Apo Calmodulin Research Articles

Impact of calmodulin missense variants associated with congenital arrhythmia on the thermal stability and the degree of unfolding.

Thermal denaturation profiles of proteins that bind several ligands may deviate from the single transition, making their thermodynamic description challenging. We report an empirical method that estimates melting temperatures (Tm) from multi-transition thermal denaturation profiles of 16 variants of calmodulin (CaM) associated with congenital arrhythmia. Differences in Tm estimated by empirical fitting correlate (for apo CaM variants) with those obtained by thermodynamic models. Most CaM variants were more stable than the wild type (WT) in the absence of Ca2+, but less stable in the presence of Ca2+, and displayed either WT-like or higher unfolding percentages in their apo-form, as evaluated by circular dichroism spectroscopy.

Allosteric inhibitors targeting the calmodulin-PIP2 interface of SK4 K+ channels for atrial fibrillation treatment

The Ca2+-activated SK4 K+ channel is gated by Ca2+-calmodulin (CaM) and is expressed in immune cells, brain, and heart. A cryoelectron microscopy (cryo-EM) structure of the human SK4 K+ channel recently revealed four CaM molecules per channel tetramer, where the apo CaM C-lobe and the holo CaM N-lobe interact with the proximal carboxyl terminus and the linker S4-S5, respectively, to gate the channel. Here, we show that phosphatidylinositol 4-5 bisphosphate (PIP2) potently activates SK4 channels by docking to the boundary of the CaM-binding domain. An allosteric blocker, BA6b9, was designed to act to the CaM-PIP2-binding domain, a previously untargeted region of SK4 channels, at the interface of the proximal carboxyl terminus and the linker S4-S5. Site-directed mutagenesis, molecular docking, and patch-clamp electrophysiology indicate that BA6b9 inhibits SK4 channels by interacting with two specific residues, Arg191 and His192 in the linker S4-S5, not conserved in SK1-SK3 subunits, thereby conferring selectivity and preventing the Ca2+-CaM N-lobe from properly interacting with the channel linker region. Immunohistochemistry of the SK4 channel protein in rat hearts showed a widespread expression in the sarcolemma of atrial myocytes, with a sarcomeric striated Z-band pattern, and a weaker occurrence in the ventricle but a marked incidence at the intercalated discs. BA6b9 significantly prolonged atrial and atrioventricular effective refractory periods in rat isolated hearts and reduced atrial fibrillation induction ex vivo. Our work suggests that inhibition of SK4 K+ channels by targeting drugs to the CaM-PIP2-binding domain provides a promising anti-arrhythmic therapy.

Exploring and Engineering the Conformational Landscape of Calmodulin through Specific Interactions.

Conformational fluctuations often play paramount role in the function and activity of proteins. Calmodulin (CaM) is a calcium sensing protein that shows significant conformational flexibility on going from a ligand-free open state to a ligand-bound closed state. By employing large-scale equilibrium molecular dynamics simulations and free energy calculations, we have shown that apo CaM frequently visits a state that is neither fully open nor fully closed and referred to as half-open half-closed (HOHC) state. Such states are functionally relevant as they structurally resemble a ligand-bound closed state. Here, we have envisaged that the inherent conformational dynamics of CaM is primarily triggered by a dual salt bridge interaction between the glutamates and lysine at two different domains (N- and C-terminal domains) of the protein. Upon abolition of the dual salt bridge interaction, conformational dynamics is restricted and centered only near the open state. When a cation-π interaction is introduced while replacing the dual salt bridge interaction, the HOHC state tends to reappear as a stable state. Since salt bridge and cation-π interactions are frequently encountered in the protein dynasty, our work may illuminate an interesting direction toward controlling the conformational landscape of proteins through the modulation of minimal specific interactions.

