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In this review, Nathan et al. discuss recent structural insights into the regulation of the Na+ channels NaV1.5 and NaV1.4, facilitated by the combination of cryo-EM and x-ray crystallography data., Voltage-gated sodium channels (NaVs) are membrane proteins responsible for the rapid upstroke of the action potential in excitable cells. There are nine human voltage-sensitive NaV1 isoforms that, in addition to their sequence differences, differ in tissue distribution and specific function. This review focuses on isoforms NaV1.4 and NaV1.5, which are primarily expressed in skeletal and cardiac muscle cells, respectively. The determination of the structures of several eukaryotic NaVs by single-particle cryo-electron microscopy (cryo-EM) has brought new perspective to the study of the channels. Alignment of the cryo-EM structure of the transmembrane channel pore with x-ray crystallographic structures of the cytoplasmic domains illustrates the complementary nature of the techniques and highlights the intricate cellular mechanisms that modulate these channels. Here, we review structural insights into the cytoplasmic C-terminal regulation of NaV1.4 and NaV1.5 with special attention to Ca2+ sensing by calmodulin, implications for disease, and putative channel dimerization.

Neuronal voltage-gated sodium channel Na(V)1.2 C-terminal domain (CTD) binds calmodulin (CaM) constitu-tively at its IQ motif. A solution structure (6BUT) and other NMR evidence showed that the CaM N domain (CaM(N)) is structurally independent of the C-domain (CaM(C)) whether CaM is bound to the Na(V)1.2(IQp) (1,901–1,927) or Na(V)1.2(CTD) (1,777–1,937) with or without calcium. However, in the CaM + Na(V)1.2(CTD) complex, the Ca(2+) affinity of CaM(N) was more favorable than in free CaM, while Ca(2+) affinity for CaM(C) was weaker than in the CaM + Na(V)1.2(IQp) complex. The CTD EF-like (EFL) domain allosterically widened the energetic gap be-tween CaM domains. Cardiomyopathy-associated CaM mutants (N53I(N54I), D95V(D96V), A102V(A103V), E104A(E105A), D129G(D130G), and F141L(F142L)) all bound the Na(V)1.2 IQ motif favorably under resting (apo) conditions and bound calcium normally at CaM(N) sites. However, only N53I and A102V bound calcium at CaM(C) sites at [Ca(2+)] < 100 μM. Thus, they are expected to respond like wild-type CaM to Ca(2+) spikes in excitable cells.

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.

Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2+-dependent inactivation; no such inactivation is observed in the cardiac Na+ channel (NaV1.5). Taken together, the crystal structures of the NaV1.4 C-terminal domain relevant complexes and thermodynamic binding data presented here provide a rationale for this isoform difference. A Ca2+-dependent CaM N-lobe binding site previously identified in NaV1.5 is not present in NaV1.4 allowing the N-lobe to signal other regions of the NaV1.4 channel. Consistent with this mechanism, removing this binding site in NaV1.5 unveils robust Ca2+-dependent inactivation in the previously insensitive isoform. These findings suggest that Ca2+-dependent inactivation is effected by CaM’s N-lobe binding outside the NaV C-terminal while CaM’s C-lobe remains bound to the NaV C-terminal. As the N-lobe binding motif of NaV1.5 is a mutational hotspot for inherited arrhythmias, the contributions of mutation-induced changes in CDI to arrhythmia generation is an intriguing possibility., Skeletal muscle voltage-gated Na+ channel (NaV1.4) activity is subject to calmodulin (CaM) mediated Ca2 +-dependent inactivation while cardiac NaV1.5 is not. Here authors use structural biology, binding and electrophysiology to parse the Ca2 +-dependent changes of CaM when bound to the NaV1.4.

The structures of the cytosolic portion of voltage activated sodium channels (CTNav) in complexes with calmodulin and other effectors in the presence and the absence of calcium provide information about the mechanisms by which these effectors regulate channel activity. The most studied of these complexes, those of Nav1.2 and Nav1.5, show details of the conformations and the specific contacts that are involved in channel regulation. Another voltage activated sodium channel, Nav1.4, shows significant calcium dependent inactivation, while its homolog Nav1.5 does not. The available structures shed light on the possible localization of the elements responsible for this effect. Mutations in the genes of these 3 Nav channels are associated with several disease conditions: Nav1.2, neurological conditions; Nav1.4, syndromes involving skeletal muscle; and Nav1.5, cardiac arrhythmias. Many of these disease-specific mutations are located at the interfaces involving CTNav and its effectors.

