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J. Biol. Chem., Vol. 279, Issue 43, 45004-45012, October 22, 2004

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Calmodulin Mediates Ca²⁺ Sensitivity of Sodium Channels^*

James Kim, Smita Ghosh, Huajun Liu, Michihiro Tateyama, Robert S. Kass, and Geoffrey S. Pitt¶||

From the Departments of Pharmacology and ^¶Medicine, Division of Cardiology, Columbia University, New York, New York 10032

Received for publication, June 29, 2004 , and in revised form, August 9, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Ca²⁺ has been proposed to regulate Na⁺ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Na_v1 C terminus. Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca²⁺ confers sensitivity are poorly understood. Studies on the homologous Ca_v1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na⁺ channels. In contrast to previous reports, we found that Ca²⁺ does not bind directly to Na⁺ channel C termini. Rather, Ca²⁺ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CaM regulation of Ca_v1.2. In Na_v1.2 or Na_v1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca²⁺-dependent conformational change seen in the Ca_v1.2·CaM complex. Furthermore, CaM binding to Na_v1 C termini lowered Ca²⁺ binding affinity and cooperativity among the CaM-binding sites compared with CaM alone. Nonetheless, we found suggestive evidence for Ca²⁺/CaM-dependent effects upon Na_v1 channels. The R1902C autism mutation conferred a Ca²⁺-dependent conformational change in Na_v1.2 C terminus·CaM complex that was absent in the wild-type complex. In Na_v1.5, CaM modulates the Cterminal interaction with the III–IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death. Together, these data offer new biochemical evidence for Ca²⁺/CaM modulation of Na⁺ channel function.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Changes in cytoplasmic Ca²⁺ fluxes are the fundamental readouts of cellular electrical signals. It has become increasingly recognized that the ion channels controlling this electrical activity are subject to feedback modulation by these very Ca²⁺ fluxes. Among the best characterized responses are Ca²⁺ activation of small conductance K⁺ (SK)¹ channels to generate slow after-hyperpolarizations (1) and Ca²⁺-dependent inactivation of L-type Ca²⁺ channels (Ca_v1.2) to limit Ca²⁺ entry during action potentials (2). For both cases, the Ca²⁺-binding protein calmodulin (CaM) is an obligate channel subunit that serves as the Ca²⁺ sensor (3–5).

Identification of CaM in these roles has helped foster a new understanding of this ubiquitous Ca²⁺-binding protein. Previously, the model for CaM action had been enzymatic activation through disinhibition; binding of Ca²⁺/CaM to an autoinhibitory peptide, usually an amphipathic -helix, exposes the active site of an enzyme (6). In contrast, biochemical, functional, and structural analyses of CaM regulation of SK and Ca_v1.2 channels revealed that these channels contain large CaM-binding pockets in their cytoplasmic C termini composed of multiple noncontiguous sequences (3, 7–10). Furthermore, although the details of CaM binding and Ca²⁺/CaM action differ between the SK and Ca_v1.2 channels, both constitutively bind apoCaM, which sits poised as a resident Ca²⁺ sensor. The recent structural determination of Ca²⁺/CaM in complex with the edema factor of Bacillus anthracis also revealed a large non-canonical CaM-binding site (11) and demonstrated a new mode of CaM action by promoting active-site remodeling (6). These and other recent studies have greatly expanded the repertoire of CaM interaction motifs and modes of CaM function.

Recognition of an "IQ" motif within the Ca_v1.2 channel CaM-binding pocket (5, 12) fostered the identification of similar motifs in homologous regions of other channels and subsequent attempts to identify whether these channels are likewise Ca²⁺/CaM-regulated. First described as the binding site in myosins for CaM-like essential light chains (13), IQ motifs were subsequently found in apoCaM-binding proteins such as neuromodulin (14). Many proteins containing CaM-binding IQ motifs have now been identified (15), providing a loose consensus sequence of IQXXXRXXXXR for identifying additional CaM-binding proteins. The voltage-gated Na⁺ channels form one family of ion channels in which an IQ motif has been identified. After identifying CaM as binding partner for the Na_v1.2 C terminus in a yeast two-hybrid screen, Mori et al. (16) recognized an IQ motif in their bait construct at a position homologous to the IQ motif in Ca_v1.2. This led to a series of studies looking for Ca²⁺/CaM regulation of Na⁺ channel function (17–19).

