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CALM binds KCNQ1. 213 / 214
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"First, it has not been shown that CaM interaction with KCNQ1 exhibits cooperativity."
37965565
sparser
"Notably, proximity between CaM and KCNQ1 tetramer does not necessitate cooperative binding."
37965565
sparser
"Taken together, our FRET assay detected robust binding between CaM and KCNQ1 WT as well as no interactions between CaM and KCNQ1-IQ/AA designed to ablate CaM binding to KCNQ1."
37965565
sparser
"To further investigate the key role of PIP 2 in KCNQ1 channel gating, we resolved KCNQ1-CaM structures in the presence of both PIP 2 and ML277."
36763058
sparser
"Cryo-EM structures of CaM-bound KCNQ1 obtained in the presence or absence of PIP2 [ xref , xref ] showed that, without PIP2, the S2–S3 linker interacts with the CaM C-lobe."
36077086
sparser
"CaM binds to the C-terminal of KCNQ1 and is essential for channel folding and assembly."
31825788
sparser
"Although no direct structure determination exists for KCNQ1-KCNE1 complexes, the structures of KCNQ1-CaM and KCNQ1-KCNE3-CaM have been solved and they also support the positioning of KCNEs in the aforementioned groove ( xref ) ( xref , xref )."
34247280
sparser
"CaM can bind and modulate Kv7.1 through interaction with two C‐terminal domains (helix A, HA and helix B, HB) in both in the absence and presence of Ca 2+ (Sachyani et al., xref ; Sun & MacKinnon, xref , xref ; Wiener et al., xref ; Yus‐Najera et al., xref )."
37428651
sparser
"PIP 2 -ML277-bound KCNQ1-CaM structures exhibited clearly two different conformations, KCNQ1-CaM ML277-PIP2-A and KCNQ1-CaM ML277-PIP2-B ( xref )."
36763058
sparser
"While the ML277-bound KCNQ1-CaM is stable in the attached conformation, the ML277-PIP 2 -bound KCNQ1-CaM seems dynamic and toggles between the attached and the detached conformations."
36763058
sparser
"The different conformations of the VSDs may be caused by different sample preparations (the detergent GDN used for KCNQ1-CaM apo vs. Digitonin for KCNQ1-CaM apo-6UZZ ) and (or) different constructs of KCNQ1 (the full-length KCNQ1 for KCNQ1-CaM apo vs. the truncated KCNQ1 with residues 76–620 for KCNQ1-CaM apo-6UZZ )."
36763058
sparser
"CaM is constitutively bound to KCNQ1 and mandatory for functional channel assembly."
36077086
sparser
"Then we sought to resolve KCNQ1-CaM structures with ML277 and PIP 2 to rescue the VSD-PD coupling."
36763058
sparser
"In the structure of KCNQ1-CaM ML277 , ML277 binds in the hydrophobic pocket formed by the S4-S5 linker and S5 from one subunit, and S5′ and S6′ from the adjacent subunit ( xref )."
36763058
sparser
"Since the CTD and CaM remain almost unchanged (attached), this ML277-induced opening of the activation gate in KCNQ1-CaM seems different from the PIP 2 -induced one in KCNQ1-CaM-KCNE3, which involves a large conformational change in the CTD of KCNQ1 and CaM (detached) ( xref )."
36763058
sparser
"Taken together, these functional studies support the ML277 binding site observed in the structure of KCNQ1-CaM ML277 ."
36763058
sparser
"HF‐associated changes may also influence interaction of KCNQ1 with CaM, a calcium‐binding second messenger that is a regulator of I Ks inactivation gating and channel assembly. xref Specifically, CaM has been shown to interact with KCNQ1 via 2 CaM‐binding domains within helices A and B of the KCNQ1 carboxyl terminus in a process that is required for channel tetramerization. xref , xref Given the essential role of CaM in regulating I Ks , reduced expression of CaM, as has been consistently demonstrated in HF, may be involved in the pathologic regulation of I Ks in HF. xref , xref "
32865116
sparser
"In comparison with those in KCNQ1-CaM apo , the CTD and CaM in KCNQ1-CaM ML277 remain almost unchanged, and KCNQ1-CaM ML277 is still in the attached conformation ( xref )."
36763058
sparser
"Here, we take a multipronged approach employing a live-cell fluorescence resonance energy transfer binding assay, fluorescence trafficking assay, and functional electrophysiology to characterize >10 arrhythmia-associated CaM variants for effect on KCNQ1 CaM binding, membrane trafficking, and channel function."
37965565
sparser
"Thus, ML277 itself is sufficient to rescue the defective VSD-PD coupling from the KCNQ1-CaM apo and induce the pore opening."
36763058
sparser
"A FRET-based assay to probe CaM interaction with full-length KCNQ1."
37965565
sparser
"We note it is possible that CaM binding to KCNQ1 may exhibit cooperativity, which was not modeled in the fitting process to estimate K D,Eff ."
37965565
sparser
"In fact, when CaM does not interact with KCNQ1 due to mutations in the channel protein, it prevents tetramerization of KCNQ1, which is directly related with aberrant channel function and long QT syndrome."
31825788
sparser
"In addition, PIP 2 in KCNQ1-CaM ML277-PIP2-A also forms a salt bridge with Arg91 in CaM, whereas in KCNQ1-CaM ML277-PIP2-B , it loses the interaction with the CaM and forms a new interaction with another residue Arg116 in S0 ( xref )."
36763058
sparser
"KCNQ1-CaM ML277-PIP2-A is virtually the same as the ML277-bound structure KCNQ1-CaM ML277 ."
36763058
sparser
"KCNQ1-CaM ML277-PIP2-B , by contrast, displays several structural differences in the CTD and CaM, although in the TMD, they all maintain the same activated VSD and opened pore ( xref and)."
36763058
sparser
"In the CTD, S6 and the HA helix, which is connected by a loop linker of residues KHFN with an ∼120° angle in KCNQ1-CaM ML277-PIP2-A , becomes a joint continuous helix in KCNQ1-CaM ML277-PIP2-B ( xref ) ."