Voltage-gated sodium channels (Nav) are essential for cardiac and skeletal muscle function. Channelopathic mutations in cardiac sodium channel (Nav1.5) and skeletal muscle sodium channel (Nav1.4), particularly in their cytoplasmic carboxy tails (CTNav), give rise to numerous arrhythmias and myotonias. In Nav regulation, CTNav partners with the Ca2+-sensing protein calmodulin (CaM). We aim to understand how CaM regulates channel function. Ca2+-free CaM (apoCaM) is a regulatory modulator of Nav, and apoCaM bound to the CTNav increases the channel's open probability. CaM also participates in the Ca2+-dependent inactivation (CDI) of Nav1.4. In contrast, Nav1.5 does not show CDI and no role of Ca2+-CaM is known in Nav1.5. To understand the Ca2+-control of CaM regulation of these sodium channels we have collected binding data of CTNavs with CaM, in the presence and absence of Ca2+. Binding data of CTNavs with CaM mutants with Ca2+-binding knocked out in either of CaM's functional domains (lobes) have also been collected to understand the distinct roles of CaM's lobes. Collectively, these binding data have allowed us to predict CTNav and CaM populations as a function of Ca2+ concentration. To gain information on the structural changes induced by Ca2+ on the CTNav-CaM complexes, we conducted small angle scattering (SAXS) experiments on CTNav1.4-CaM and CTNav1.5-CaM in the presence and absence of Ca2+. The distance distribution function, P(r), shows changes in both complexes upon addition of Ca2+. Computational modeling of flexible forms of the complexes reveals conformations of CTNav-CaM, in the presence and absence of Ca2+, that are compatible with our SAXS data. The molecular envelope of the CTNav1.5-apoCaM matches well with the apoCaM-CTNav1.5 crystal structure previously determined by our lab.

Calcium-mediated regulation of early stages of heart development critically depends on calcineurin (CaN), a heterodimeric, calcium-activated phosphatase. CaN acts by dephosphorylating NF-AT, promoting its nuclear localization to launch transcription that controls cardiac muscle growth. Vulnerability of the embryonic heart to fluctuations in CaN activity propels our studies to understand calcium-dependent regulation of CaN. Calcium partially activates CaN by binding to CaNB, its intrinsic 4-EF-Hand subunit. Full activity occurs upon binding of calmodulin (CaM, another 4-EF-Hand protein) which recognizes a CaM-binding domain (CaMBD) near the CaNA auto-inhibitory domain. To probe sequential calcium-mediated activation of CaN, we found CaM binding to CaNA caused a thousand-fold increase in calcium-binding affinity of each domain of CaM, while preserving a 10-fold difference between the domains. To explore the molecular basis for calcium-triggering of CaN, we studied site-knockout mutants of CaNB and CaM, and mutagenized CaMBD, a 1-5-8-14 basic, amphipathic alpha-helix with picomolar affinity for calcium-saturated CaM. CaM binding to an auto-fluorescent biosensor (donor-CFP-CaMBD-acceptor-YFP) allows estimates of equilibrium constants. Site-knockout mutants of CaM had variable effects on the CaN-CaM interface. Mutagenized CaMBDs indicated energetic contributions of the 1-5-8-14 positions were dramatically different: Ile>Ala at position 5 lowered binding energy by 3 kcal/mol more than Ile>Ala at position 1 or 8. Conventional motif classifications for CaMBDs clearly do not indicate energetic contributions at interfaces. Further, Phe>Ala (substitution of the sole aromatic residue) only modestly affected WT CaM binding, indicated that Phe410 is not an “anchor residue”. We are pursuing connections between these protein-protein interfaces and enzymatic activity of CaN to understand how congenital defects may lead to faulty development of the cardiac muscle. NIH R01 GM57001, AHA 12GRNT12050395 to MAS, and AHA Predoctoral Fellowship to SEO.