Because Na⁺ channels initiate the rapid upstroke of the cardiac and neuronal action potentials, Ca²⁺/CaM regulation of Na⁺ channel activity offers an intriguing way for the fine-tuning of membrane excitability. The clinical implications of such regulation are underscored by findings that mutations within or near the IQ motif are pathogenic. Mutations in the major cardiac sodium channel (Na_v1.5) are arrhythmogenic, placing patients at risk for sudden cardiac death (20, 21), and a mutation in a neuronal sodium channel (Na_v1.2) is associated with a familial form of autism (22). Because of the homology between Na⁺ and Ca²⁺ channels with respect to their IQ motifs and the location of these motifs within the channels, the search for a defined role for Ca²⁺/CaM in Na⁺ channel function followed the model originally developed for Ca_v1.2, in which interactions of Ca²⁺/CaM with the IQ motif in Ca_v1.2 accelerate channel inactivation (2). Studies with similar approaches on three different Na⁺ channels yielded inconsistent results, however (17–19). Although it has been proposed that Ca²⁺/CaM may regulate Na⁺ channels in an isoform-specific manner (19), this cannot explain the conflicting results with common isoforms among these studies. Direct binding of Ca²⁺ to a paired EF hand motif in Na_v1.5 channels has recently been proposed as an alternative mode of Ca²⁺ regulation (23). Structural modeling of the proximal portion of the Na_v1.5 C terminus had predicted overall homology to the N-terminal lobe of CaM, but unlikely binding capacity for Ca²⁺, since many key acidic residues used for specific Ca²⁺ binding in CaM are not conserved in the Na_v1.5 C terminus (24). Nevertheless, the new model presented by Wingo et al. (23) and their functional analysis of a pro-arrhythmic mutation within this region, if correct, could offer an explanation for the differences reported among the studies focused on CaM.

New information on CaM interaction with and regulation of Ca²⁺ channels (8, 25) has offered a mirror with which to reexamine CaM interaction with Na⁺ channels. The specific rationale for this approach derives from the appreciation that CaM interaction domains cannot be predicted from the primary amino acid sequence, as demonstrated by the crystal structures of CaM in complex with SK channels and B. anthracis edema factor (9, 10) and highlighted by the recent work on CaM regulation of Ca_v1.2 (8). If sequences outside the IQ motif are essential for CaM binding and regulation of Na_v1 channels, as they are with Ca_v1.2, then a more systematic approach than the original focus on the IQ motifs might yield more definitive results. Another motivation for this approach came from the specific biochemical tools developed for understanding Ca²⁺/CaM regulation of Ca_v1.2 (8), which offered new opportunities for exploring Ca²⁺/CaM interaction with Na_v1 channels. With this framework, we explored the biochemistry of CaM interaction with the C termini of both Na_v1.2 and Na_v1.5. These are two potentially informative isoforms because both have pathogenic mutations in or near the IQ motif that offer the possibility to further define a role for CaM regulation of channel function.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Computational Analysis—Sequence alignment of the C terminus of Ca_v1.2 (GenBank^TM/EBI Data Bank accession number X15539 [GenBank] ), Na_v1.2 (accession number M94055 [GenBank] ), and Na_v1.5 (accession number M77235 [GenBank] ) was performed using Proteinsolve (Stratagene) based on a modified version of the Boyko weight matrix (26). The secondary structure prediction of the Ca_v1.2 C terminus was determined using Proteinsolve based on the consensus prediction from multiple methods (homology, hydrophobic moment, GOR (40), Chou-Fasman (41, 42), and motifbased), and prediction of Na_v1.2 and Na_v1.5 C termini was based on previous work by Cormier et al. (24).

Construction of cDNA Plasmids—A cDNA encoding the C terminus of Na_v1.2 was purchased from Open Biosystems after identifying an appropriate expressed sequence tag. DNA sequences corresponding to amino acids 1777–1937 of Na_v1.2 and amino acids 1773–1940 of Na_v1.5 were amplified by PCR with primers containing endonuclease restriction sites. Products were digested with the appropriate enzymes and ligated into pET28a⁺ (Novagen) to produce cDNAs encoding His₆-tagged proteins. The CaM expression plasmid has been described (8). Mutations were generated using QuikChange (Stratagene).

