17).
www.landesbioscience.com Channels 5
Figure 1. For figure legend, see page 4.
6 Channels Volume 4 Issue 1
A decrease in the channel affinity to PIP2 is expected to increase
sensitivity to changes in membrane PIP2 levels, as seen for a num-
ber of PIP2 sensitive channels.19-22 Our results are consistent with
both R366 and R555 being PIP2-interacting sites.
LQT1 related mutations affect IKs regulation by PKA. To
assess the PKA effects on the channel, we applied forskolin (50
μM), which activates PKA through direct stimulation of adenylyl
cyclase, and measured its effects on the activity KCNQ1/KCNE1
channels (Fig. 2A and B). KCNQ1/KCNE1 currents were poten-
tiated by forskolin. To determine the voltage dependence of IKs,
we constructed isochronal (t = 10 s) activation curves. Forskolin
applications significantly shifted the voltage of activation of the
IKs channel to more hyperpolarizing voltages. These findings are
consistent with data published for the PKA regulation of the IKs
channels.5,14,31 In addition, direct phosphorylation by PKA has
been shown biochemically for KCNQ1/KCNE1 channels.5
We tested whether the four LQT1 associated mutations affected
PKA regulation of the channel. Note the effects of the mutations
shown in Figure 1B are opposite to the effect of channel regula-
tion by PKA (Fig. 2A). As in Figure 2A we applied forskolin (50
μM) and measured its effects on the activity of wild-type and
mutant channels subunits co-expressed with KCNE1. For the
two mutations present in the cytoplasmic loops of the channel,
R174C and R243C (Fig. 5), there was no significant increase in
current after forskolin treatment (Fig. 2E). In addition, there was
no shift in the voltage dependence of activation caused by for-
skolin (Fig. 2F). For the two mutations in the c-terminus of the
channel, R366Q and R555C, the shift in the voltage dependence
of activation caused by forskolin was not affected by the muta-
tion, and activation by forskolin was increased when compared to
wild-type (Fig. 2B and C).
LQT1 related mutations affect IKs regulation by PKC. To
assess the PKC-mediated effects on the channel, we co-expressed
KCNQ1 and KCNE1 subunits with muscarinic type 1 (M1) recep-
tors. Stimulation of the M1 showed two phases of regulation: first, a
decrease of the current, followed by current increase (Fig. 3C). The
activation phase of this response was significantly reduced by the
PKC inhibitor calphostin C (2 μM) (Fig. 3E). These findings are
consistent with data showing that PKC phosphorylation underlies
IKs activation upon GqPCR stimulation.13 For these experiments
we co-expressed KCNQ1, KCNE1 and the M1 receptor with IP3-
phosphatase (IP3phosp) to inhibit intracellular Ca2+-release and
endogenous Ca2+-dependent Cl- currents present in oocytes.
In order to test whether the mutations also affected PKC-
mediated activation upon GqPCR stimulation of the channel, we
co-expressed M1 receptors together with KCNE1 and KCNQ1
mutant subunits. We measured channel activation upon stimula-
tion by ACh for three LQT1 mutants. For KCNQ1(R174C) and
KCNQ1(R243C) the activation phase was blunted (Fig. 3A–D).
For the KCNQ1(R366Q) mutant channel the activation was
potentiated. PKC activation effects paralleled the effect of the
mutation on the PKA activation for the three mutants tested.
PKC and PIP2 regulationof IKs crosstalk. We previously
showed that PKA regulation can alter response of the IKs chan-
nel to PIP2 in both heterologous and native systems.14 To test
whether PKC-mediated activation of the channel also depended
of IKs is dependent on the length of the depolarizing pulse, but
relative shifts in the voltage dependence persist, independent of
the length of the pulse.27 A typical response to depolarizing volt-
ages is shown in Figure 1A for each of the mutants tested. All
four mutants showed a decrease in function when compared to
the wild-type channel (Fig. 1B). In addition, all mutants tested
showed a significant right shift in the voltage dependence of acti-
vation and a decrease in Gmax (Fig. 1B).