Interaction of NaV1.2 IQ Motif with Disease-Causing Mutants of Calmodulin

Calmodulin (CaM) binds constitutively to an IQ motif (residues 1901-1927, NaV1.2IQp) in the cytosolic C-terminal tail of the a-subunit of the neuronal voltage-gated sodium channel NaV1.2. Solution structures of CaM C-domain (CaMC) bound to NaV1.2IQp showed calcium binding reverses the orientation of (Ca2+)2-CaMC (2M5E.pdb) relative to apo CaMC (2KXW.pdb). Our structure of full-length apo human CaM bound to human NaV1.2IQp showed that only CaMc interacts with NaV1.2IQp. Thermodynamic and NMR analysis of the CaM-NaV1.2IQp complex showed no evidence for CaMN-NaV1.2IQp interactions, and indicated nearly identical affinity for calcium binding to the 4 EF-hand sites of CaM. Residues in NaV1.2IQp had different chemical environments when bound to apo and (Ca2+)4-CaM, consistent with the calcium-trigged change observed in the (Ca2+)2-CaMC-NaV1.2IQp interface (2M5E.pdb). Apo human CaM carrying cardiomyopathy-causing mutations [N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)] all bound tightly to NaV1.2IQp. However, mutations in CaMC inhibited Ca2+-mediated conformational changes of CaM bound to NaV1.2IQp. This suggests that high cytosolic Ca2+ levels would not be sufficient to trigger reversal of the orientation of these CaMC mutants. The effects of other recently described disease-causing CaM mutations are being investigated. NIH R01 GM57001 (MAS), NIH T32 NS045549 (RM), UI CCOM FUTURE in Biomedicine (AMK)

Backbone resonance assignments of complexes of apo human calmodulin bound to IQ motif peptides of voltage-dependent sodium channels NaV1.1, NaV1.4 and NaV1.7.

Human voltage-gated sodium (NaV) channels are critical for initiating and propagating action potentials in excitable cells. Nine isoforms have different roles but similar topologies, with a pore-forming α-subunit and auxiliary transmembrane β-subunits. NaV pathologies lead to debilitating conditions including epilepsy, chronic pain, cardiac arrhythmias, and skeletal muscle paralysis. The ubiquitous calcium sensor calmodulin (CaM) binds to an IQ motif in the C-terminal tail of the α-subunit of all NaV isoforms, and contributes to calcium-dependent pore-gating in some channels. Previous structural studies of calcium-free (apo) CaM bound to the IQ motifs of NaV1.2, NaV1.5, and NaV1.6 showed that CaM binding was mediated by the C-domain of CaM (CaMC), while the N-domain (CaMN) made no detectable contacts. To determine whether this domain-specific recognition mechanism is conserved in other NaV isoforms, we used solution NMR spectroscopy to assign the backbone resonances of complexes of apo CaM bound to peptides of IQ motifs of NaV1.1, NaV1.4, and NaV1.7. Analysis of chemical shift differences showed that peptide binding only perturbed resonances in CaMC; resonances of CaMN were identical to free CaM. Thus, CaMC residues contribute to the interface with the IQ motif, while CaMN is available to interact elsewhere on the channel.

Effect of Ca2+ on the promiscuous target-protein binding of calmodulin.

Calmodulin (CaM) is a calcium sensing protein that regulates the function of a large number of proteins, thus playing a crucial part in many cell signaling pathways. CaM has the ability to bind more than 300 different target peptides in a Ca2+-dependent manner, mainly through the exposure of hydrophobic residues. How CaM can bind a large number of targets while retaining some selectivity is a fascinating open question. Here, we explore the mechanism of CaM selective promiscuity for selected target proteins. Analyzing enhanced sampling molecular dynamics simulations of Ca2+-bound and Ca2+-free CaM via spectral clustering has allowed us to identify distinct conformational states, characterized by interhelical angles, secondary structure determinants and the solvent exposure of specific residues. We searched for indicators of conformational selection by mapping solvent exposure of residues in these conformational states to contacts in structures of CaM/target peptide complexes. We thereby identified CaM states involved in various binding classes arranged along a depth binding gradient. Binding Ca2+ modifies the accessible hydrophobic surface of the two lobes and allows for deeper binding. Apo CaM indeed shows shallow binding involving predominantly polar and charged residues. Furthermore, binding to the C-terminal lobe of CaM appears selective and involves specific conformational states that can facilitate deep binding to target proteins, while binding to the N-terminal lobe appears to happen through a more flexible mechanism. Thus the long-ranged electrostatic interactions of the charged residues of the N-terminal lobe of CaM may initiate binding, while the short-ranged interactions of hydrophobic residues in the C-terminal lobe of CaM may account for selectivity. This work furthers our understanding of the mechanism of CaM binding and selectivity to different target proteins and paves the way towards a comprehensive model of CaM selectivity.