Protein Expression and Purification—BL21 cells were transformed by electroporation with the appropriate plasmid(s). A 10-ml starter culture was grown with the appropriate antibiotic(s) for 2 h at 37 °Cand then transferred to 1-liter flasks, where the culture was grown until A₆₀₀ = 0.3. The culture flasks were cooled in an ice-water bath, and protein expression was induced with 1.0 mM isopropyl -D-thiogalactopyranoside for 72 h at 16 °C. Cells were harvested and resuspended in 500 mM NaCl, 20 mM Tris, 5 mM imidazole, and 25% glycerol (pH 7.5) supplemented with EDTA-free Complete protease inhibitor mixture (Roche Applied Science), and bacterial cell lysates were prepared by passage through a French pressure cell. The lysates were centrifuged at 100,000 x g for 90 min, and the supernatants were then applied to Talon metal affinity resin (Clontech). The proteins were eluted with 250 mM imidazole, aliquoted, and stored at -20 °C in 25% glycerol for further use.

Gel Filtration Analysis—Gel filtration was performed over a Superdex 200 HR 10/30 column on an AKTA FPLC (Amersham Biosciences) in 500 mM NaCl and 20 mM Tris (pH 7.5) supplemented with CaCl₂ (10 µM) or EGTA (5 mM). The following protein standards (Amersham Biosciences) were used to calibrate the elutions: aldolase (158 kDa), albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen (26 kDa).

Protein Concentration Determination—The concentration of CaM was determined by UV absorbance at 277 nm using a molar extinction coefficient of 3300 M^-1 cm^-1. The concentration of CaM in complex with fusion proteins of Na_v1 C termini was determined based on a standard of known CaM concentrations, as described above, on a Coomassie Blue-stained gel.

Fluorescence Spectroscopy—Before obtaining fluorescence spectra, all solutions and proteins were treated with Chelex (Bio-Rad) to remove any Ca²⁺ present. Fluorescence spectra were obtained on a Photon Technology International QuantaMaster spectrofluorometer in a 2-ml quartz cuvette (Hellma) in buffer containing 20 mM Tris (pH 7.4) and 100 mM NaCl. Intrinsic tyrosine fluorescence spectra were obtained at _ex = 280 nm and monitored for fluorescent emission between 290 and 390 nm. Spectra for 5 µM CaM alone and in complex with the fusion proteins of Na_v1 C termini were obtained upon sequential addition of 100 µM EGTA, 140 µM CaCl₂, and 400 µM EGTA. Ca²⁺ titration experiments were carried out with Ca²⁺ buffers containing 20 mM MOPS (pH 7.2), 100 mM KCl, 1 mM MgCl₂, and various concentrations of CaCl₂ and EGTA. Free Ca²⁺ concentrations were precalibrated in buffers obtained from Molecular Probes, Inc. or calculated using WEBMAXC STANDARD (www.stanford.edu/~cpatton/maxc.html) to obtain buffers with a range of free Ca²⁺ concentrations not well represented by the available buffer set.

Glutathione S-Transferase (GST) Pull-down Assays—The GST-Na_v1.5 III–IV linker fusion protein and the GST control were bound to glutathione-Sepharose 4B (Amersham Biosciences). The Na_v1 C terminus·CaM fusion protein complex was incubated with bound GST or GST-Na_v1.5 III–IV linker for 60 min at 25 °C in 150 mM NaCl, 50 mM Tris, and 0.1% Triton X-100 (pH 7.4) supplemented with 1 mM Ca²⁺ or 1 mM EGTA. The bound complexes were then washed extensively with the appropriate buffer, eluted in SDS sample buffer, separated by SDS-PAGE, and visualized by Coomassie Blue staining.

Expression of Recombinant Na⁺ Channels and Electrophysiology—Na⁺ channels were expressed in human embryonic kidney 293 cells at 22 °C as described previously (27). CD8-positive cells were identified using Dynabeads (M-450, Dynal, Inc.) and were patch-clamped 48 h after transfection. Membrane currents were measured using whole cell patch-clamp procedures with Axopatch 200B amplifiers (Axon Instruments, Inc., Foster City, CA). Protocols and solutions for measurement of Na⁺ channel current tetrodotoxin-sensitive Na⁺ channel currents and sustained caret (I_sus) are described in detail in a previous publication (28).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
As a first step in using insights from the recent work on Ca_v1.2·CaM (8) to analyze CaM binding to Na_v1 channels, we aligned the primary sequences of Na_v1.2 and Na_v1.5 C termini with the Ca_v1.2 C terminus and looked for similarities within the regions found important for CaM interaction with the Ca_v1.2 C terminus (Fig. 1A). We especially focused on homology outside the IQ motif, as identification of additional regions that contribute to CaM interaction in the Na_v1 channels could provide important insight into CaM interaction and function. The Na_v1.2 and Na_v1.5 sequences are very similar to each other (76% identical) in this proximal portion of the C terminus. Compared with the Ca_v1.2 sequence, they show significant similarity (18 and 19% identical, respectively) that extends throughout the entire region. Secondary structure predictions of the Na⁺ and Ca²⁺ channel C termini also showed remarkable correlations (Fig. 1A). These results led us to hypothesize that CaM interaction with Na⁺ channels may also involve multiple noncontiguous sequences in addition to the IQ motifs.