In order to test whether mutations associated with LQT1
affect channel regulation by PIP2, we used two approaches.
First, we co-expressed the GqPCR bradykinin type 2 (BK2)
receptor together with KCNE1 and KCNQ1 mutant subunits.
Bradykinin-mediated inhibition of IKs was shown to be medi-
ated by PIP2 depletion.13 For other GqPCRs, a PKC-mediated
activation follows the inhibition phase (Fig. 3). This PKC-
mediated activation is strongly decreased for the BK2 receptor,
allowing the study of the PIP2 contribution in isolation (Fig.
1C).13 We measured channel regulation upon stimulation by BK
for four LQT1 mutants (Fig. 1D and E). The inhibition mea-
sured for each mutant was compared to the inhibition observed
in wild-type channels expressed in the same batch of oocytes. For
three of the mutant channels tested, BK regulation was affected.
Mutations had similar effects on agonist-induced inhibition
observed after stimulation of another GqPCR, the muscarinic
type 1 receptor (M1) (data not shown). Agonist-inhibition could
not be assessed for the R366Q mutant after M1 receptor stimula-
tion because it was masked by the dramatic increase in PKC acti-
vation (Fig. 3). For KCNQ1(R174C)/E1, the inhibitory phase
was blunted. For KCNQ1(R243C)/E1 there was no significant
effect on the inhibition phase. For the KCNQ1(R366Q)/E1
and KCNQ1(R555C)/E1 mutant channels, the inhibition phase
was potentiated. KCNQ1(R555) residue has been suggested to
directly interact with PIP2.28 Our results show that mutations
in the R366 and R555 increased channel sensitivity to agonist
As a second approach to assess changes in channel regulation
by PIP2 in the mutant channels, we increased membrane PIP2 lev-
els by expressing PI(4)5-kinase (PI5-kinase), one of the enzymes
responsible for PIP2 production. Overexpression of PI5-kinase
in atrial myocytes or in sympathetic neurons tonically increases
membrane PIP2 levels and dramatically reduces PIP2 mediated
regulation for PIP2-sensitive channels. It decreases desensitization
of Kir3 channels and muscarinic modulation of endogenous M
current.29,30 Overexpression of PI5-kinase increased channel cur-
rent for wild-type, R243C, R366Q and R555C mutant channels,
but not R174C, suggesting that the latter is tonically saturated by
basal PIP2 levels (Fig. 1H). R366Q and R555C channels show a
larger PI5 kinase-mediated increase in current when compared
to wild-type channels. Increased sensitivity to changes in mem-
brane PIP2 levels indicate a decrease in the apparent affinity of
PIP2 for the R366Q and R555C channels. The changes observed
are not consistent with an exclusive decrease in PIP2 efficacy, but
our results do not preclude efficacy changes in addition to affinity
changes. Binding to PIP2 is necessary for IKs channel activity.22
Mutations in residues interacting with PIP2 are not expected to
abolish lipid binding but to decrease PIP2 affinity to the channel.
www.landesbioscience.com Channels 7
on PIP2, we studied dependence of PKC activa-
tion on membrane PIP2 levels. PIP2 is produced
in the plasma membrane by sequential phospho-
rylation of PI by PI4-kinase and PI(4)5-kinase
(PI5-kinase). Altering either the expression level
or activity of either enzyme will markedly affect
membrane PIP2 levels. We measured IKs chan-
nel regulation by ACh and studied the effect
of changes of PIP2 levels in the PKC-mediated
activation (Fig. 4). Treatment of the cells with
the PI4-kinase inhibitor wortmannin (5 μM)
(WMN) for 30–60 min has been shown to
inhibit the IKs channel by decreasing mem-
brane PIP2 levels.13 Here we show that 5 μM
WMN, for 30–60 min potentiated the PKC-
mediated activation phase (Fig. 4). WMN
treatment inhibited channel current by 48 ±
13% (n = 6).