36763058
sparser
"Functionally, IKs is regulated by a number of interacting proteins and post‐translational modifications. xref – xref The IKs currents and the channel subunits KCNQ1 and KCNE1 proteins are regulated by the β‐adrenergic‐mediated protein kinase A– dependent phosphorylation, a process that might be pathogenic in heart failure. xref , xref – xref Likewise, IKs are calcium‐responsive currents, partly because of interaction of the KCNQ1 with calmodulin, which is a constitutive component of the K + channels. xref One might speculate that altered interactions of KCNQ1 and calmodulin, in the presence of KCNQ1 mutations, could perturb intracellular calcium homeostasis and affect the contractile performance of cardiac myocytes."
25616976
sparser
"We showed that KCNE2, when associated with KCNQ1, exerts a drastic sustained inhibition of the Q1E2 channel availability, and that KCNQ1-bound CaM requires high Ca 2+ to effectively relieve this inhibition."
36077086
sparser
"Our finding that CaM binding to KCNQ1 is not significantly affected by raising intracellular [Ca 2+ ] is consistent with prior studies demonstrating that I Ks ionic current is maximally activated over physiological ranges of intracellular [Ca 2+ ] ( xref , xref )."
37965565
sparser
"HB, which sits at the right side of HA in KCNQ1-CaM ML277-PIP2-A , moves to the left side of HA in KCNQ1-CaM ML277-PIP2-B ( xref andand)."
36763058
sparser
"This finding indicates that the CaM G114W likely does not associate with KCNQ1 at any point during the cardiac cycle."
37965565
sparser
"This suggests that CaM F142L may interact with KCNQ1 in a Ca 2+ -dependent manner with variable binding depending on intracellular [Ca 2+ ] during the cardiac cycle."
37965565
sparser
"In summary, like KCNQ1-CaM ML277 , KCNQ1-CaM ML277-PIP2-A is still in the attached conformation ( xref and), whereas KCNQ1-CaM ML277-PIP2-B is in the detached conformation due to the dissociation of CaM and HA/HB from VSD and their following rotation ( xref )."
36763058
sparser
"Although the KCNQ1–CaM complex constitutes a fully functional voltage-dependent channel, KCNQ1 additionally interacts with the auxiliary subunit KCNE1 in cardiomyocytes to conduct the slow delayed rectifier current (I Ks ) ( xref , xref , xref )."
37965565
sparser
"Using ITC, we showed that in the absence of Ca 2+ , no measurable interaction between CaM‐WT and Kv7.1‐HA 370‐389 was observed (Fig.  xref )."
37428651
sparser
"In particular, the CaM G114W effect on increasing KCNQ1 trafficking is a surprising finding in light of our finding that CaM G114W binds KCNQ1 very poor affinity."
37965565
sparser
"CaM binding to KCNQ1 is required for channel assembly and membrane trafficking ( xref , xref )."
37965565
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"In KCNQ1-CaM ML277-PIP2-A , the phosphate at position 5 of the inositol in PIP 2 interacts with the S2-S3 linker and the phosphate at position 4 binds in CaM ( xref ), whereas in KCNQ1-CaM ML277-PIP2-B , both phosphate groups interact with the S2-S3 linker and S0 ( xref ), which induces a 1–2 Å movement of the S2-S3 linker closer to the 1,4,5 trisphosphate head group of PIP 2 ( xref )."
36763058
sparser
"Accordingly, CaM G114W is not expected to cause KCNQ1 current dysfunction, as all KCNQ1 expressed on the cardiomyocyte membrane are likely associated with CaM WT."
37965565
sparser
"CaM variant interaction with KCNQ1 in relation to other CaM targets."
37965565
sparser
"LQTS‐associated mutations impair interaction of CaM with the helix B domain of Kv7.1."
37428651
sparser
"In this study, we report four structures of the KCNQ1-CaM complex, one in the apo closed state, one in the ML277-bound open state, and two in the ML277-PIP 2 -bound open states."
36763058
sparser
"These different conformations of the KCNQ1-CaM complex provide mechanistic insights into the ligand activation of the channel."
36763058
sparser
"Functional and biochemical analysis of LQTS mutations in the KCNQ1 C-terminus showed that disruption of CaM interaction with KCNQ1 interfered with subunit assembly and that the resultant channels generated very small currents."
19333131
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"CaM is constitutively bound to KCNQ1 and mandatory for functional channel assembly."
36077086
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"Cryo-EM structures of CaM-bound KCNQ1 obtained in the presence or absence of PIP2 [13,14] showed that, without PIP2, the S2–S3 linker interacts with the CaM C-lobe."
36077086
sparser
"However, we did not observe any significant impairment of Kv7.1 trafficking to the plasma membrane for any of the LQTS‐associated CaM variants assayed, consistent with other studies of mutant CaM‐Kv7.1 trafficking (Kato et al., xref )."
37428651
sparser
"Here, the ML277-bound KCNQ1-CaM ML277 structure provides a second activation mode, which shows that this large structural rearrangement of the CTD and the CaM is not essential for the activation of KCNQ1."
36763058
sparser
"Since ML277 exclusively changes the gating properties of the AO state ( xref , xref ), ML277-induced conformational changes, especially the upward movement at the N-terminal of the S4-S5 linker, is more likely to correlate with the AO state gating process, and the KCNQ1-CaM ML277-PIP2-B represents the AO state."
36763058
sparser
"While the apo‐CaM:Kv7.1‐HA 370‐389 could not be fully characterised, crystal structures of the CaM‐KCNQ1 complex at HA and HB consistently reveal the apo C‐lobe of CaM bound to HA, even in high molar excesses of Ca 2+ (Sachyani et al., xref )."
37428651
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"This model is an extension of a five-state kinetic KCNQ1 model [34] with parameters adapted to KCNQ1 currents recorded in CHO cells.The structure models of the KCNQ1-CaM complex in the PIP2-free and the PIP2-bound states (Figure A2 in Appendix B) are based on pdb 6V00 and pdb 6V01 [14] and were generated using the program UCSF Chimera 1.14 (San Francisco, CA, USA)."