View larger version (59K): [in this window] [in a new window]   FIG. 1. Nav1.2 and Nav1.5 CTs form stable complexes with CaM and apoCaM. A, sequence alignment of the calmodulin-binding regions of Cav1.2, Nav1.2, and Nav1.5. Amino acids conserved among the three are shaded; amino acids identical among the three are shown in boldface. The IQ motif is boxed. Shown below the Nav1.5 sequence are the locations of the six predicted -helices (H1–H6) from the structural model proposed by Cormier et al. (24). Shown above the Cav1.2 sequence are the locations of -helices predicted by computational methods as described under "Experimental Procedures." The amino acids mutated in this study are circled. B, Coomassie Blue-stained gels showing purification of the Nav1.2 CT·CaM (left panel) and Nav1.5 CT·CaM (right panel) complexes. First and second lanes, in the absence of CaM coexpression, the Nav1 CT was present in the crude extract (Ex), but little remained in the supernatant (Sup) after ultracentrifugation at 100,000 x g. Third and fourth lanes, coexpression with CaM resulted in soluble Nav1 protein. An equivalent amount of the bacterial culture was loaded in the first through fourth lanes. Fifth lane, shown is the purified (P) Nav1 CT·CaM complex. C, gel filtration analyses of Nav1.2 CT·CaM (left panel) and Nav1.5 CT·CaM (right panel) using a Superdex 200 HR 10/30 column run in the presence of 10 µM Ca2+ (black) or 5 mM EGTA (gray). There was no Ca2+-dependent change for either complex.

We next tested whether the Na_v1.2 CT·CaM or Na_v1.5 CT·CaM complex displayed an altered mobility on a gel filtration column. Since the Ca²⁺-induced mobility shift of the Ca_v1.2·CaM complex was interpreted as the conformational change that underlies Ca²⁺-dependent gating and provided an important part of the model for CaM regulation of Ca_v1.2 (8), a similar Ca²⁺-dependent conformational change in Na_v1 CT·CaM complexes would be informative. For the Na_v1 CT·CaM complexes, we did not detect any difference in mobility in either EGTA- or 10 µM Ca²⁺-containing buffer (Fig. 1C). This result raised the possibility that the Na_v1 CT·CaM complexes are Ca²⁺-insensitive. Alternatively, Ca²⁺ interaction with the Na_v1 CT·CaM complexes might induce a change not detectable by gel filtration chromatography.

We therefore looked for Ca²⁺ enhancement of intrinsic Tyr fluorescence as a more sensitive test of Ca²⁺ binding to CaM. This well characterized property reports changes in the hydrophobicity of the environment near Tyr⁹⁹ and Tyr¹³⁸ in the CaM C-terminal lobe that occur as a result of a local conformational change induced by Ca²⁺ binding (29), as demonstrated in Fig. 2C. For the Ca²⁺-insensitive CaM₁₂₃₄ mutant, there was no Ca²⁺ enhancement of Tyr fluorescence (Fig. 2F). By this measure, Ca²⁺ clearly bound to the C-terminal lobe of CaM when in complex with either the Na_v1.2 or Na_v1.5 CT (Fig. 2, A and B). Ca²⁺ enhanced the intrinsic Tyr fluorescence (black circles; _em = 308 nm) of both the Na_v1.2 CT·CaM and Na_v1.5 CT·CaM complexes, which was completely reversed by the addition of EGTA. Sequential addition of Ca²⁺ and EGTA to the same sample ensured that the increase in intensity was a property of the protein and not a result of changes in protein concentration due to pipetting errors. To rule out that the Tyr fluorescence enhancement was confounded by Ca²⁺-induced changes in the fluorescent properties of the five additional Tyr residues in Na_v1.2 or the three in Na_v1.5, we repeated these studies with Na_v1 CT·CaM₁₂₃₄ complexes and found no Ca²⁺-enhanced Tyr fluorescence (Fig. 2, D–F). For CaM in complex with either the Na_v1.2 or Na_v1.5 CT, the apparent K_0.5 for Ca²⁺ binding to the C-terminal lobes showed an 6-fold increase (K_0.5 = 12.6 ± 2.8 µM for Na_v1.2 CT·CaM, 12.6 ± 3.9 µM for Na_v1.5 CT·CaM, and 1.9 ± 0.2 µM for CaM alone; n = 3) and a loss of the cooperativity between the Ca²⁺-binding sites (Fig. 2I).