We assessed effects of increasing membrane
PIP2 levels by overexpressing PI5-kinase. An
inactive, truncated PI5-kinase was used as a
negative control. Overexpression of PI5-kinase,
but not the inactive PI5-kinase mutant, and
consequent increase in membrane PIP2 levels,
blunted PKC-induced IKs activation (Fig. 4B).
Currents were also larger when PI5-kinase was
expressed (Fig. 4C) suggesting the unstimu-
lated channels in the absence of PI5-kinase
expression, are not saturated by PIP2.
We studied the effect of PKC activation on
the PIP2-mediated WMN inhibition and the
PI5-kinase activation of the wild-type chan-
nel. Both inhibition by WMN and activation
by PI5-kinase were blunted in PKC-activated
channels (Fig. 4C). These results suggest a
crosstalk between PIP2 and PKC regulation of
IKs as previously seen for the PKA and PIP2
signaling. PIP2 activates the channel in a dose
dependent manner. For a given membrane PIP2
level, a channel with higher PIP2 affinity will be
closer to saturation and less sensitive to changes
in membrane PIP2 level. For channel with a
weaker apparent affinity for PIP2, both depletion
of membrane PIP2 and increase in membrane
PIP2 levels are expected to cause an increase
in the fractional of change of the current. For
channels with only changes in efficacy of PIP2
activation, the fractional WMN-inhibition and
activation by PI5-kinase are expected to be the
same. Our data is consistent with the PKC acti-
vated channel having an increase in the apparent
affinity to PIP2, and it is compatible with PKC
strengthening KCNQ1 interactions with PIP2.
To test whether the PKC regulation of response
to PIP2 was abolished for the PKC-insensitive
mutant R243C, we measured the effect of
Figure 2. PKA regulation is modified by LQT1 mutants. (A) Typical response to forskolin
treatment in oocytes expressing KCNQ1/KCNe1 subunits. Currents were measured to de-
polarized steps from -80 to +80 mV from a -80 mV holding potential followed by a -40 mV
step. (B) Left: summary data for current activation by forskolin measured after 4 s depolar-
ization to +40 mV and voltage dependence of activation for cells either treated or untreated
with 50 μM forskolin for 60–90 min (untreated: 1.00 ± 0.05, n = 25; treated 1.33 ± 0.05, n =
24). Forskolin was present during the measurement of the currents for treated cells. Right:
V1/2 for current activation for cells either treated or untreated with forskolin (as in a) (un-
treated: 17 ± 2 mV, n = 25; treated 5 ± 1 mV, n = 25). V1/2 was calculated from a Boltzmann
fit of the tail current data. Tail currents were measured at -40 mV after 6 s depolarizing
pulses from -60 mV to +80 mV from -80 mV holding potential. (C) Typical KCNQ1(R366Q)
response to forskolin treatment. Currents were measured to 6 s depolarized steps from
-80 to +80 mV from a -80 mV holding potential followed by a -40 mV step. (D) summary
currents for KCNQ1/KCNe1 wild-type and mutant channels (n = 7–21). Whole-oocytes
currents were measured after 4 s depolarization to +40 mV from a -80 mV holding potential
for wild-type channels and each of the mutants indicated. Foskolin potentiation: WT: 1.38 ±
0.05, n = 24; R174C: 1.20 ± 0.12, n = 7; R243C: 1.21 ± 0.12, n = 19; R366Q: 2.11 ± 0.18, n =
16; R555C: 1.68 ± 0.10, n = 16. Currents were normalized to the average untreated current.
(e) shift in V1/2 for current activation for cells treated with forskolin compared to untreated
cells (as in a). V1/2 was calculated from a Boltzmann fit of the tail current data measured
at -40 mV (in mV: WT: -13.3 ± 1.4, n = 25; R174C: 1.0 ± 0.7, n = 7; R243C: 4.6 ± 7.1, n = 8;
R366Q: -13.3 ± 2.0, n = 13; R555C: -11.1 ± 3.5, n = 11). Channels expressing R174C and
R243C mutant subunits had no significant increase in current or shift in V1/2 after forskolin
treatment. Regulation of wild type channels were compared to mutant channel regulation in
the same batches of oocytes.