A Novel Mechanism for Calmodulin-Dependent Inactivation of Transient Receptor Potential Vanilloid 6.

The paralogues TRPV5 and TRPV6 belong to the vanilloid subfamily of the transient receptor potential (TRP) superfamily of ion channels, and both play an important role in overall Ca2+ homeostasis. The functioning of the channels centers on a tightly controlled Ca2+-dependent feedback mechanism in which the direct binding of the universal Ca2+-binding protein calmodulin (CaM) to the channel's C-terminal tail is required for channel inactivation. We have investigated this interaction at the atomic level and propose that under basal cellular Ca2+ concentrations CaM is constitutively bound to the channel's C-tail via CaM C-lobe only contacts. When the cytosolic Ca2+ concentration increases charging the apo CaM N-lobe with Ca2+, the CaM:TRPV6 complex rearranges and the TRPV6 C-tail further engages the CaM N-lobe via a crucial interaction involving L707. In a cellular context, mutation of L707 significantly increased the rate of channel inactivation. Finally, we present a model for TRPV6 CaM-dependent inactivation, which involves a novel so-called "two-tail" mechanism whereby CaM bridges two TRPV6 monomers resulting in closure of the channel pore.

Conformational Ensembles of Calmodulin Revealed by Nonperturbing Site-Specific Vibrational Probe Groups.

Seven native residues on the regulatory protein calmodulin, including three key methionine residues, were replaced (one by one) by the vibrational probe amino acid cyanylated cysteine, which has a unique CN stretching vibration that reports on its local environment. Almost no perturbation was caused by this probe at any of the seven sites, as reported by CD spectra of calcium-bound and apo calmodulin and binding thermodynamics for the formation of a complex between calmodulin and a canonical target peptide from skeletal muscle myosin light chain kinase measured by isothermal titration. The surprising lack of perturbation suggests that this probe group could be applied directly in many protein–protein binding interfaces. The infrared absorption bands for the probe groups reported many dramatic changes in the probes’ local environments as CaM went from apo- to calcium-saturated to target peptide-bound conditions, including large frequency shifts and a variety of line shapes from narrow (interpreted as a rigid and invariant local environment) to symmetric to broad and asymmetric (likely from multiple coexisting and dynamically exchanging structures). The fast intrinsic time scale of infrared spectroscopy means that the line shapes report directly on site-specific details of calmodulin’s variable structural distribution. Though quantitative interpretation of the probe line shapes depends on a direct connection between simulated ensembles and experimental data that does not yet exist, formation of such a connection to data such as that reported here would provide a new way to evaluate conformational ensembles from data that directly contains the structural distribution. The calmodulin probe sites developed here will also be useful in evaluating the binding mode of calmodulin with many uncharacterized regulatory targets.

Inference of Calmodulin's Ca2+-Dependent Free Energy Landscapes via Gaussian Mixture Model Validation.

A free energy landscape estimation method based on the well-known Gaussian mixture model (GMM) is used to compare the efficiencies of thermally enhanced sampling methods with respect to regular molecular dynamics. The simulations are carried out on two binding states of calmodulin, and the free energy estimation method is compared with other estimators using a toy model. We show that GMM with cross-validation provides a robust estimate that is not subject to overfitting. The continuous nature of Gaussians provides better estimates on sparse data than canonical histogramming. We find that diffusion properties determine the sampling method effectiveness, such that diffusion-dominated apo calmodulin is most efficiently sampled by regular molecular dynamics, while holo calmodulin, with its rugged free energy landscape, is better sampled by enhanced sampling methods.

Quantifying Secondary Structure Changes in Calmodulin Using 2D-IR Spectroscopy.