View larger version (24K): [in this window] [in a new window]   FIG. 2. Fluorescence spectroscopy shows that Ca2+ binds to CaM in the Nav1 CT·CaM complexes and not to the Nav1 CTs. A–F, intrinsic fluorescence spectra (ex = 280 nm) for the indicated complexes, CaM, and CaM1234 were obtained before (red) or after (black) the addition of Ca2+. A third spectrum was then obtained after the addition of EGTA (blue) to demonstrate return to starting conditions. Black circles, peak of intrinsic tyrosine fluorescence from CaM ( = 308 nm); gray circles, peak of intrinsic tryptophan fluorescence from Nav1 ( = 325 nm), demonstrated in G and H. These data demonstrate a Ca2+-induced increase in tyrosine fluorescence dependent on the presence of WT CaM. G and H, intrinsic fluorescence spectra for Nav1.5 at ex = 280 nm or at ex = 295, respectively, for selective excitation of tryptophan show no Ca2+-induced increase. The line colors represent the same conditions as described for A–F. I, shown is the increase in intrinsic tyrosine fluorescence (ex = 280 nm and em = 308 nm) for CaM, Nav1.2 CT·CaM, and Nav1.5 CT·CaM at different concentrations of free Ca2+ (n = 3 for each).

These dissimilarities led us to examine more closely the contributions of sequences outside the IQ motifs to CaM interaction with the Na_v1 CTs. Although the ability of CaM to promote solubilization of the Na_v1 CTs (Fig. 1) supported our hypothesis that these proteins, like Ca_v1.2, contain a CaM-binding pocket consisting of multiple noncontiguous sequences, the lack of a conformational change on the gel filtration column suggested that the details of CaM interaction with the Na_v1 CTs likely differed from those of CaM interaction with Ca_v1.2. Our specific approach was to examine whether mutations outside the Na_v1 IQ motifs would disrupt CaM interaction, similar to what we observed for Ca_v1.2. Long QT syndrome subtype 3 (LQTS3) and Brugada syndrome (BrS) mutations in the C terminus were chosen as candidates for Na_v1.5 because these mutations, which place patients at risk for arrhythmogenic sudden cardiac death (30), are within the Na_v1.5 CT that we tested and alter channel gating behavior; if the mutations also alter CaM interaction, this would provide an important correlation between CaM interaction and channel function and would therefore parallel the changes in channel gating resulting from disruption of CaM interaction in Ca_v1.2 (8). We chose for testing a set of three mutations that reside in distinct regions of the predicted structure of the Na_v1.5 CT and for which the electrophysiological effects have been well characterized: the BrS mutation Y1795H (31), in the first of the paired EF hands; the LQTS mutation L1825P (20), in the second paired EF hand; and the BrS mutation A1924T (21), just C-terminal to the predicted helix containing the IQ motif. None of these mutations in the Na_v1.5 CT affected the generation of a complex with CaM in the bacterial expression system (Fig. 3A). Furthermore, the migration of the resultant complexes on the gel filtration column in either Ca²⁺ or EGTA was indistinguishable from that of the WT complex (data not shown). The lack of an effect with A1924T was particularly surprising, as this mutation within a 20-amino acid peptide alters CaM mobility in a gel shift assay (17) (but see "Discussion"). Although these data do not rule out a functional role for CaM, they suggest that these LQTS and BrS mutations do not alter channel gating through disruption of CaM interaction.