8 Channels Volume 4 Issue 1
both blunting and potentiating regula-
tion. Our data indicates that mutations
that alter channel sensitivity to PIP2 can
be rescued by regulatory inputs and that
one mutation in the S2-S3 and another in
the S4-S5 loop impair channel regulation.
We suggest that effects on channel regula-
tion can be important in the risk strati-
fication of LQT patients and may have
important implications for disease treat-
ment. In addition, this work is the first to
suggest that changes in Gq-coupled regu-
lation underlie the pathophysiology of this
The role of PIP2 as a second messenger
molecule has become increasingly impor-
tant through its modulation of a growing
number of ion channels and transporters
(reviewed in ref. 32). For Kir channels,
several stimuli exert their effects through
modifying interactions of the channel with
PIP2.19,23,33 Mutations of the inward recti-
fiers Kir2.1 and Kir1.1 have been shown
to cause disruptions in the channel-PIP2
interactions leading to both Andersen’s and
Bartter’s syndromes.21 The activity of the
channels formed by KCNQ1 and KCNE1
subunits, by KCNQ1 subunits alone, and
by all other members of the KCNQ fam-
ily have been shown to be critically depen-
dent on PIP2.22,34,35 KCNQ channels were
shown to be activated by PIP2 and chan-
nel run down was linked to PIP2 hydro-
lysis.22 In addition, for channels formed
by the KCNQ2 and KCNQ3 subunits,
agonist-induced depletion of PIP2 has
been associated with channel inhibition.22
PIP2-dependent rundown and exogenous
PIP2 application were shown to shift the
voltage dependence of IKs activation.28,36
We have shown that GqPCRs inhibit the
IKs channel by depleting membrane PIP2
Decreases in IKs channel-PIP2 interac-
tion have been suggested to be associated
with LQT1.28 We tested whether LQT mutants modify PKA
and PKC regulation of IKs. We tested four putative PIP2 interac-
tion sites of the channel (R174C, R243C, R366Q and R555C),
which are highly conserved in other KCNQ channels, also shown
to interact with PIP2. R174 is located in the S2-S3 intracellular
loop, R243 in the S4-S5 loop, R366 and R555 in the c-terminus
of the channel. Park and colleagues,13 by adding PIP2 directly
to excised patches, suggested less PIP2 was needed to further
activate endogenous currents for three LQT1 mutant channels
(R243H, R539W and R555C). In addition, they showed that the
charge of the R555C residue was important for maintenance of
WMN treatment and PI5-kinase expression in PKC-stimulated
and unstimulated R243C mutant channel. Currents were nor-
malized to cells not expressing PI5kinase (left) or cells without
WMN treatment. For this mutant channel, sensitivity to changes
in PIP2 levels was maintained after PKC-activation (Fig. 4D).
Here we show that several mutations associated with the LQT1
syndrome affect both PKA and GqPCR regulation of IKs.
Different mutations can alter regulation in a diverse manner,
Figure 3. PKC regulation is modified by LQT1 mutants. Effect of M1 receptor stimulation on the
KCNQ1(R174C)/KCNe1, KCNQ1(R243C)/KCNe1 and KCNQ1(R366Q)/KCNe1 mutant channel
currents when compared to WT regulation. (a–D) Left: typical current response to aCh 10 μM
before (a), during the inhibition (b) and activation (c) phase induced by agonist stimulation. Right:
time course of the effects of agonist stimulation measured at the end of the depolarizing pulse.
Dashed line represents initial normalized current level. Depolarizing step to +40 mV from -80 mV.