Revealing the details of biomolecular processes in solution needs tools that can monitor structural dynamics over a range of time and length scales. We assess the ability of 2D-IR spectroscopy in combination with multivariate data analysis to quantify changes in secondary structure of the multifunctional calcium-binding messenger protein Calmodulin (CaM) as a function of temperature and Ca2+ concentration. Our approach produced quantitative agreement with circular dichroism (CD) spectroscopy in detecting the domain melting transitions of Ca2+-free (apo) CaM (reduction in α-helix structure by 13% (CD) and 15% (2D)). 2D-IR also allows accurate differentiation between melting transitions and generic heating effects observed in the more thermally stable Ca2+-bound (holo) CaM. The functionally relevant random-coil-α-helix transition associated with Ca2+ uptake that involves just 7-8 out of a total of 148 amino acid residues was clearly detected. Temperature-dependent Molecular Dynamics (MD) simulations show that apo-CaM exists in dynamic equilibrium with holo-like conformations, while Ca2+ uptake reduces conformational flexibility. The ability to combine quantitative structural insight from 2D-IR with MD simulations thus offers a powerful approach for measuring subtle protein conformational changes in solution.

Backbone and side chain chemical shift assignments of apo calmodulin bound to the NaV1.2 IQ motif peptide

Backbone and side chain chemical shift assignments of apo calmodulin bound to the NaV1.2 IQ motif peptide

Backbone resonance assignments of complexes of human voltage-dependent sodium channel NaV1.2 IQ motif peptide bound to apo calmodulin and to the C-domain fragment of apo calmodulin.

Human voltage-gated sodium channel NaV1.2 has a single pore-forming α-subunit and two transmembrane β-subunits. Expressed primarily in the brain, NaV1.2 is critical for initiation and propagation of action potentials. Milliseconds after the pore opens, sodium influx is terminated by inactivation processes mediated by regulatory proteins including calmodulin (CaM). Both calcium-free (apo) CaM and calcium-saturated CaM bind tightly to an IQ motif in the C-terminal tail of the α-subunit. Our thermodynamic studies and solution structure (2KXW) of a C-domain fragment of apo 13C,15N- CaM (CaMC) bound to an unlabeled peptide with the sequence of rat NaV1.2 IQ motif showed that apo CaMC (a) was necessary and sufficient for binding, and (b) bound more favorably than calcium-saturated CaMC. However, we could not monitor the NaV1.2 residues directly, and no structure of full-length CaM (including the N-domain of CaM (CaMN)) was determined. To distinguish contributions of CaMN and CaMC, we used solution NMR spectroscopy to assign the backbone resonances of a complex containing a 13C,15N-labeled peptide with the sequence of human NaV1.2 IQ motif (NaV1.2IQp) bound to apo 13C,15N-CaM or apo 13C,15N-CaMC. Comparing the assignments of apo CaM in complex with NaV1.2IQp to those of free apo CaM showed that residues within CaMC were significantly perturbed, while residues within CaMN were essentially unchanged. The chemical shifts of residues in NaV1.2IQp and in the C-domain of CaM were nearly identical regardless of whether CaMN was covalently linked to CaMC. This suggests that CaMN does not influence apo CaM binding to NaV1.2IQp.

Allosteric Mechanism of Calmodulin Revealed by Targeted Molecular Dynamics Simulation**Supported by the Natural Science Fund for Distinguished Young Scholars of Hebei Province under Grant Nos C2015202340 and C2013202244, the Fund for Outstanding Talents of Hebei Province under Grant No C201400305, the National Natural Science Fund of China under Grant Nos 11247010, 11175055, 11475053, 11347017, 31600594, 31400711 and 11647121, the Fund

Calmodulin (CaM) is involved in the regulation of a variety of cellular signaling pathways. To accomplish its physiological functions, CaM binds with Ca2+ at its EF-hand Ca2+ binding sites which induce the conformational switching of CaM. However, the molecular mechanism by which Ca2+ binds with CaM and induces conformational switching is still obscure. Here we combine molecular dynamics with targeted molecular dynamics simulation and achieve the state-transition pathway of CaM. Our data show that Ca2+ binding speeds up the conformational transition of CaM by weakening the interactions which stabilize the closed state. It spends about 6.5 ns and 5.25 ns for transition from closed state to open state for apo and holo CaM, respectively. Regarding the contribution of two EF-hands, our data indicate that the first EF-hand triggers the conformational transition and is followed by the second one. We determine that there are two interaction networks which contribute to stabilize the closed and open states, respectively.