View larger version (27K): [in this window] [in a new window]   FIG. 3. Identification of critical regions for CaM interaction in the Nav1.2 and Nav1.5 CTs. A, LQTS and BrS mutations do not disrupt CaM interaction with the Nav1.5 CT. Shown are the extracts (Ex) and high speed supernatants (Sup), demonstrating that, unlike in the absence of CaM coexpression, the Nav1.5 CT remains soluble. B, diagrams of the proximal regions of the Nav1.2 and Cav1.2 C termini, with the indicated H1–H6 helices as described in the legend to Fig. 1 (H6 is labeled as "IQ" to emphasize the location of the IQ motif), and the mutations tested. All proteins were successfully coexpressed with CaM and purified as described in the legend to Fig. 1. The resulting effects of the mutations are summarized to the right. Solubility (Sol.) was scored as + if >75% of the Nav1.2 CT in the extract was recovered in the high speed supernatant. Aggregation state (Agg. State) was scored as + if the complex migrated as a single species on the gel filtration column with an elution volume consistent with the predicted molecular mass of the complex or - if the protein eluted in the void volume. N/A, not applicable. C, mutation of the IQ motif in the Nav1.2 CT disrupts CaM interaction, but makes the Nav1.2 CT soluble. Nav1.2 with the IQ/AA mutation was expressed with or without CaM and then processed as described in the legend to Fig. 1. Shown are the extracts (Ex), high speed supernatants (Sup), flow-through fraction (FT) from the metal affinity column, and purified protein (P). None of the CaM coexpressed with Nav1.2 remained associated, and all of it was found in the flow-through fraction. D, the familial autism-associated R1902C mutation in Nav1.2 makes the Nav1.2 CT·CaM complex Ca2+-sensitive as shown by gel filtration analysis of the purified Nav1.2 CT·CaM complex (shown in the inset, as described in the legend to Fig. 1), demonstrating the Ca2+-dependent shift in mobility.

The lack of an apparent contribution of the sequences outside the IQ motif to CaM binding focused our attention back on the IQ motif. Mutation of IQ to AA in Na_v1.5 and in the highly homologous Na_v1.4 (19) completely disrupts CaM interaction in GST pull-down assays (18), providing a candidate for testing in our coexpression system. Consistent with those results, we found that no CaM co-purified with either the Na_v1.2 or Na_v1.5 CT bearing the IQ/AA mutation (Figs. 3C and 4E). These proteins remained soluble and non-aggregated after purification by metal affinity chromatography, migrating as a single species with an apparent molecular of 22 kDa on the gel filtration column. This was a surprising result since the WT proteins were mostly insoluble in the absence of CaM coexpression and always co-purified stoichiometrically with CaM. We next tested whether coexpression of CaM is necessary to obtain soluble material from the IQ/AA mutants, even though CaM did not co-purify, and found that it was not. Thus, the IQ/AA mutation disrupts CaM interaction and obviates the need for CaM coexpression to obtain soluble material. This result also offers another point of departure in the comparison of CaM interactions between Ca_v1.2 and Na_v1 CTs since the identical mutation in the Ca_v1.2 motif does not disrupt CaM interaction; data with an isolated IQ motif-containing peptide from Ca_v1.2 suggest, in contrast, that the mutation increases CaM affinity (7).

View larger version (72K): [in this window] [in a new window]   FIG. 4. Interaction of Nav1.5 CT with III–IV linker is enhanced by Ca2+ and CaM. Shown are Coomassie Blue-stained gels of GST pull-down assays performed with GST or GST-III–IV linker (III–IV) and Nav1.5 CT·CaM (A and B), Nav1.2 CT·CaM (C), Nav1.5 CT (D), and the IQ/AA mutant Nav1.5 CT (E). Assays were performed in the presence of Ca2+ (1 mM) or EGTA (1 mM), except in B, in which the free [Ca2+] is indicated.

Does CaM also regulate Na_v1.5 function? Although the arrhythmogenic mutations were not revealing in the assays above, localization of the CaM interaction site to the putative H6 in Na_v1.5 raises the possibility that CaM might participate in a recently described function of the C terminus: stabilization of the inactivation gate (33). Certain mutations in the intracellular III–IV linker, which serves as the "inactivation particle" mediating fast inactivation, lead to a very small sustained Na⁺ current that is nevertheless associated with arrhythmia and sudden death (30). Truncation of H6 also causes sustained current on the same order of magnitude as those induced by inherited mutations, leading to the hypothesis and subsequent demonstration that the C terminus and III–IV linker physically interact and possibly contribute to stabilizing Na_v1.5 inactivation (33). We therefore explored whether the constitutive CaM binding with H6 in Na_v1.5 influences interaction with III–IV linker. Using a GST-III–IV linker fusion protein, we performed a pull-down assay with the Na_v1.5 CT·CaM complex. As shown in Fig. 4 (A and B), the Na_v1.5 CT·CaM complex interacted with III–IV linker in the presence of submicromolar Ca²⁺, but only weakly in the presence of EGTA. This interaction is specific since there was no interaction with the GST control, and the highly homologous Na_v1.2 CT·CaM complex did not bind the Na_v1.5 III–IV linker well (Fig. 4C). Interestingly, CaM was not pulled down with the Na_v1.5 CT, suggesting that binding of the C terminus to III–IV linker may displace CaM. We therefore tested whether CaM is necessary for this interaction using the Na_v1.5 CT purified in the absence of CaM. As shown in Fig. 4D, this protein did not bind. Interpretation of the role for CaM in this experiment must be made with caution, however, since the yield of the soluble Na_v1.5 CT was markedly reduced in the absence of CaM coexpression even though the expression level was unaffected (Fig. 1B). The reduction in solubility may imply improper folding of the Na_v1.5 CT in the absence of CaM and therefore compromise the interaction with III–IV linker. We further tested this requirement for CaM using the Na_v1.5 CT with the IQ/AA mutation, from which higher yields of peptide were obtained without CaM coexpression. This mutant protein bound only weakly to III–IV linker (Fig. 4E). These data suggest that, even though CaM does not remain bound when the C terminus binds to III–IV linker, its presence may be necessary for the interaction.

