A PIP2 substitute mediates voltage sensor-pore coupling in KCNQ activation.
ABSTRACT: KCNQ family K+ channels (KCNQ1-5) in the heart, nerve, epithelium and ear require phosphatidylinositol 4,5-bisphosphate (PIP2) for voltage dependent activation. While membrane lipids are known to regulate voltage sensor domain (VSD) activation and pore opening in voltage dependent gating, PIP2 was found to interact with KCNQ1 and mediate VSD-pore coupling. Here, we show that a compound CP1, identified in silico based on the structures of both KCNQ1 and PIP2, can substitute for PIP2 to mediate VSD-pore coupling. Both PIP2 and CP1 interact with residues amongst a cluster of amino acids critical for VSD-pore coupling. CP1 alters KCNQ channel function due to different interactions with KCNQ compared with PIP2. We also found that CP1 returned drug-induced action potential prolongation in ventricular myocytes to normal durations. These results reveal the structural basis of PIP2 regulation of KCNQ channels and indicate a potential approach for the development of anti-arrhythmic therapy.
Project description:Retigabine (RTG) is a first-in-class antiepileptic drug that suppresses neuronal excitability through the activation of voltage-gated KCNQ2-5 potassium channels. Retigabine binds to the pore-forming domain, causing a hyperpolarizing shift in the voltage dependence of channel activation. To elucidate how the retigabine binding site is coupled to changes in voltage sensing, we used voltage-clamp fluorometry to track conformational changes of the KCNQ3 voltage-sensing domains (VSDs) in response to voltage, retigabine, and PIP2. Steady-state ionic conductance and voltage sensor fluorescence closely overlap under basal PIP2 conditions. Retigabine stabilizes the conducting conformation of the pore and the activated voltage sensor conformation, leading to dramatic deceleration of current and fluorescence deactivation, but these effects are attenuated upon disruption of channel:PIP2 interactions. These findings reveal an important role for PIP2 in coupling retigabine binding to altered VSD function. We identify a polybasic motif in the proximal C terminus of retigabine-sensitive KCNQ channels that contributes to VSD-pore coupling via PIP2, and thereby influences the unique gating effects of retigabine.
Project description:KCNQ2-5 (Kv7.2-Kv7.5) channels are strongly influenced by an emerging class of small-molecule channel activators. Retigabine is the prototypical KCNQ activator that is thought to bind within the pore. It requires the presence of a Trp side chain that is conserved among retigabine-sensitive channels but absent in the retigabine-insensitive KCNQ1 subtype. Recent work has demonstrated that certain KCNQ openers are insensitive to mutations of this conserved Trp, and that their effects are instead abolished or attenuated by mutations in the voltage-sensing domain (VSD). In this study, we investigate the stoichiometry of a VSD-targeted KCNQ2 channel activator, ICA-069673, by forming concatenated channel constructs with varying numbers of drug-insensitive subunits. In homomeric WT KCNQ2 channels, ICA-069673 strongly stabilizes an activated channel conformation, which is reflected in the pronounced deceleration of deactivation and leftward shift of the conductance-voltage relationship. A full complement of four drug-sensitive subunits is required for maximal sensitivity to ICA-069673-even a single drug-insensitive subunit leads to significantly weakened effects. In a companion article (see Yau et al. in this issue), we demonstrate very different stoichiometry for the action of retigabine on KCNQ3, for which a single retigabine-sensitive subunit enables near-maximal effect. Together, these studies highlight fundamental differences in the site and mechanism of activation between retigabine and voltage sensor-targeted KCNQ openers.
Project description:In voltage-activated ion channels, voltage sensor (VSD) activation induces pore opening via VSD-pore coupling. Previous studies show that the pore in KCNQ1 channels opens when the VSD activates to both intermediate and fully activated states, resulting in the intermediate open (IO) and activated open (AO) states, respectively. It is also well known that accompanying KCNQ1 channel opening, the ionic current is suppressed by a rapid process called inactivation. Here we show that inactivation of KCNQ1 channels derives from the different mechanisms of the VSD-pore coupling that lead to the IO and AO states, respectively. When the VSD activates from the intermediate state to the activated state, the VSD-pore coupling has less efficacy in opening the pore, producing inactivation. These results indicate that different mechanisms, other than the canonical VSD-pore coupling, are at work in voltage-dependent ion channel activation.