Calcium triggers reversal of calmodulin on nested anti-parallel sites in the IQ motif of the neuronal voltage-dependent sodium channel NaV1.2

Several members of the voltage-gated sodium channel family are regulated by calmodulin (CaM) and ionic calcium. The neuronal voltage-gated sodium channel NaV1.2 contains binding sites for both apo (calcium-depleted) and calcium-saturated CaM. We have determined equilibrium dissociation constants for rat NaV1.2 IQ motif [IQRAYRRYLLK] binding to apo CaM (~3nM) and (Ca2+)4-CaM (~85nM), showing that apo CaM binding is favored by 30-fold. For both apo and (Ca2+)4-CaM, NMR demonstrated that NaV1.2 IQ motif peptide (NaV1.2IQp) exclusively made contacts with C-domain residues of CaM (CaMC). To understand how calcium triggers conformational change at the CaM-IQ interface, we determined a solution structure (2M5E.pdb) of (Ca2+)2-CaMC bound to NaV1.2IQp. The polarity of (Ca2+)2-CaMC relative to the IQ motif was opposite to that seen in apo CaMC-Nav1.2IQp (2KXW), revealing that CaMC recognizes nested, anti-parallel sites in Nav1.2IQp. Reversal of CaM may require transient release from the IQ motif during calcium binding, and facilitate a re-orientation of CaMN allowing interactions with non-IQ NaV1.2 residues or auxiliary regulatory proteins interacting in the vicinity of the IQ motif.

FRET Based Mapping of Calmodulin within the Ryanodine Receptor

Using time-resolved fluorescence resonance energy transfer (TR-FRET) and multi-site-directed labeling of calmodulin (CaM) and FKBP12.6, we further resolve the structural basis underlying CaM regulation of skeletal and cardiac ryanodine receptor isoforms (RyR1 and RyR2) at relaxed and contracting [Ca2+]. Although cryo-EM and FRET-based mapping place Ca2+-bound CaM in a similar location on RyR1 and RyR2, the position of Ca2+-free (apo) CaM remains controversial - cryo-EM suggests a large shift in the centers of mass between apo- and Ca-CaM states, but this was not observed in previous FRET studies. Using TR-FRET, we resolved structural states of CaM bound to RyR1 and RyR2 in isolated sarcoplasmic reticulum membranes at 0.03 and 30 micromolar Ca2+. Donor fluorophores were targeted to the cytoplasmic assembly of RyR1 or RyR2 by pre-incubating sarcoplasmic reticulum vesicles with fluorescent FKBP12.6 labeled at one of five well dispersed sites (D-FKBP). FRET was measured in the presence of CaM labeled with an acceptor fluorophore attached to one of four sites (A-CaM) for each distance measurement. The donor-acceptor distances, calculated from fluorescence decay waveforms, were used to trilaterate the acceptor probe loci within RyR. On both RyR1 and RyR2, we detect a Ca2+-driven decrease in separation between CaM lobes, suggesting a more compact Ca2+-bound CaM, relative to apo-CaM, structure. However, these Ca2+-driven shifts in fluorophore positions are not as dramatic as the large shift previously proposed. We also observe isoform specific differences, as CaM's binding location appears to be rotated down and slightly inward on RyR2 relative to RyR1. This work was supported by NIH grants R01HL092097 (to RLC/DMB) and R37AG26160 (to DDT), and by a Postdoctoral Fellowship from the American Heart Association 16POST31010019 (to RTR).

PEP-19 modulates calcium binding to calmodulin by electrostatic steering.

PEP-19 is a small protein that increases the rates of Ca2+ binding to the C-domain of calmodulin (CaM) by an unknown mechanism. Although an IQ motif promotes binding to CaM, an acidic sequence in PEP-19 is required to modulate Ca2+ binding and to sensitize HeLa cells to ATP-induced Ca2+ release. Here, we report the NMR solution structure of a complex between PEP-19 and the C-domain of apo CaM. The acidic sequence of PEP-19 associates between helices E and F of CaM via hydrophobic interactions. This allows the acidic side chains in PEP-19 to extend toward the solvent and form a negatively charged surface that resembles a catcher's mitt near Ca2+ binding loop III of CaM. The topology and gradients of negative electrostatic surface potential support a mechanism by which PEP-19 increases the rate of Ca2+ binding to the C-domain of CaM by ‘catching' and electrostatically steering Ca2+ to site III.