36077086
reach
"We showed that KCNE2, when associated with KCNQ1, exerts a drastic sustained inhibition of the Q1E2 channel availability, and that KCNQ1-bound CaM requires high Ca to effectively relieve this inhibition."
36077086
sparser
"Due to the lack of high-resolution structure for KCNQ1-CaM PIP2 , the structural basis for this single-channel amplitude change requires further study."
36763058
sparser
"By contrast, ML277 alone is able to activate KCNQ1 efficiently, and the open-state structure of KCNQ1-CaM ML277 was readily obtained."
36763058
sparser
"Since structures of KCNQ1-CaM mentioned above were resolved at 0 mV, the voltage sensor of those channels was proposed to be in the activated conformation."
33322401
sparser
"This KCNE3-induced conformational change of S5 and S6 would likely prevent the binding of ML277, as revealed by the structural alignment of KCNQ1-CaM-KCNE3 apo-6V00 and KCNQ1-CaM ML277 , where the side chain of Phe335 in S6 in KCNQ1-CaM-KCNE3 apo-6V00 would form clashes with ML277 ()."
36763058
sparser
"The recent cryo-EM structure of the KCNQ1-CaM complex [ xref ] supported the idea that in the absence of PIP 2 , the voltage sensor still moves but the pore remains closed."
33322401
sparser
"Structural Comparison of ML277-Bound Human and Xenopus KCNQ1-CaM."
36763058
sparser
"The three-dimensional cryo-EM structures of KCNQ1-CaM, KCNQ1/KCNE3-CaM and KCNQ2 channels have helped to explain some functional studies of KCNQ channels with or without different KCNE subunits."
33322401
sparser
"During the review of this paper, the Fedida Laboratory reported the structure of ML277-bound Xenopus KCNQ1-CaM (PBD 7CTI, xKCNQ1-CaM ML277-7CTI ) ( xref )."
36763058
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"The structure of the Kv7.1-CaM complex (PDB code: 6UZZ) reveals that CaM contacts Kv7.1 in the VSD and the proximal C terminus at helix A and helix B (26)."
33355140
sparser
"Comparing the structures between KCNQ1-CaM and KCNQ1/KCNE3-CaM, Mackinnon et al. [ xref ] found that KCNE3 lies in between S1, S4, S5 and S6 from three different subunits, suggesting KCNE1 and KCNE3 might share a similar location when associated with KCNQ1."
33322401
sparser
"Structural comparison of KCNQ1-CaM-KCNE3 apo-6V00 and KCNQ1-CaM apo-6UZZ shows that the binding of KCNE3 between S1 and S6 induces a 2∼3 Å shift of both S5 and S6 at the outer leaflet of the membrane () ( xref )."
36763058
sparser
"The proximal Kv7.1 C-terminus binds calmodulin (CaM) and phosphatidylinositol-4,5-bisphosphate (PIP 2 ) and recently we revealed the competition of PIP 2 with the calcified CaM N-lobe to a previously unidentified site in Kv7.1 helix B, also known to harbor a LQT mutation."
28976808
sparser
"According to the recent cryo-EM structure of KCNQ1-CaM, the lower S6 bends at the Pro-Ala-Gly motif."
33322401
sparser
"Generally, structures of KCNQ1-CaM ML277 and xKCNQ1-CaM ML277-7CTI reveal the same binding site of ML277."
36763058
sparser
"In the xKCNQ1-CaM ML277-7CTI , the carbonyl in ML277 points to S6 and introduces electrostatic repulsion with the mainchain carbonyl of Phe335, whereas in our KCNQ1-CaM ML277 structure, it points outwards ()."
36763058
sparser
"The xKCNQ1-CaM ML277-7CTI structure remains in a closed state, while the human KCNQ1-CaM ML277 is in an open state ()."
36763058
sparser
"Our observations in the structures of KCNQ1-CaM ML277-PIP2-A and KCNQ1-CaM ML277-PIP2-B not only recapitulate the large structural rearrangement of KCNQ1 CTD and CaM ( xref and xref ), but also provide insight into the PIP 2 -mediated gating processes."
36763058
sparser
"In our experiment, by adding PIP 2 alone, we captured two low-resolution structures of KCNQ1-CaM PIP2 , one in the attached conformation and the other likely in an intermediate state between attached and detached conformations ()."
36763058
sparser
"Formation of functional tetramers requires CaM interaction with the KCNQ1 C-terminus."
16556866
sparser
"In this regard, high-resolution cryoelectron microscopy studies of the full-length Kv7.1–calmodulin complex have revealed intricated interactions between Kv7.1–VSD, calmodulin and the PIP2 complex [ xref , xref ]."
38256028
sparser
"The ML277 in the structure of KCNQ1-CaM ML277-PIP2-B also helps to stabilize the open state."
36763058
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"Functional and biochemical analysis of LQTS mutations in the KCNQ1 C-terminus showed that disruption of CaM interaction with KCNQ1 interfered with subunit assembly and that the resultant channels generated very small currents."
19333131
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"To test whether CaM binding to KCNQ1 was dissociated by PIs, we fused each of the two C-terminal KCNQ1 CaM binding regions (CBS1 and CBS2) to MBP and performed competition assays."
17317623
reach
"Other mutations that affect PIP 2 or CaM binding to KCNQ1 underlie certain forms of the long QT syndrome and can result in sudden cardiac death (Park et al., 2005; Ghosh et al., 2006; Shamgar et al., [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
17317623
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"While it is clearly established that CaM binds to A and B helices in KCNQ1, its mode of interaction with this domain is unknown."