Project description:Voltage-gated potassium channels of the KCNQ (Kv7) subfamily are essential for control of cellular excitability and repolarization in a wide range of cell types. Recently, we and others found that some KCNQ channels functionally and physically interact with sodium-dependent solute transporters, including myo-inositol transporters SMIT1 and SMIT2, potentially facilitating various modes of channel-transporter signal integration. In contrast to indirect effects such as channel regulation by SMIT-transported, myo-inositol-derived phosphatidylinositol 4,5-bisphosphate (PIP<sub>2</sub>), the mechanisms and functional consequences of the physical interaction of channels with transporters have been little studied. Here, using co-immunoprecipitation with different channel domains, we found that SMIT1 binds to the KCNQ2 pore module. We next tested the effects of SMIT1 co-expression, in the absence of extracellular myo-inositol or other SMIT1 substrates, on fundamental functional attributes of KCNQ2, KCNQ2/3, KCNQ1, and KCNQ1-KCNE1 channels. Without exception, SMIT1 altered KCNQ ion selectivity, sensitivity to extracellular K<sup>+</sup>, and pharmacology, consistent with an impact on conformation of the KCNQ pore. SMIT1 also altered the gating kinetics and/or voltage dependence of KCNQ2, KCNQ2/3, and KCNQ1-KCNE1. In contrast, SMIT1 had no effect on Kv1.1 (KCNA1) gating, ion selectivity, or pharmacology. We conclude that, independent of its transport activity and indirect regulatory mechanisms involving inositol-derived increases in PIP<sub>2</sub>, SMIT1, and likely other related sodium-dependent solute transporters, regulates KCNQ channel ion selectivity, gating, and pharmacology by direct physical interaction with the pore module.
Project description:All subtypes of KCNQ channel subunits (KCNQ1-5) require calmodulin as a co-factor for functional channels. It has been demonstrated that calmodulin plays a critical role in KCNQ channel trafficking as well as calcium-mediated current modulation. However, how calcium-bound calmodulin suppresses the M-current is not well understood. In this study, we investigated the molecular mechanism of KCNQ2 current suppression mediated by calcium-bound calmodulin. We show that calcium induced slow calmodulin dissociation from the KCNQ2 channel subunit. In contrast, in homomeric KCNQ3 channels, calcium facilitated calmodulin binding. We demonstrate that this difference in calmodulin binding was due to the unique cysteine residue in the KCNQ2 subunit at aa 527 in Helix B, which corresponds to an arginine residue in other KCNQ subunits including KCNQ3. In addition, a KCNQ2 channel associated protein AKAP79/150 (79 for human, 150 for rodent orthologs) also preferentially bound calcium-bound calmodulin. Therefore, the KCNQ2 channel complex was able to retain calcium-bound calmodulin either through the AKPA79/150 or KCNQ3 subunit. Functionally, increasing intracellular calcium by ionomycin suppressed currents generated by KCNQ2, KCNQ2(C527R) or heteromeric KCNQ2/KCNQ3 channels to an equivalent extent. This suggests that a change in the binding configuration, rather than dissociation of calmodulin, is responsible for KCNQ current suppression. Furthermore, we demonstrate that KCNQ current suppression was accompanied by reduced KCNQ affinity toward phosphatidylinositol 4,5-bisphosphate (PIP2) when assessed by a voltage-sensitive phosphatase, Ci-VSP. These results suggest that a rise in intracellular calcium induces a change in the configuration of CaM-KCNQ binding, which leads to the reduction of KCNQ affinity for PIP2 and subsequent current suppression.
Project description:Phosphatidylinositol 4,5-bisphosphate (PIP2) regulates Shaker K+ channels and voltage-gated Ca2+ channels in a bimodal fashion by inhibiting voltage activation while stabilizing open channels. Bimodal regulation is conserved in hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, but voltage activation is enhanced while the open channel state is destabilized. The proposed sites of PIP2 regulation in these channels include the voltage-sensor domain (VSD) and conserved regions of the proximal cytoplasmic C terminus. Relatively little is known about PIP2 regulation of Ether-á-go-go (EAG) channels, a metazoan-specific family of K+ channels that includes three gene subfamilies, Eag (Kv10), Erg (Kv11), and Elk (Kv12). We examined PIP2 regulation of the Elk subfamily potassium channel human Elk1 to determine whether bimodal regulation is conserved within the EAG K+ channel family. Open-state stabilization by PIP2 has been observed in human Erg1, but the proposed site of regulation in the distal C terminus is not conserved among EAG family channels. We show that PIP2 strongly inhibits voltage activation of Elk1 but also stabilizes the open state. This stabilization produces slow deactivation and a mode shift in voltage gating after activation. However, removal of PIP2 has the net effect of enhancing Elk1 activation. R347 in the linker between the VSD and pore (S4-S5 linker) and R479 near the S6 activation gate are required for PIP2 to inhibit voltage activation. The ability of PIP2 to stabilize the open state also requires these residues, suggesting an overlap in sites central to the opposing effects of PIP2 on channel gating. Open-state stabilization in Elk1 requires the N-terminal eag domain (PAS domain + Cap), and PIP2-dependent stabilization is enhanced by a conserved basic residue (K5) in the Cap. Our data shows that PIP2 can bimodally regulate voltage gating in EAG family channels, as has been proposed for Shaker and HCN channels. PIP2 regulation appears fundamentally different for Elk and KCNQ channels, suggesting that, although both channel types can regulate action potential threshold in neurons, they are not functionally redundant.