(e) activation is inhibited by the pKC inhibitor calphostin-C. summary data: current activation
upon application of 10 μM aCh in the presence and absence of pKC inhibitor calphostin-C (2
μM). Cells were pre-treated with calphostin-C for one hour and the drug was present during the
course of the experiment. activation was calculated as % increase with respect to the minimum
current. (F) Mutant activation compared to wild-type. The activation phase of the regulation was
disrupted for all mutants tested. summary data: regulation for each mutant was compared to
wild-type regulation measured in same oocyte batches. activation for R174C (87 ± 15%, n = 32)
was lower than wild-type (160 ± 26%, n = 33); activation for R243C (27 ± 6%, n = 13) was lower
than wild-type (191 ± 49%, n = 14) and activation of R366Q (406 ± 67%, n = 25) was stronger than
wild-type (148 ± 35%, n = 21).
www.landesbioscience.com Channels 9
channel activity, strongly suggesting R555 was a PIP2 interacting
site. Our results agree with these results for R555C. The residues
tested here affected channel regulation by distinct mechanisms.
R174C decreased PKA, PKC and PIP2 regulation, suggesting that
it is important for the transduction of regulatory inputs. R243C
decreased PKA and PKC activation, suggesting that the S4-S5
region may be necessary to transduce PKA and PKC regulation
to the channel gate. Indeed, the S4-S5 linker region has been sug-
gested to interact with KCNE1.37 We also previously showed the
residue in KCNE1 (S102) is important for channel regulation by
PKC.13 In addition, KCNE1 mutations have been shown to affect
IKs regulation by PKA without affecting channel phosphoryla-
tion.38 For the R366Q mutant channel, a stronger inhibition by
PIP2-depletion and activation by increases in membrane PIP2
Figure 4. Changes in pIp2 levels modulate pKC-mediated activation. Current activation induced by 10 μM aCh measured as in Figure 3. (a) Typical
trace of aCh regulation in cells after 30–60 min WMN treatment and co-expressing pI5kinase. Depolarizing step to +40 mV from -80 mV. (B) activa-
tion phase is stronger with after WMN treatment (control: 152 ± 17%, n = 10; WMN: 298 ± 77%, n = 6), presumably because of lower levels of mem-
brane pIp2. activation phase is lower when pI5-kinase was overexpressed, presumably because of higher membrane pIp2 levels (control: 152 ± 14%, n =
16; pI5-Kinase: 2.9 ± 1.3%, n = 15; pI5-kinaseΔ1-238: 181 ± 23%, n = 6). Significant difference from WT regulation was indicated. In all experiments IP-
3phosp was co-expressed to block Cai
pI5kinase are abolished after pKC-mediated channel activation. Basal current is measured before aCh stimulation. pKC-stimulated current is measured
800 s after aCh stimulation. Currents were measured after 4 s depolarization to +40 mV from -80 mV. (D) Comparison of regulation of wild-type and
R243C mutant channel. pKC-induced effects were abolished for the mutant channel. Left: pI5kinase activation is preserved after pKC activation for
R243C channels. Right: WMN inhibition is preserved for the R243C mutant channel (n at least 6 for all experiments).
2+-release. (C) Data as in (B) normalized to wild-type control current. Both inhibition by WMN and activation by
Figure 5. scheme depicting LQT1 mutation location.
10 Channels Volume 4 Issue 1
levels was observed, consistent with the mutant channel having
weaker interactions with PIP2 than the wild-type channel. We
also tested the R555C channel, suggested by Park et al. to interact
with PIP2, which also shows stronger PIP2-mediated inhibition
and PI5-kinase-mediated activation. Our data suggest that muta-
tions in R366 and R555 increase channel sensitivity to changes
in membrane PIP2 levels, suggesting a decrease in the apparent
affinity of the channel to PIP2 for these mutant channels. That is
not the case for R174 and R243.
For the four mutants tested, regulation of the channel was
altered. A decrease in the activation both by PKA and PKC such
as the one observed for cytoplasmic-loop KCNQ1(R174C) and
KCNQ1(R243C) may contribute to the disease phenotype by
decreasing normal channel activation upon receptor stimulation.