24721657
reach
"Knowing the differences in the role played by Ca 2+ –CaM in the signaling of the various KCNQ channel subtypes, it remains to determine whether CaM interaction with KCNQ1 is similar to that described [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
24721657
sparser
"Knowing the differences in the role played by Ca 2+ –CaM in the signaling of the various KCNQ channel subtypes, it remains to determine whether CaM interaction with KCNQ1 is similar to that described [MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
24721657
sparser
"In the presence of KCNE3 the architecture of human KCNQ1-CaM is similar to that of the Xenopus complex ( xref ) and thus we use the same nomenclature for secondary structure elements ( xref )."
31883792
sparser
"Similarly, we determined the activator ML277- and PIP 2 -bound open-state structures of KCNQ1-CaM xref ."
37857637
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"One possibility is that Ca 2+ binding to KCNQ1 bound calmodulin exerts conformational effects that can be transmitted to co-assembled SMITs to Ca 2+ -dependently regulate myo-inositol intake."
28718502
sparser
"The overall RMSD between the human and Xenopus KCNQ1-CaM monomer is about 1.1 Å ( xref )."
31883792
sparser
"The potential relevance of interactions between KCNQ1 and CaM is underscored by the important role that CaM plays in channel modulation as well as its connection to human disease mutations."
28575668
sparser
"Altogether, these data suggest that mutations located at this region could disrupt Kv7.1-calmodulin interactions resulting in defective trafficking."
33693037
sparser
"The interface between KCNE3 and KCNQ1-CaM consists of two parts: cytosolic interactions between KCNE3 and CaM ( xref ) and transmembrane interactions between KCNE3 and KCNQ1 ( xref )."
31883792
sparser
"CaM binds to the proximal C-terminus of KCNQ1 and is thought to compete with PIP 2 to stabilize the KCNQ1 open state; this also occurs in physiological relevant complexes in which KCNQ1-CaM co-assembles with the KCNE1 ancillary subunit to form I Ks complexes (see below) ( xref )."
32655402
sparser
"Recent biophysical studies and a cryo-EM structure of the KCNQ1-calmodulin complex have provided new insights into K V 7.1 channel function, and how interactions between KCNQ1 and KCNE subunits alter the gating properties of heteromultimeric channels."
35138621
sparser
"R555 is located on the HC helix of KCNQ1, which interacts with the CaM C-lobe in the PIP2-bound state."
31883792
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"Earlier reports of constitutive association of the Ca 2+ -binding protein calmodulin (CaM) with the C-terminus of KCNQ1 prompted us to consider intracellular Ca 2+ as a putative ligand for KCNQ1 and CaM complex."
22866036
sparser
"The gating variants identified in our data include well-studied variants like D76N and W87R xref and novel variants in the transmembrane-proximal cytosolic domain that interact with KCNQ1 and calmodulin."
37162834
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"The recently published cryo-EM structure of the KCNQ1 and CaM complex [12] falls within the conformational space of possible structures in the library."
30292722
sparser
"We also conducted a similar analysis on the inactive conformation of Kv7.1-CaM structure without PIP2, designated as Kv7.1-PIP2(–), derived from the published structure ( xref )."
37648450
sparser
"CaM binding to Kv7.1 is also required for appropriate folding of the C-terminus and it is also necessary for correct channel trafficking to the plasma membrane [ xref , xref ]."
30022004
sparser
"A recent cryo-EM study showed that each Kv7.1 subunit associates with one calmodulin molecule [ xref ]."
29451064
sparser
"Significantly, this arrangement is also displayed on the crystallographic complexes of CaM with Kv7.1 [ xref ], Kv7.4, Kv7.5 [ xref ] and a chimera between hA of Kv7.3 and hB of Kv7.2 [ xref ]."
30022004
sparser
"On the other hand, helices A‐B in C‐terminal mediate the binding of KCNQ1 and CaM in charge of gating, folding, and membrane trafficking of the channel (Kinoshita et al., xref )."
31565860
sparser
"We next conducted studies of docking to the cryo-electron microscopy (cryo-EM)-derived human KCNQ1-calmodulin structure xref ."
32641720
sparser
"Helix A (residues 370–386) and helix B (residues 504–531) mediate binding of KCNQ1 with CaM, which is required for normal KCNQ1 channel gating, folding and membrane trafficking [9–11] ."
24713462
sparser
"Our study highlights the importance of Gly262′, Phe325′, and Leu256 on xKCNQ1-CaM in not only forming a binding pocket for ML277, but also creating steric clashes that would inhibit the binding of ML213 and retigabine to KCNQ1-CaM (Fig.  xref )."
35768468
sparser
"To understand the actions of ML277 on the open human KCNQ1-CaM channel, in the presence of PIP2, and in the absence of KCNE3, MD simulations were carried out on the open hKCNQ1-CaM-PIP2 (PDB-ID: 6V01) structure xref ."
35768468
reach
"By interacting with both the voltage sensors and the HA-HB helices, CaM could provide an alternative functional linkage between voltage sensors and the pore of KCNQ1 EM separate from the S4-S5 linker.[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
28575668
sparser
"To test whether CaM binding to KCNQ1 was dissociated by PIs, we fused each of the two C-terminal KCNQ1 CaM-binding regions (CBS1 and CBS2) to MBP and performed competition assays."
17317623
sparser
"Structurally, CaM binds the KCNQ1 C-terminal domain (CTD) such that CaM N-lobe associates with KCNQ1 HB and CaM C-lobe interacts with KCNQ1 HA ( xref ) ( xref , xref , xref , xref , xref )."
33310856
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"The KCNQ1 cryo-EM structure model utilized for this work, had bound calmodulin (CAM), no PIP and represented a decoupled state with activated voltage sensor domain and a closed pore domain [36]."
35442947
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"CaM binding to Kv7.1 is also required for appropriate folding of the C-terminus and it is also necessary for correct channel trafficking to the plasma membrane [47,48]."
30022004
sparser
"Other mutations that affect PIP 2 or CaM binding to KCNQ1 underlie certain forms of the long QT syndrome and can result in sudden cardiac death ( Park et al., 2005; Ghosh et al., 2006; Shamgar et al.[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
17317623
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"5 Å resolution cryo-EM structures of human KCNQ1-CaM complex in the apo closed, ML277-bound open, and two ML277-PIP -bound open states."