Voltage-dependent sodium channels (VDSCs) control the rising phase of action potentials in excitable cells. Channelopathies associated with these transmembrane proteins include Dravet syndrome, epilepsy, Long QT syndrome type 3, ventricular fibrillation, autism, and pain insensitivity. This family of ion channels is regulated by calmodulin (CaM), a small essential eukaryotic calcium sensor that contains two domains (N and C) with different affinity for calcium. CaM recognizes at least two regions of the Nav channels: the inactivation gate between transmembrane domains III and IV, and an IQ motif located in the intracellular C-terminal tail of the alpha subunit of Nav. In a genetic screen of Paramecium, the Kung laboratory discovered the first biological role for differences in the N and C domains of CaM. They identified N-domain mutations that specifically altered calcium-dependent regulation of sodium channels. However, the molecular mechanism is not understood. Our laboratory has quantitatively characterized the interactions of both domains of mammalian and Paramecium CaM with the IQ motif of NaV1.2. Under apo conditions, both mCaM and PCaM bind to the IQ motif of NaV1.2 exclusively via the CaM C-domain; Ca2+ binding to CaM lowers its affinity for the IQ motif. To understand calcium–mediated feedback control, we conducted thermodynamic analyses of CaM binding to IQ motif sequences from 9 members of the human Nav family. CaM association was detected as fractional disruption of FRET in a biosensor containing the NaV IQ motif sandwiched between YFP and CFP. We see two classes of IQ motifs in human sodium channels that differ in their relative binding affinity for apo and calcium-saturated CaM, and the roles of the two CaM domains. Support: NIH R01 GM57001 & Carver Charitable Trust Grant 01-224

Voltage-dependent sodium channels in the NaV1 family control action potentials in neurons and muscle. They are regulated by calmodulin (CaM), an essential eukaryotic calcium sensor that contains two highly homologous domains (N and C). NaV channelopathies include epilepsy, Long QT syndrome, ventricular fibrillation, familial autism, and pain insensitivity. Some mutations are in or near an IQ motif (IQxxxBGxxxB, B = K, R) in the intracellular C-terminal tail of the pore-forming alpha subunit where CaM binds tightly. In the 11 residues of each NaV IQ motif, only the Q is completely conserved. In NaV1.2 (2KXW) and NaV1.5 (2L53), both apo and Ca2+-saturated CaM anchor to the IQ motif via C. However, Ca2+ binding to CaM changes its conformation and lowers its affinity for the IQ motif. To determine how CaM translates changes in intracellular [Ca2+] into conformational work, and how it regulates multiple isoforms of NaV, we are using NMR and fluorescence to test how CaM discriminates between IQ motifs of NaV and how disease-causing mutations uniquely affect binding of apo and calcium-saturated N and C. To determine energetic differences in CaM binding to IQ motifs, we embedded each sequence into an auto-fluorescent biosensor (YFP-IQ-CFP) to determine their affinity for CaM, and each CaM domain individually. Biosensor quantum yield permits resolution of dissociation constants close to 1 nM from equilibrium titrations. Based on thermodynamic and structural studies of (Ca2+)4-CaM bound to IQ motifs in the CaV family, a homology model is proposed for (Ca2+)4-CaM bound to NaV1.2. NIH R01 GM57001 and Carver Charitable Trust Grant 01-224.91

Voltage-gated sodium channels (NaV) are integral membrane proteins, part of a macromolecular complex that is central to signaling in the heart and other excitable tissues. Regulation of the essential functions of the channel are complex and may differ among tissue-specific isoforms nevertheless, mechanistic understanding of the molecular regulation of the channel is beginning to emerge. We and others have demonstrated the importance of the carboxyl terminus (CT) in the regulation of the channel. The CT is a hot spot for mutations that produce inherited cardiac arrhythmias, myotonias, epilepsy and autism. In the case of NaV1.5, the cardiac channel, mutations of critical structural motifs in the CT (including an EF hand-like motif and an IQ motif) result in disease conditions such as Brugada and LQT syndromes. Also, altered NaV channel trafficking and function with consequent intracellular Na+ overload contributes to the development of dilated cardiomyopathy. The structure of the CT of the Nav1.5 channel in complex with calmodulin (CaM), determined to 2.9 A resolution, shows that many of the mutations associated with disease states occur at CTNav1.5-CaM interfaces. Based on this structure a mechanism for the transition to the non-inactivated state of the channel is proposed.