This would be expected to be independent of the QTc prolonga-
tion measured at rest for these patients. An increase in the activa-
tion such as the one observed for the c-terminal KCNQ1(R366Q)
and KCNQ1(R555C) mutants may be protective and decrease
the burden of the mutation. Indeed, patients with mutations in
the c-terminus region of the KCNQ1 subunit have been found
to be less prone to cardiac arrhythmias than patients with muta-
tions in the transmembrane regions, independently of the effect
of the mutation on QT interval.24 In addition, in contrast to most
LQT1 patients, patients with KCNQ1(R555C) mutation have
been found not to have exercise induced syncopes.39 Most c-ter-
minus mutations linked to LQT1 occur in or in close proximity
to conserved positive amino acids, suggesting this may be a com-
mon feature of LQT1 C-terminus mutations. Our data provides
a possible mechanistic explanation for the milder clinical pheno-
type of any mutant that decreases channel sensitivity to PIP2.
For the R366Q and R555C channels, which our results sug-
gest interact with PIP2, regulation of the channel was potentiated,
partially rescuing the mutant phenotype. We recently showed that
PKA modulated channel response to PIP2 for the IKs channel.14
In addition, for Kir channels, PKC has been shown to regulate
the channels through modifying channel-PIP2 interactions.23,40
Our results indicate that PKC regulation of IKs is dependent
of membrane PIP2 levels. Our data is compatible with a model
where PIP2 binding to the C-terminus of the channel maintains
channel activity and both PKA and PKC activate the channel by
strengthening KCNQ1 interactions with PIP2.
Our results suggest that there is altered GPCR regulation
for several mutant channels linked to LQT1. IKs regulation by
GqPCRs may prove to be one of the causes of cardiac arrhythmias
both in heart failure, where Gq-coupled receptors are particularly
important in regulating inotropy, and for long-QT patients. The
understanding IKs regulation may allow us to devise innovative
strategies to treat patients with these conditions.
Materials and Methods
Molecular biology. Human KCNQ1, KCNE1, M1, AT1a and
BK2 clones were subcloned in to the pGEMsh vector (modified
from PGEMHE vector25) for oocyte expression.21,26 Site direct
mutagenesis was performed using PFU ultra DNA polymerase
(Stratagene). Construct sequences were confirmed by DNA
sequencing (Cornell, Ithaca). cRNAs were transcribed using the
“message-machine” kit (Ambion) and RNA concentrations were
estimated using RNA markers (Gibco).
Electrophysiology. Xenopus oocytes were harvested, dissoci-
ated and defolliculated by collagenase type I (Sigma) treatment.
cRNA was injected at the approximately concentrations: 2 ng for
KCNQ1, 0.4 ng for KCNE1, 2 ng for IP3-phosphatase, AT1a,
M1 or BK2 receptors. Wild-type and mutant PI5-kinase RNA
were injected into oocytes one day after receptor and channel
subunits at a concentration of 0.7 ng/oocyte.
Oocytes were constantly superfused with (in mM): 82.5
NaCl, 2 KCl, 1 MgCl2, 1.8 CaCl2, 5 NaOH/HEPES (pH
7.5). Currents were evaluated 5–10 min after oocytes impale-
ment. Cl--free solutions were used to inhibit endogenous Ca2+-
activated Cl- currents for experiments where BK2 receptor was
expressed and Cai
Na-acetate, 2 KOH, 1 Mg-sulfate, 1.8 Ca-acetate, 5 NaOH/
HEPES, pH 7.5. IKs currents were measured after 2 s depolar-
ization to +60 mV from -80 mV holding potential unless oth-
Error bars represented standard-error of the mean. All experi-
ments were performed in at least 3–6 oocytes from the same batch
and at least 2–3 oocyte batches were used. Test conditions and
control experiments were always done on oocytes from the same
batch. Student t-test (two groups) or one-way ANOVA (more
than two groups) were applied for the assessment of statistical
significance. The investigation conforms with the Guide for the
Care and Use of Laboratory Animals published by the US National
Institutes of Health (NIH Publication No. 85-23, revised 1996).
Protocol approval was granted by a university ethics review board
2+-release occur, and contained (in mM): 82.5
We thank Jeffrey Le for preparing oocytes, as well as Daniel Gray
for useful comments on the manuscript. This work was supported
by a Scientist Development Grant (0430052N) from American
Heart Association (AHA) and NIH (R01HL033843).
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