The disruption of both the CaM and III–IV linker interactions with the Na_v1.5 CT by the IQ/AA mutation provided an opportunity for an initial test of a possible role for these interactions in stabilizing the inactivation gate. In previous experiments, disruption of the C terminus/III–IV linker interactions has been shown to correlate with subtle changes in Na⁺ channel activity that promote a small increase in sustained current during prolonged depolarization (33). Although only a fraction of the peak whole cell current, these subtle changes in gating are associated with fatal arrhythmias (28) and epilepsy (34). We thus designed experiments to test for functional effects of the IQ/AA mutation on Na⁺ channel gating. In low gain recordings, both WT and IQ/AA mutant channels expressed in human embryonic kidney cells showed a large inward current that rapidly inactivated with little mutation-altered gating differences (Fig. 5A), similar to results obtained by Deschenes et al. (18). However, comparison of the current traces from the IQ/AA mutant and WT channels at high gain revealed a mutation-induced increase in sustained current (Fig. 5A, right panel, inset, arrow). Analysis of population data (Fig. 5B) indicated that this change in sustained current (measured as percent peak current) was significant and, although small (0.07 ± 0.03% for WT channels and 0.48 ± 0.05% for IQ/AA mutant channels; 10 nM Ca²⁺_i), was on the same order of magnitude reported for other mutations that disrupt C terminus/III–IV linker interactions (33). Modulating free [Ca²⁺]_i within the range of 10 nM to 1 µM did not affect the sustained current in these experiments (Fig. 4B). Thus, disruption of CaM and III–IV linker interactions with the C terminus destabilizes the inactivation gate and results in subtle but significant changes in sustained current measured during prolonged depolarization.

View larger version (14K): [in this window] [in a new window]   FIG. 5. IQ/AA mutation destabilizes the Nav1.5 inactivation. A, shown are the normalized (to peak current) and averaged (n = 11) whole cell recordings of Na+ currents in human embryonic kidney 293 cells expressing WT and IQ/AA mutant channels illustrated at low and high (insets) gain in response to voltage pulses to -10 mV. The IQ/AA mutation promoted sustained channel activity (arrow). B, the bars summarize the sustained current (mean ± S.E.) measured at 150 ms during test pulses to -10 mV and normalized to peak current for WT and IQ/AA mutant channels recorded under conditions in which free [Ca2+]i = 10 nM (white bars) and 1 µM (gray bars). The number of experiments was as follows: for WT channels, n = 11 at 10 nM Ca2+i and n = 3 at 1 µM Ca2+i; and for IQ/AA mutant channels, n = 19 at 10 nM Ca2+i and n = 5 at 1 µM Ca2+i. Under each recording condition, the means were significantly different. *, p < 0.01; #, p < 0.02. Scale bars = 5 ms (low gain) and 50 ms (high gain) (0.05%).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The primary motivation behind our investigation was our assumption that a better understanding of the biochemistry of CaM interaction with Na⁺ channels could guide future studies to resolve the conflicting results of the published work on CaM regulation of Na⁺ channel function (17–19). We therefore took a more systematic approach to define the CaM interaction domain(s) in the Na_v1 CTs using the homology between Ca_v1.2 and Na_v1 and our development of a bacterial coexpression system (8) as a template.