36763058
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"Different from the previously reported structures of the human KCNQ1 complexes (apo KCNQ1-CaM complex, PDB 6UZZ, KCNQ1-CaM ; apo KCNQ1-CaM-KCNE3 complex, PDB 6V00, KCNQ1-CaM-KCNE3 ; PIP -bound KCNQ1-CaM-KCNE3 complex, PDB 6V01, KCNQ1-CaM-KCNE3 ) that were obtained from a truncated KCNQ1 sample (residues 76–620) (8), we determined cryo-EM structures of the full-length human KCNQ1 channel in three different conditions: 1) the apo-state structure of the KCNQ1-CaM complex (KCNQ1-CaM ) at 3."
36763058
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"By contrast, all three ligand-bound structures of KCNQ1-CaM complex are in an open state, with the pore radii expending to ∼2."
36763058
reach
"Helix A (residues 370-386) and helix B (residues 504-531) mediate binding of KCNQ1 with CaM, which is required for normal KCNQ1 channel gating, folding and membrane trafficking [9-11]."
24713462
sparser
"Other studies found that CaM C-lobe bound to KCNQ1 does not coordinate Ca 2+ ions under 1 mM EGTA or 5 mM Ca 2+ conditions ( xref )."
33310856
reach
"In addition, we observe two conformations of KCNQ1-CaM based on different interactions between CaM and KCNQ1."
36763058
reach
"In this study, we report four structures of the KCNQ1-CaM complex, one in the apo closed state, one in the ML277-bound open state, and two in the ML277-PIP -bound open states."
36763058
reach
"These different conformations of the KCNQ1-CaM complex provide mechanistic insights into the ligand activation of the channel."
36763058
sparser
"Here, we combine electrophysiology experiments and network analysis of molecular dynamics (MD) simulations to show that CaM interacts with the KCNQ1 VSD during voltage-dependent activation to modulate ionic current, despite PIP 2 presence in the membrane."
33310856
sparser
"Together, the combined functional and simulation results suggest CaM interacts with the KCNQ1 S2-S3 linker during gating."
33310856
sparser
"Our data indicate that in healthy individuals, CaM binding to KCNQ1 is essential for correct channel folding and assembly and for conferring Ca(2+)-sensitive IKS-current stimulation, which increases the cardiac repolarization reserve and hence prevents the risk of ventricular arrhythmias."
16556865
sparser
"Additionally, LQT1-linked mutations can disrupt the CaM–Kv7.1 interaction, uncover inactivation of IKs and decrease IKs similar to other defined loss-of-function mutations (Ghosh et al. 2006; Shamgar et al. 2006)."
28008618
sparser
"Mutation in the CaM-binding motifs of KCNQ1 [29] is associated with LQTS."
26164367
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"In KCNQ1 (46) and the I (KCNQ/KCNE1) channel complex (47), allosteric activation models are also required to accurately simulate the activation kinetics and steady-state current and fluorescence relationships with membrane voltage.The lack of positive allosteric regulation in KCNQ2 opening [present in KCNQ1 (36)] may be explained from the distinctive structural arrangements of the VSD subunits and associated calmodulin (CaM) proteins between KCNQ2/CaM and KCNQ1/CaM complexes (fig."
40043113
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"As shown in the recent cryo-EM structures of KCNQ2 and KCNQ4 bound to CaM [KCNQ2/CaM (59) and KCNQ4/CaM (60)], there appears to be more space between VSD subunits in the C terminus for the A-B helices and bound CaM than the narrow spaces between activating subunits found in the KCNQ1/CaM complex (fig."
40043113
reach
"Our data indicate that in healthy individuals, CaM binding to KCNQ1 is essential for correct channel folding and assembly and for conferring Ca (2+)-sensitive IKS-current stimulation, which increases the cardiac repolarization reserve and hence prevents the risk of ventricular arrhythmias."
16556865
reach
"Here, we take a multipronged approach employing a live-cell fluorescence resonance energy transfer binding assay, fluorescence trafficking assay, and functional electrophysiology to characterize >10 arrhythmia-associated CaM variants for effect on KCNQ1 CaM binding, membrane trafficking, and channel function."
37965565
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"The KCNQ1/CaM complex exists in a 4:4 stoichiometry (35, 38)."
37965565
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"We fitted the E readouts to a 1:1 (equivalent to 4:4) binding model to derive a FRET binding curve with an apparent dissociation constant (K ) related to the binding affinity between CaM and KCNQ1 (Fig. 1F; also see Extended Methods)."
37965565
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"To test if the mutant M520R disrupts channel function by impairing the interaction of Kv7.1 with CaM, we performed yeast two-hybrid experiments ( Fig. 4 B)."
17482572
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"ML277 (solubilized in 100% v/v DMSO at 50 mM) was added to a solution containing 1 mg/mL of purified KCNQ1/CaM complex, to a final concentration of 100 μM ML277 (resulting in 0.2% v/v final DMSO concentration) 20 min prior to grid preparation."
35768468
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"This analysis assumed CaM and KCNQ1 binds identically and independently in a 4:4 fashion."
37965565
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"Therefore, we considered the M520R mutation to affect the CaM-Kv7.1 interaction."
17482572
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"7 It is thought that specific CALM variants result in differing phenotypes of LQTS and CPVT owing to divergent effects on the various molecular targets.The IK potassium channel is formed by the assembly of KCNQ1 and KCNE1 subunits and the associated IK current is an important part of cardiac repolarization.8 CaM binding to KCNQ1 is essential for correct channel folding and assembly.9 Thus loss of CaM function can result in a prolonged QT interval."
38204837
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"We note it is possible that CaM binding to KCNQ1 may exhibit cooperativity, which was not modeled in the fitting process to estimate K ."
37965565
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"First, it has not been shown that CaM interaction with KCNQ1 exhibits cooperativity."
