Here we show that Gq-coupled receptors regulate IKs in a biphasic manner. Although downstream signaling molecules from GqPCR activation have been suggested to activate the channel [9
], their physiological role in GqPCR stimulation has not been established. We studied the role of intracellular Ca2+
-release, PKC phosphorylation and PIP2
depletion for Gq-coupled receptor regulation of IKs. We show that the activation phase is mediated by PKC phosphorylation and that PIP2
depletion underlies channel inhibition. This is the first time that evidence for a molecular mechanism is presented to explain Gq-coupled receptor inhibition of IKs. The work we present here is the first to study regulation by Gq-coupled receptors on channels formed by KCNQ1 and KCNE1 subunits in intact cells and suggests that this is an important pathway of heart rhythm regulation.
The kinetics of channel regulation observed in and for the stimulation of the three receptors is complex. Using a simple model we can simulate the basic kinetic parameters and magnitude of activation and inhibition of the channel with 3 components: one inhibitory component and two activation components (). The inhibitory component corresponds to PIP2 depletion, modeled by an exponential inhibition. The activation components correspond to a fast and a slow/delayed activation, modeled by sigmoidal functions. Our data suggest that most of the activation phase is mediated by PKC, but they do not preclude a small contribution from other signaling pathways.
Schematic model of IKs current regulation by M1, AT1 and BK2 receptors
The change in kinetics of activation observed when comparing and requires the faster activation component to be more prominent in the presence of intracellular Ca2+ release (). Changes in the inhibitory component are not necessary to fit the data, but are not precluded. Nonetheless, activation of endogenous Ca2+-dependent Cl− currents were the same for the receptors tested (data not shown), suggesting equivalent Ca2+-release upon receptor activation and hydrolysis of PIP2. For simplicity, the model assumes a full inhibition by PIP2. A strong inhibition by PIP2 is necessary to explain the average inhibition observed in the concomitant presence of an activating signal. A complete inhibition is likely not achieved because of activation of lipid kinases in response to PIP2 depletion.
The lack of activation observed with BK stimulation can be explained by a smaller contribution of the fast activation component for this receptor ( right panel). A decrease in the fast activation component also explains the faster kinetics observed for BK inhibition when compared to ACh and angII in the absence of intracellular Ca2+-release (). The model also explains the slower kinetics of inhibition observed for BK stimulation in the presence of Ca2+ when compared to the absence of Ca2+ ( A and B right panels). Our data does not preclude that the fast-activation component is transient, but that is not necessary to explain the overall data.
We hypothesize that a faster channel response to Ca2+
-dependent PKCs occurs in the presence of intracellular Ca2+
release. In the absence of intracellular Ca2+
-release, a slower response to Ca2+
-independent PKCs is more prominent. Our data is consistent with the translocation of the Ca2+
-dependent isoforms of PKC being impaired with bradykinin stimulation. Indeed, bradykinin has been shown to activate PKC δ and PKCε but not PKCα in neonatal ventricular cardiomyocytes [31
], while angiotensin has been shown to mediate redistribution of PKCε and the Ca2+
dependent PKC α and βII in guinea pig heart [32
PKC is a serine/threonine kinase that has been implicated in many pathological conditions, such as cardiac hypertrophy, arrhythmias, heart failure and ischemic preconditioning [33
]. Knowledge of the arrhythmogenesis mechanism of PKC is very limited. Use of specific PKC blockers have been suggested as novel treatments for heart failure [36
]. Channels formed with rat, mouse or human KCNE1 protein together with the endogeneous KCNQ subunit in oocytes have been shown to be inhibited by PMA [4
], a PKC activator. Phosphorylation of the KCNE1 S102 residue, also present in human KCNE1 has been implicated in this effect. More recent experiments on channels formed by human KCNQ1 and KCNE1 subunits showed current activation by PMA. The effect was blocked by the PKC inhibitor calphostin-C [10
]. In addition, angiotensin II was suggested to potentiate atrial IKs through activation of PKC [39
]. Phosphorylation of the KCNQ1 subunit has also been suggest to be involved in PMA regulation of the IKs channel [40
]. Our data does not preclude the phosphorylation of the KCNQ1 subunit, but it suggests that KCNE1(S102) phosphorylation accounts for about half of the PKC-mediated activation observed upon Gq agonist stimulation for the human channel.
-release controls cardiac contraction. Abnormal release of Ca2+
from sarcoplasmic reticulum contributes to contractile dysfunction and arrhythmogenesis in heart failure. In heart failure the sarcoplasmic reticulum has reduced Ca2+
content so that the amount of Ca2+
released is smaller than under normal conditions [41
]. Channels formed by co-expression of the KCNQ1 and KCNE1 subunits have been reported to be activated by increases in intracellular calcium with use of the Ca2+
ionophore ionomycin [11
]. Work done with KCNE1 and endogenous oocyte KCNQ subunits showed that this channel is inhibited by decreases and enhanced by increases in intracellular Ca2+
. Phosphorylation of the KCNE1 subunit by calmodulin-dependent kinases were implicated in this effect [4
] (for review see [45
]). However the phosphorylated residue (S40) is not present in the human KCNE1. Recently two papers suggested that basal intracellular Ca2+
affects both gating and assembly of IKs channels [16
]. Our work does not contradict these data, here we studied whether increases of Ca2+
above resting level regulate channel gating and does not preclude regulation of channel gating by basal Ca2+
. Nonetheless, our data suggests that the activation phase is mediated by PKC and main effect of IP3-dependent Ca+2
-release upon GqPCR stimulation is not direct. We suggest that intracellular Ca2+
-release regulates PKC-mediated activation, possibly through a Ca2+
-dependent PKC isoform. A minor effect of Ca2+
-Calmodulin in the early activation phase was not precluded.
The role of PIP2
as a second messenger molecule has become increasingly important through its modulation of a growing number of ion channels and transporters (for review see [46
]). For Kir channels, several stimuli exert their effects through modifying interactions of the channel with PIP2
]. Mutations of the inward rectifiers 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 [50
]. The activity of the channels formed by KCNQ1 and KCNE1 subunits, by KCNQ1 subunits alone, and by all other members of the KCNQ family have been shown to be critically dependent on PIP2
]. KCNQ channels were shown to be activated by PIP2
and channel run down was linked to PIP2
]. In addition, for channels formed by the KCNQ2 and KCNQ3 subunits, agonist-induced depletion of PIP2
has been associated with channel inhibition [12
-dependent rundown/exogenous PIP2
application was shown to shift the voltage dependence of IKs activation [19
]. Our work indicates that PIP2
depletion, upon Gq-coupled receptor activation, inhibits channel activity.
Understanding the signaling pathways underlying IKs regulation provides insight into the molecular basis of electrical signaling in the normal heart and the pathophysiology of heart failure and long-QT syndrome. Our results suggest that 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 Long-QT syndrome. Understanding IKs regulation may allow us to devise innovative strategies to treat cardiac arrhythmias.