Structural Differences in Calmodulin Bound to Voltagate-Gated Sodium Channel IQ Motifs

Voltage-gated sodium channels (NaV) control the influx of Na+ and the rising phase of action potentials in excitable cells. They bind calmodulin (CaM) at an IQ motif in their C-terminal domain (CTD), but calcium-mediated regulation is poorly understood. Five high-resolution structures of apo (calcium-depleted) CaM bound to the IQ motif of NaV1.2, NaV1.5 and NaV1.6 show the NaV primarily contacts the C-domain of CaM (CaMC), which is in a “semi-open” tertiary conformation. In these structures, the IQ motif adopts the same orientation relative to apo CaMC. In two structures (4JPZ/Xray and 2M5E/NMR) of Ca2+-CaM bound to IQ-containing fragments of NaV1.2, Ca2+-CaM primarily makes contacts with residues 1905-1921. However, the orientation of the IQ motif relative to CaMC in 2M5E is opposite to that of 4JPZ. In 4JPZ (X-ray), the NaV1.2 CTD (residues 1777-1937, including the EF-like domain and IQ region), binds “semi-open” CaMC, whereas in 2M5E (NMR), CaMC is “open” when bound to NaV1.2 residues 1901-1927 (I1912 and Q1913 are the IQ residues). These extremely different CaM-IQ complexes may both be populated in the cell. To explore how the NaV1.2 sequence outside the IQ motif may determine these differences, we used solution (NMR) studies to compare full-length human Ca2+-CaM bound to two NaV1.2 fragments - residues 1777-1937 and 1901-1927. In both complexes, Ca2+-CaMC adopted the “open” conformation (observed in 2M5E and other Ca2+-CaMstructures), suggesting that differences in the sequences flanking the NaV1.2 IQ motif are not sufficient to explain the differences between 4JPZ and 2M5E. Future structural studies will explore how different solution conditions used in the 4JPZ (X-ray) and 2M5E (NMR) structures may select for different conformations of CaM bound to IQ motifs. Supported by NIH R01 GM57001.

FRET-Based Mapping of Calmodulin within the Ryanodine Receptor

Using time-resolved fluorescence resonance energy transfer (TR-FRET) and multi-site-directed labeling of calmodulin (CaM) and FKBP12.6, we further resolve the structural basis underlying CaM regulation of skeletal and cardiac ryanodine receptor isoforms (RyR1 and RyR2) at relaxed and contracting [Ca2+]. Although cryo-EM and FRET-based mapping place Ca2+-bound CaM in a similar location on RyR1 and RyR2, the position of Ca2+-free (apo) CaM remains controversial - cryo-EM suggests a large shift in the centers of mass between apo- and Ca-CaM states, but this was not observed in previous FRET studies. Using TR-FRET, we resolved structural states of CaM bound to RyR1 and RyR2 in isolated sarcoplasmic reticulum membranes at 0.03 and 30 micromolar Ca2+. Donor fluorophores were targeted to the cytoplasmic assembly of RyR1 or RyR2 by pre-incubating sarcoplasmic reticulum vesicles with fluorescent FKBP12.6 labeled at one of five well dispersed sites (D-FKBP). FRET was measured in the presence of CaM labeled with an acceptor fluorophore attached to one of four sites (A-CaM) for each distance measurement. The donor-acceptor distances, calculated from fluorescence decay waveforms, were used to trilaterate the acceptor probe loci within RyR. On both RyR1 and RyR2, we detect a Ca2+-driven decrease in separation between CaM lobes, suggesting a more compact Ca2+-bound CaM, relative to apo-CaM, structure. However, these Ca2+-driven shifts in fluorophore positions are not as dramatic as the large shift previously proposed. We also observe isoform specific differences, as CaM's binding location appears to be rotated down and slightly inward on RyR2 relative to RyR1. This work was supported by NIH grants R01HL092097 (to RLC/DMB) and R37AG26160 (to DDT), and by a Postdoctoral Fellowship from the American Heart Association 16POST31010019 (to RTR).