37965565
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"Taken together, our FRET assay detected robust binding between CaM and KCNQ1 WT as well as no interactions between CaM and KCNQ1-IQ/AA designed to ablate CaM binding to KCNQ1."
37965565
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"We note that fluorophore fusion to KCNQ1 and CaM may induce possible steric effects on KCNQ1/CaM interactions."
37965565
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"We next applied our FRET-based assay to probe whether CaM variants bind to the full-length KCNQ1 differently compared with CaM WT."
37965565
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"Their positions suggest that CaM E46K and G114W likely disrupt CaM/KCNQ1 binding by altering N-lobe interaction with HB and C-lobe interaction with HA, respectively."
37965565
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"A redox motif flanking Cys(445) and the interaction of KCNQ1 with calmodulin are required for preferential S-nitrosylation of Cys(445)."
19124472
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"We note that we do not explicitly measure intracellular [Ca ] with this method, and it is possible for local microdomain [Ca ] to affect CaM binding to KCNQ1."
37965565
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"Our finding that CaM binding to KCNQ1 is not significantly affected by raising intracellular [Ca ] is consistent with prior studies demonstrating that I ionic current is maximally activated over physiological ranges of intracellular [Ca ] (40, 41)."
37965565
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"Although the KCNQ1–CaM complex constitutes a fully functional voltage-dependent channel, KCNQ1 additionally interacts with the auxiliary subunit KCNE1 in cardiomyocytes to conduct the slow delayed rectifier current (I ) (36, 37, 56)."
37965565
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"In contrast, although CaM also associates and regulates KCNQ1 channel membrane trafficking and function, relatively little is known regarding whether KCNQ1 or I dysfunction may also contribute to calmodulinopathy."
37965565
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"Because CaM is also known to interact with multiple KCNQ1 domains including the voltage-sensing domain, a key strength of our assay is quantification of CaM interaction with the full-length KCNQ1 channel."
37965565
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"Our FRET-based screen demonstrates that most CaM variants (10/14 screened) interact with the full-length KCNQ1 with similar or better affinity compared with CaM WT under both resting and elevated intracellular [Ca ] conditions, suggesting that most CaM variants can preassociate with KCNQ1 in cardiomyocytes expressing both CaM variants and WT (Figs."
37965565
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"Our finding that most CaM variants bind to KCNQ1 with similar affinity as CaM WT represents the first step to contextualize KCNQ1's contribution to calmodulinopathy, as arrhythmia ultimately arises from cardiac action potential pathologies that are triggered by aberrant ionic currents."
37965565
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"As we found that CaM variants can bind KCNQ1 with varying affinities, the relative ratio of CaM variants to CaM WT within cardiomyocytes figures critically in whether CaM variants can exert dominant negative effect on KCNQ1 function."
37965565
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"CaM binding to KCNQ1 is required for channel assembly and membrane trafficking (5, 6)."
37965565
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"CaM variant interaction with KCNQ1 in relation to other CaM targets."
37965565
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"For example, KCNQ1 may act as a protective “sink” by sequestering CaM variants that bind KCNQ1 with high affinity without affecting I current, effectively chelating these variants from affecting alternate targets.Taken together, our study furnishes extensive characterization of CaM variant interaction and effect on KCNQ1 channels, delineating the CaM variants that cause KCNQ1 dysfunction to play a role in arrhythmogenesis."
37965565
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"In the cardiac cycle, CaM can coordinate Ca 2+ ions and change conformation when intracellular [Ca 2+ ] rises during systole, thereby potentially changing CaM interaction with KCNQ1."
37965565
sparser
"In follow-up studies, Roderick Mackinnon's group determined structures of the human KCNQ1-CaM in the apo state (HsKCNQ1-CaM apo ) and KCNQ1-CaM-KCNE3 complex in the apo and PIP 2 -bound state (HsKCNQ1[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
37976835
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"In this regard, high-resolution cryoelectron microscopy studies of the full-length Kv7.1–calmodulin complex have revealed intricated interactions between Kv7.1–VSD, calmodulin and the PIP2 complex [13,29]."
38256028
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"These structures, recently determined by high-resolution cryoelectron microscopy, comprise the full-length human KCNQ1–calmodulin complex [13]."
38256028
sparser
"We note that we do not explicitly measure intracellular [Ca 2+ ] with this method, and it is possible for local microdomain [Ca 2+ ] to affect CaM binding to KCNQ1."
37965565
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"Recently, Mackinnon's group further determined structures of human KCNQ1-CaM that was reconstituted into liposomes with polarization of the membrane potential ( Table 1 ) [ 60 ]."
37976835
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"Meanwhile, our group determined three open-state structures of human KCNQ1-CaM, one in complex with ML277 alone (HsKCNQ1-CaM ML277 ), and two with both ML277 and PIP 2 (HsKCNQ1-CaM ML277-PIP2-A and Hs[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
37976835
sparser
"Earlier reports of constitutive association of the Ca 2+ -binding protein calmodulin (CaM) with the C-terminus of KCNQ1 (Yus-Najera et al., xref ; Ghosh et al., xref ; Shamgar et al., xref ; Ciampa et al., xref ) prompted us to consider intracellular Ca 2+ as a putative ligand for KCNQ1-CaM complex."
22866036
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"The gating variants identified in our data include well-studied variants like D76N and W87R [ xref ] and novel variants in the cytosolic region adjacent to the transmembrane domain that interact with KCNQ1 and calmodulin."
38816749
sparser
"Therefore, although we cannot definitively identify the mechanism of action, we conclude that many of these variants may disrupt function through their interactions with KCNQ1 or calmodulin."
38816749
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"CaM binding to KCNQ1 is essential for correct channel folding and assembly and for conferring Ca 2+ -sensitive I Ks stimulation, which increases the cardiac repolarization reserve. xref Mutations associated with LQT syndrome located near the IQ motif mediate Ca 2+ -free CaM binding to KCNQ1 at the COOH termini, which impairs CaM binding to KCNQ1 , alters channel assembly, and stabilizes inactivation, which results in a decrease in current density. xref "
34113909
sparser
"These studies were motivated by the observed similarities between the suppression of KCNQ1 function by pharmacological disruption of KCNQ1-CaM interactions and the effects of KCNE4 co-expression on the channel."