Voltage-gated sodium channels (Na(v)) underlie the rapid upstroke of action potentials in excitable tissues. Binding of channel-interactive proteins is essential for controlling fast and long-term inactivation. In the structure of the complex of the carboxy-terminal portion of Na(v)1.5 (CTNa(v)1.5) with calmodulin (CaM)-Mg(2+) reported here, both CaM lobes interact with the CTNa(v)1.5. On the basis of the differences between this structure and that of an inactivated complex, we propose that the structure reported here represents a non-inactivated state of the CTNa(v), that is, the state that is poised for activation. Electrophysiological characterization of mutants further supports the importance of the interactions identified in the structure. Isothermal titration calorimetry experiments show that CaM binds to CTNa(v)1.5 with high affinity. The results of this study provide unique insights into the physiological activation and the pathophysiology of Na(v) channels.

The neuronal voltage-gated sodium channel (NaV1.2) is regulated by calmodulin (CaM), a highly conserved, ubiquitous eukaryotic protein that mediates many calcium-triggered signaling events. Fast inactivation of the channel depends on CaM-mediated feedback transduction of calcium flux during the repolarization phase of an action potential. CaM binds to an intracellular loop (the III-IV linker) and an IQ motif [IQRAYRRYLLK] in the cytosolic C-terminal tail of the channel. The NaV1.2 IQ motif binds only to the C-domain of CaM with high affinity to both its calcium-free (apo) and calcium-saturated states. However, the IQ motif binds more favorably to apo CaM than to calcium-saturated CaM. To determine the molecular basis for this calcium-dependent difference in association, mutational perturbations of residues in the NaV1.2 IQ motif were designed to disrupt close contacts observed in our solution (NMR) structure of the semi-open C-domain of apo-CaM bound to the IQ motif (2KXW.pdb). The contributions of these residues to binding energetics were determined by monitoring CaM-induced disruption of FRET in biosensors containing wild-type or mutant sequences of the IQ motif bracketed by auto-fluorescent proteins YFP and CFP. All mutations lowered affinity for calcium-saturated CaM, but they had uniformly more deleterious effects on the binding of apo CaM. Furthermore, the decrease in affinity for apo CaM caused by loss of the Ile-Gln pair was 30-fold greater than that observed for loss of the Tyr-Tyr pair. Thus, the energy of interaction between the NaV1.2 IQ motif and semi-open apo CaM is not accounted for primarily by the classical “aromatic anchors” that dominate interactions of calcium-saturated CaM with its target sequences in kinases, receptors and other channels. Support: NIH R01 GM57001, Carver Charitable Trust Grant 01-224.

NaV1.2 is regulated by calmodulin (CaM), an essential calcium sensor with two homologous domains (N and C). CaM binds tightly to an IQ motif in the intracellular C-terminal tail of the pore-forming alpha subunit of NaV1.2 as well as to the inactivation gate. The IQ motif interacts with the “semi-open” cleft of apo CaM: I inserts into the cleft, while Q contacts the FG-turn of CaM (2KXW). To learn how CaM-IQ responds to increases in intracellular [Ca2+], we determined equilibrium constants for apo and calcium-saturated CaM binding to biosensors containing mutated IQ motif sequences sandwiched between YFP and CFP. Their quantum yields permit resolution of Kd values close to 1 nM from equilibrium titrations. Changes of NaV1.2 residues making close contacts with apo CaM were anticipated to diminish binding of both apo and calcium-saturated CaM. However, the quantitative effects differed by orders of magnitude. The affinity for calcium-saturated CaM dropped by factors of 10-100, while effects on apo CaM binding were more severe. Thus, the CaM-IQ interface differs dramatically depending on calcium-saturation of CaM. NMR studies of a complex of (Ca2+)2-CaM-C-domain bound to the IQ motif showed that calcium binding opens CaM but also causes it to pivot by 180° so that it binds to the IQ motif in the opposite direction. The I of the IQ motif contacts the “open” hydrophobic cleft of (Ca2+)2-CaM, but the Q points towards the linker between the N- and C-domains of CaM. In conjunction with 2KXW, this new structure provides the first pair of high resolution structures for apo and calcium-saturated CaM bound to a single IQ motif. Support: NIH R01 GM57001 and Carver Charitable Trust Grant 01-224.