Recruitment of Calmodulin to the Tail of the Voltage-Gated Sodium Channel Nav1.2

Voltage-gated sodium channels (NaV) found in excitable cells are responsible for the rising phase of action potentials. These multi-domain transmembrane proteins are regulated by calmodulin (CaM), a highly conserved eukaryotic protein that mediates many calcium-triggered signaling events. Inactivation of sodium channels depends on CaM-mediated feedback during repolarization. In the neuronal sodium channel NaV1.2, CaM binds at least two well-separated sites: an intracellular “inactivation” loop between domains DIII and DIV, and an IQ motif [IQRAYRRYLLK] in the cytosolic C-terminal tail. The IQ motif is hypothesized to recruit calcium-free (apo) CaM, making it available to move to the III-IV linker after an influx of calcium. Despite a high degree of sequence identity, the equilibrium constants for CaM binding to nine human NaV IQ motifs span more than 3 orders of magnitude. Apo CaM binds to the NaV1.2 IQ motif with a dissociation constant (Kd) of ∼6 nM, while the Kd for binding the NaV1.9 IQ motif is ∼ 4 μM. Mutational analysis within the IQ motif has not been sufficient to explain the full range of CaM-binding affinities observed for human NaV sequences. Thus, we hypothesized that isoform-specific differences in upstream sequences were making energetically significant contributions to the free energy of binding CaM to NaV1.2. The roles of these residues are being investigated by monitoring CaM binding to biosensors containing mutant sequences of sodium channels bracketed by auto-fluorescent proteins YFP and CFP. Residue-specific information obtained by NMR will provide structural insight into the contributions of residues in the binding interface formed by NaV IQ motif sequences binding to calmodulin from multiple eukaryotes. NIH R01 GM57001.

Polarized axonal surface expression of neuronal KCNQ potassium channels is regulated by calmodulin interaction with KCNQ2 subunit.

KCNQ potassium channels composed of KCNQ2 and KCNQ3 subunits give rise to the M-current, a slow-activating and non-inactivating voltage-dependent potassium current that limits repetitive firing of action potentials. KCNQ channels are enriched at the surface of axons and axonal initial segments, the sites for action potential generation and modulation. Their enrichment at the axonal surface is impaired by mutations in KCNQ2 carboxy-terminal tail that cause benign familial neonatal convulsion and myokymia, suggesting that their correct surface distribution and density at the axon is crucial for control of neuronal excitability. However, the molecular mechanisms responsible for regulating enrichment of KCNQ channels at the neuronal axon remain elusive. Here, we show that enrichment of KCNQ channels at the axonal surface of dissociated rat hippocampal cultured neurons is regulated by ubiquitous calcium sensor calmodulin. Using immunocytochemistry and the cluster of differentiation 4 (CD4) membrane protein as a trafficking reporter, we demonstrate that fusion of KCNQ2 carboxy-terminal tail is sufficient to target CD4 protein to the axonal surface whereas inhibition of calmodulin binding to KCNQ2 abolishes axonal surface expression of CD4 fusion proteins by retaining them in the endoplasmic reticulum. Disruption of calmodulin binding to KCNQ2 also impairs enrichment of heteromeric KCNQ2/KCNQ3 channels at the axonal surface by blocking their trafficking from the endoplasmic reticulum to the axon. Consistently, hippocampal neuronal excitability is dampened by transient expression of wild-type KCNQ2 but not mutant KCNQ2 deficient in calmodulin binding. Furthermore, coexpression of mutant calmodulin, which can interact with KCNQ2/KCNQ3 channels but not calcium, reduces but does not abolish their enrichment at the axonal surface, suggesting that apo calmodulin but not calcium-bound calmodulin is necessary for their preferential targeting to the axonal surface. These findings collectively reveal calmodulin as a critical player that modulates trafficking and enrichment of KCNQ channels at the neuronal axon.