21118809
reach
"The recent cryo-EM structure of the KCNQ1 and CaM complex [XREF_BIBR] supported the idea that in the absence of PIP 2, the voltage sensor still moves but the pore remains closed."
33322401
sparser
"When Kv7.1–CaM interactions are disrupted, IKs is largely reduced and the voltage dependence of activation is positively shifted."
28008618
reach
"Other studies found that CaM C-lobe bound to KCNQ1 does not coordinate Ca 2+ ions under 1 mM EGTA or 5 mM Ca 2+ conditions."
33310856
reach
"Recent cryo-electron microscopy (EM) structural studies of the full-length KCNQ1 and CaM complex revealed intricate interactions between CaM, the KCNQ1 VSD, and the membrane lipid PIP 2."
33310856
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"In this study, utilizing two VSD-PD coupling enhancers, namely, the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) and a small-molecule ML277, we determined 2.5–3.5 Å resolution cryo-electron microscopy structures of full-length human KCNQ1-calmodulin (CaM) complex in the apo closed, ML277-bound open, and ML277-PIP 2 -bound open states."
36763058
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"First, the MD trajectories of the KCNQ1 and CaM complex were converted into a residue interaction network representation (XREF_FIG)."
33310856
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"In this network, each node corresponded to an individual amino acid residue within the KCNQ1 and CaM complex."
33310856
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"The final network thus encodes all residues (nodes) and interactions (edges) within the KCNQ1 and CaM complex."
33310856
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"The underlying idea is that a perturbation of residue interactions, such as those induced by the movements of S4 gating charges (the source), spreads to other residues (nodes) via diffusion along the KCNQ1 and CaM complex (network) before they arrive at the pore (the sink)."
33310856
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"The results were close to identical to the analysis of the human KCNQ1 and CaM complex, reinforcing the findings from the human KCNQ1 and CaM simulations."
33310856
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"Interestingly, co-immunoprecipitation experiments in yeast cells expressing wild-type Kv7.1 or mutated Kv7.1 with truncated α-helices showed that calmodulin can bind to the C-terminus of Kv7.1 ( xref )."
24600393
sparser
"To capture the KCNQ1-CaM structure in the depolarized membrane potential, Roderick Mackinnon's group first reconstituted the KCNQ1-CaM protein into liposome in 300 mM KCl and exchanged the solution to[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
37976835
reach
"Similar results were obtained with the same analysis on the Xenopus KCNQ1 and CaM complex, further validating the conserved importance of Q1-R360 and Q1-P535."
33310856
sparser
"Using the KCNQ1-CaM sample in liposome in hyperpolarization conditions, they captured structures of KCNQ1-CaM with VSDs in up (activated, HsKCNQ1-CaM up ), down (resting, HsKCNQ1-CaM down ), and inter[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
37976835
sparser
"In the case of KCNQ1-CaM, PIP 2 –I does not directly interact with S6, suggesting that PIP 2 –I may only involve in disrupting the VSD-CaM interactions ( Fig. 4 B)."
37976835
sparser
"Different from the previously reported structures of the human KCNQ1 complexes (apo KCNQ1-CaM complex, PDB 6UZZ, KCNQ1-CaM apo-6UZZ ; apo KCNQ1-CaM-KCNE3 complex, PDB 6V00, KCNQ1-CaM-KCNE3 apo-6V00 ; PIP 2 -bound KCNQ1-CaM-KCNE3 complex, PDB 6V01, KCNQ1-CaM-KCNE3 PIP2-6V01 ) that were obtained from a truncated KCNQ1 sample (residues 76–620) ( xref ), we determined cryo-EM structures of the full-length human KCNQ1 channel in three different conditions: 1) the apo-state structure of the KCNQ1-CaM complex (KCNQ1-CaM apo ) at 3.5 Å resolution ( xref and); 2) the ML277-bound KCNQ1-CaM structure (KCNQ1-CaM ML277 ) at 2.6 Å resolution ( xref and); and 3) the PIP 2 - and ML277-bound KCNQ1-CaM structures in two different conformations, which were designated as KCNQ1-CaM ML277-PIP2-A at 3.1 Å resolution and KCNQ1-CaM ML277-PIP2-B at 2.5 Å resolution ( xref and)."
36763058
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"For example, based on mutational, electrophysiological, and computational studies, Jianmin Gui's group proposed that in KCNQ1 VSD-CaM interactions work as a state-dependent switch to control the chann[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
37976835
sparser
"We also endeavored to capture the structure of KCNQ1-CaM in the PIP 2 -only condition."
36763058
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"In all the three ligand-bound structures, i.e., KCNQ1-CaM ML277 , KCNQ1-CaM ML277-PIP2-A , and KCNQ1-CaM ML277-PIP2-B , the density for the ML277 molecule was unambiguously resolved in the EM map, and therefore its binding configuration was confidently assigned ( xref and)."
36763058
sparser
"We present 2.5–3.5 Å resolution cryo-EM structures of human KCNQ1-CaM complex in the apo closed, ML277-bound open, and two ML277-PIP 2 -bound open states."
36763058
sparser
"Structural Determination of KCNQ1-CaM with Different VSD-PD Coupling Enhancers."
36763058
sparser
"Thus, upon PIP2 depletion, calcium-bound CaM can bind to Kv7.1, mimic the PIP2 binding effect, and assist in channel activation ( xref )."
34149352
sparser
"To reveal activation mechanisms of human KCNQ1 by these VSD-PD coupling enhancers, we determined cryo-EM structures of KCNQ1-CaM using protein samples in the absence and presence of ML277 and/or PIP 2 ( xref and)."