Project description:Upon membrane depolarization, the KCNQ1 potassium channel opens at the intermediate (IO) and activated (AO) states of the stepwise voltage-sensing domain (VSD) activation. In the heart, KCNQ1 associates with KCNE1 subunits to form I<sub>Ks</sub> channels that regulate heart rhythm. KCNE1 suppresses the IO state so that the I<sub>Ks</sub> channel opens only to the AO state. Here, we tested modulations of human KCNQ1 channels by an activator ML277 in <i>Xenopus</i> oocytes. It exclusively changes the pore opening properties of the AO state without altering the IO state, but does not affect VSD activation. These observations support a distinctive mechanism responsible for the VSD-pore coupling at the AO state that is sensitive to ML277 modulation. ML277 provides insights and a tool to investigate the gating mechanism of KCNQ1 channels, and our study reveals a new strategy for treating long QT syndrome by specifically enhancing the AO state of native I<sub>Ks</sub> currents.
Project description:KCNQ1 is the pore-forming subunit of cardiac slow-delayed rectifier potassium (IKs) channels. Mutations in the kcnq1 gene are the leading cause of congenital long QT syndrome (LQTS). Here, we present the cryoelectron microscopy (cryo-EM) structure of a KCNQ1/calmodulin (CaM) complex. The conformation corresponds to an "uncoupled," PIP2-free state of KCNQ1, with activated voltage sensors and a closed pore. Unique structural features within the S4-S5 linker permit uncoupling of the voltage sensor from the pore in the absence of PIP2. CaM contacts the KCNQ1 voltage sensor through a specific interface involving a residue on CaM that is mutated in a form of inherited LQTS. Using an electrophysiological assay, we find that this mutation on CaM shifts the KCNQ1 voltage-activation curve. This study describes one physiological form of KCNQ1, depolarized voltage sensors with a closed pore in the absence of PIP2, and reveals a regulatory interaction between CaM and KCNQ1 that may explain CaM-mediated LQTS.
Project description:Mutations in members of the KCNQ channel family underlie multiple diseases affecting the nervous and cardiovascular systems. Despite their clinical relevance, research into these channels is limited by the lack of subtype-selective inhibitors, making it difficult to differentiate the physiological function of each family member in vivo. We have proposed that KCNQ channels might partially underlie the calcium-activated slow afterhyperpolarization (sAHP), a neuronal conductance whose molecular components are uncertain. Here, we investigated whether 3-(triphenylmethylaminomethyl)pyridine (UCL2077), identified previously as an inhibitor of the sAHP in neurons, acts on members of the KCNQ family expressed in heterologous cells. We found that 3 μM UCL2077 strongly inhibits KCNQ1 and KCNQ2 channels and weakly blocks KCNQ4 channels in a voltage-independent manner. In contrast, UCL2077 potentiates KCNQ5 channels at more positive membrane potentials, with little effect at negative membrane potentials. We found that the effect of UCL2077 on KCNQ3 is bimodal: currents are enhanced at negative membrane potentials and inhibited at positive potentials. We found that UCL2077 facilitates KCNQ3 currents by inducing a leftward shift in the KCNQ3 voltage-dependence, a shift dependent on tryptophan 265. Finally, we show that UCL2077 has intermediate effects on KCNQ2/3 heteromeric channels compared with KCNQ2 and KCNQ3 homomers. Together, our data demonstrate that UCL2077 acts on KCNQ channels in a subtype-selective manner. This feature should make UCL2077 a useful tool for distinguishing KCNQ1 and KCNQ2 from less-sensitive KCNQ family members in neurons and cardiac cells in future studies.
Project description:Calmodulin (CaM) and phosphatidylinositol 4,5-bisphosphate (PIP<sub>2</sub>) are potent regulators of the voltage-gated potassium channel KCNQ1 (K<sub>V</sub>7.1), which conducts the cardiac <i>I</i> <sub>Ks</sub> current. Although cryo-electron microscopy structures revealed intricate interactions between the KCNQ1 voltage-sensing domain (VSD), CaM, and PIP<sub>2</sub>, the functional consequences of these interactions remain unknown. Here, we show that CaM-VSD interactions act as a state-dependent switch to control KCNQ1 pore opening. Combined electrophysiology and molecular dynamics network analysis suggest that VSD transition into the fully activated state allows PIP<sub>2</sub> to compete with CaM for binding to VSD. This leads to conformational changes that alter VSD-pore coupling to stabilize open states. We identify a motif in the KCNQ1 cytosolic domain, which works downstream of CaM-VSD interactions to facilitate the conformational change. Our findings suggest a gating mechanism that integrates PIP<sub>2</sub> and CaM in KCNQ1 voltage-dependent activation, yielding insights into how KCNQ1 gains the phenotypes critical for its physiological function.