Calcineurin (CaN) is a heterodimeric Ser/Thr phosphatase. Calcineurin regulates diverse biological functions, from T-cell activation to critical events in early heart development. The enzymatic activity of the A subunit (CaNA) is regulated by direct binding of Ca2+ to its B subunit (CaNB), and Ca2+ binding of the two-domain calcium-binding protein calmodulin (CaM) that is required for full activity. CaNA contains an auto-inhibitory domain that prevents full activation of CaN even when CaNB is Ca2+ saturated.Interactions between CaM and CaN occur through a CaM-binding domain (CaMBD). This sequence (391-414 of α CaNA) is a BAA-motif (basic amphipathic alpha-helix) in a region that becomes disordered when CaNB binds calcium. (Ca2+)4-CaM has picomolar affinity for CaN, as determined by thermodynamic linkage (O'Donnell et al, Proteins 2011). The Creamer Lab (Rumi-Masante et al, JMB 2012) has shown that CaM-binding induces secondary structuring of the full regulatory region of CaNA that extends beyond this CaMBD.We have also determined that calcium binding to CaM bound to the CaMBD is sequential, with the C-domain sites saturating at lower calcium than the N-domain sites. Thus, we are investigating a hypothesis that the mechanism of calcium-induced activation of CaN requires at least three steps that utilize the two domains of CaM (N and C) differently. We are using “knockout” mutants of CaM with modifications in one or more calcium-binding sites, and monitoring their binding to biosensor proteins containing the CaMBD embedded between auto-fluorescent proteins. Without CaM binding the fluorescent proteins undergo FRET, while CaM binding to the BAA motif reduces FRET efficiency and allows calculations to be made on CaM-biosensor interaction kinetics. We will report domain-specific effects of these mutations on recognition of this region. Support: NIH R01 GM57001, UI Helen Johnson Scholar Award, American Heart Association 12GRNT12050395.

Voltage-dependent sodium channels (Nav1.X) control the rising phase of action potentials in excitable cells. Mutational pathologies include epilepsy, Long QT syndrome, familial autism, and pain insensitivity. This family of ion channels is regulated by calmodulin (CaM), a small, essential eukaryotic calcium sensor that contains two highly homologous domains whose affinities for calcium differ by an order of magnitude. CaM is known to recognize at least two regions of NaV: the highly conserved inactivation gate between transmembrane domains III and IV, and an IQ motif, located in the intracellular C-terminus of the Nav. The IQ motif, which may serve as a CaM “sink”, holds CaM available to quickly re-associate with the inactivation gate upon calcium binding. High-resolution structures of apo CaM bound to the IQ motifs of NaV1.2, 1.5 and 1.6 show many similarities. However, the free energies of CaM binding to these motifs are markedly different. In our studies of CaM binding to the IQ motif of NaV1.2, the C-domain of both apo (calcium-free) and calcium-saturated CaM bind and calcium binding to CaM lowers its affinity for the IQ motif. To better understand calcium-mediated feedback control, we conducted thermodynamic analyses of CaM binding to the IQ motif sequences representing all 9 members of the human Nav family and the consensus inactivation gate. CaM binding was detected by monitoring the loss of FRET intensity of a biosensor containing the IQ motif sandwiched between two YFP and CFP. The nine sodium channels split into two classes based on their binding affinity for apo CaM at the IQ motif. We are exploring the roles of individual residues to determine the positions that are necessary and sufficient to confer preferential binding of apo or calcium-saturated CaM. Support: NIH R01 GM57001.