36763058
sparser
"In the case of KCNQ4-CaM ML213-PIP2 , while PIP 2 –I may play a similar role as that in KCNQ1-CaM ML277-PIP2-B , PIP 2 -II has a second activation mechanism – it “pulls” S6 up and stabilizes the chann[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
37976835
sparser
"In the structures of KCNQ1-CaM ML277-PIP2-A and KCNQ1-CaM ML277-PIP2-B , the density for the inositol 1,4,5-trisphosphate head group of PIP 2 were well defined in the EM map, whereas the density for the flexible fatty acid chains of PIP 2 was poorly resolved, likely due to their relatively weak interactions with the protein ( xref and)."
36763058
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"In KCNQ1-CaM ML277 , ML277 lies in a hydrophobic pocket in PD of KCNQ1, formed by the S4–S5 linker, S5, S6, and S5 and S6 from the adjacent subunit ( Fig. 5 C) [ 59 , 61 ]."
37976835
reach
"Recent biophysical studies and a cryo-EM structure of the KCNQ1-calmodulin complex have provided new insights into K V 7.1 channel function, and how interactions between KCNQ1 and KCNE subunits alter the gating properties of heteromultimeric channels."
35138621
sparser
"The KCNQ1-CaM apo structure is in the closed state, as indicated by the <1 Å pore radius at the activation gate of the channel calculated by the program HOLE ( xref ) ( xref )."
36763058
sparser
"This disturbed calmodulin- Kv7.1 interaction may be important for channel expression."
24600393
sparser
"By contrast, all three ligand-bound structures of KCNQ1-CaM complex are in an open state, with the pore radii expending to ∼2.5 Å at the activation gate ( xref ), similar to that in the previously reported PIP 2 -bound open-state structure KCNQ1-CaM-KCNE3 PIP2-6V01 ( xref )."
36763058
sparser
"Unfortunately, so far only ML277 meets these requirements: ML277 itself is able to stabilize KCNQ1 in an open conformation (KCNQ1-CaM ML277 ) [ 59 ]."
37976835
sparser
"In addition, we observe two conformations of KCNQ1-CaM based on different interactions between CaM and KCNQ1."
36763058
sparser
"In the structures of KCNQ1-CaM apo , KCNQ1-CaM ML277 , and KCNQ1-CaM ML277-PIP2-A , CaM forms direct contact with VSD; therefore, we call this an “attached conformation” ( xref )."
36763058
sparser
"In addition, the CaM- Kv7.1 interaction may also be relevant for the regulation of I Ks gating."
24600393
sparser
"Our KCNQ1-CaM structures were determined under 0 mV, a voltage that allows the VSD to activate in both the intermediate and the fully activated states ( xref , xref )."
36763058
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"In the structure of KCNQ1-CaM ML277-PIP2-B , however, CaM is detached from VSD of KCNQ1; we designate KCNQ1-CaM ML277-PIP2-B a “detached conformation” ( xref )."
36763058
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"The KCNQ1-CaM apo Structure Shows Activated VSDs but Closed Pore."
36763058
sparser
"The structure of the Kv7.1-CaM complex (PDB code: 6UZZ) reveals that CaM contacts Kv7.1 in the VSD and the proximal C terminus at helix A and helix B ( xref )."
33355140
sparser
"Thus, the interaction of calmodulin and Kv7.1 appears to be critical for expression and function of I Ks , with the intriguing possibility that regulatory mechanisms could also involve some form of CaMKII interaction with calmodulin and Kv7.1 or KCNE1."
24600393
sparser
"The overall structure of KCNQ1-CaM apo is similar to the previously reported KCNQ1-CaM apo-6UZZ structure ( xref and) ( xref )."
36763058
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"Likewise, we previously characterized long QT mutations impairing CaM binding to the Kv7.1 proximal C terminus (helix A), which lead to channel inactivation I1 ( xref )."
33355140
sparser
"Consistent with this, the S4 in KCNQ1-CaM apo seems dynamic and is resolved at a relatively lower resolution than PD ()."
36763058
sparser
"Thus, KCNQ1-CaM apo adopts a decoupled conformation, with activated VSDs and a closed gate."
36763058
sparser
"Mechanisms that cause LOF in variants exhibiting intact cell surface expression include: deleterious effects on ion conduction, altered voltage sensitivity, disrupted KCNQ1-KCNE1, KCNQ1-calmodulin, or KCNQ1-PIP 2 interactions, or altered rate/capacity to undergo the conformational changes required for channel opening ( xref )."
39969993
sparser
"Similarly, the previously reported KCNQ1-CaM apo-6UZZ structure is also in a decoupled conformation with activated VSDs and a closed gate ( xref )."
36763058
sparser
"The structural data presented here, namely the Kv7.1 proximal C terminus bound to CaM, provide us with a near atomic resolution picture of this module."
25441029
sparser
"Structural alignment of KCNQ1-CaM apo-6UZZ and our KCNQ1-CaM apo reveals almost-identical structures except for a major difference in the VSDs at the outer leaflet of the membrane ()."
36763058
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"Relevant to this question, CaM’s binding target can affect CaM’s affinity for Ca 2+ , so that it is possible that when CaM associates with the Kv7.1 proximal C terminus, CaM N lobe Ca 2+ affinity inc[MISSING/INVALID CREDENTIALS: limited to 200 char for Elsevier]"
25441029
reach
"CaM can bind and modulate Kv7.1 through interaction with two C‐terminal domains (helix A, HA and helix B, HB) in both in the absence and presence of Ca (Sachyani et al., 2014; Sun & MacKinnon, 2017, 2020; Wiener et al., 2008; Yus‐Najera et al., 2002)."
37428651
reach
"While the apo‐CaM:Kv7.1‐HA could not be fully characterised, crystal structures of the CaM‐KCNQ1 complex at HA and HB consistently reveal the apo C‐lobe of CaM bound to HA, even in high molar excesses of Ca (Sachyani et al., 2014)."
37428651
sparser
"Different from the VSD in KCNQ1-CaM apo that forms a large extracellular-facing cavity, the VSD in KCNQ1-CaM apo-6UZZ tightly packs, with the S4 clearly resolved in the fully activated state ( xref )."
36763058