Consistent with this homology, our results demonstrate significant similarities in CaM interaction. In both cases, generation of a fusion protein of the soluble C terminus was compromised without coexpression of CaM, suggesting that CaM is an obligate subunit necessary for proper folding of the C terminus. More evidence that CaM is required for proper folding of the Na_v1 CTs came from pull-down assays showing a substoichiometric interaction of CaM with the Na_v1 CTs if CaM was not coexpressed (data not shown). Another similarity between the Ca_v1.2 C terminus and the Na_v1 CTs is that the apoCaM and Ca²⁺/CaM interaction sites appear to be inseparable, although the basis for this conclusion is different between the Ca_v1.2 and Na_v1 proteins. For Ca_v1.2, mutations throughout the CaM-binding pocket that affected apoCaM interaction also affected Ca²⁺/CaM function. For Na_v1.2, the lack of a detectable Ca²⁺-dependent conformational change as assayed by gel filtration chromatography with all the WT constructs tested, even with the shortest construct assayed (amino acids 1868–1937), suggests that CaM binds the same determinants when Ca²⁺-free or Ca²⁺-loaded. Furthermore, the complete loss of CaM binding to the IQ/AA mutant suggests that the IQ motif contributes an essential determinant, consistent with the finding of others (18, 19). Although the specific amino acids in Na_v1 that contribute to these determinants await identification, the contribution of the consensus Ile and Gln residues in the IQ motif are clear.

Less apparent are the contributions of sequences outside the IQ motif to CaM interaction with the Na_v1 CTs, representing a distinct difference in how CaM binds Na_v1.2 and Na_v1.5 compared with Ca_v1.2. Amino acids conserved between the Na_v1 proteins and Ca_v1.2 do not appear to serve the same roles in CaM interaction. Mutations homologous to those that disrupt CaM interaction with Ca_v1.2 were without effect in the Na_v1 CTs. Furthermore, the CaM interaction domain in the Ca_v1.2 channel extends far beyond the apparently limited domain required in the Na_v1 CTs, as indicated by the retention of CaM interaction after deletion of the N-terminal region of the Na_v1.2 CT constructs (Fig. 3B). Sequences distal to the IQ motif in Na_v1.5 may participate in CaM binding since a oligopeptide bearing a A1924T mutation migrates differently from a WT peptide on a nondenaturing nonreducing gel (17). With the longer Na_v1.5 CT used in our assays, however, we could not detect a difference in CaM interaction between the WT and A1924T mutant peptides (Fig. 2A). Although this may represent a detection issue in our assays (see below), it is also likely that the smaller peptides behave differently from the intact CaM-binding domain, as shown with Ca_v1.2 (8), and therefore do not accurately reflect CaM interaction with the intact channel.

The contribution of sequences outside the IQ motif in Ca_v1.2 to Ca²⁺/CaM regulation of channel function, in addition to CaM binding, represents another significant difference between Na_v1 and Ca_v1.2 channels. In Ca_v1.2, these other domains bring the Ca_v1.2 CaM-binding pocket into direct contact with or within close proximity to other C-terminal domains, such as the Ca_v1.2 EF hand motif. These additional interactions appear to be critical for transduction of the Ca²⁺ signal to the inactivation machinery in Ca_v1.2 (8, 35). This does not appear to be the case in Na_v1 channels. Although the paired EF hand motif in Na_v1.5 also plays a critical role in the regulation of open state inactivation, as demonstrated by the Tyr¹⁷⁹⁵ mutations in LQTS and BrS (36), the Na⁺ channel may have coopted this signal transducer for a purpose other than transducing a signal from CaM.

The lack of apparent functional coupling between the CaM-binding determinants and the paired EF hand motifs in the Na_v1 proteins may reflect the markedly reduced conformational change seen with the Na_v1·CaM complexes compared with the Ca_v1.2·CaM complex. CaM mediates its effects by inducing a conformational change in its target proteins (6); indeed, the conformational change detected when Ca_v1.2·CaM binds Ca²⁺ correlates well with the Ca²⁺-dependent gating mediated by CaM (8). Thus, the smaller conformational change in CaM in complex with the Na_v1.2 or Na_v1.5 CT implies that CaM may mediate a different form of Ca²⁺-dependent signaling.

A possible role for CaM in Na⁺ channel function is suggested by our finding that CaM may mediate the interaction between the Na_v1.5 CT and III–IV linker. This extends the original observation that these two domains of the cardiac Na⁺ channel interact to stabilize the inactivation gate (33) to include CaM as an essential component. Disruption of CaM binding to Na_v1.5 by the IQ/AA mutation also markedly reduced the interaction between the Na_v1.5 CT and III–IV linker. The functional effects of the IQ/AA mutation on Na⁺ channel gating are subtle, resulting in only a very small sustained Na⁺ channel current, in contrast to the marked effects of CaM in the control of Ca_v1.2 channel inactivation. Nevertheless, these small changes in sustained current are on the same order of magnitude of previously reported LQTS3 mutations (37) and suggest that similar mutations could underlie some forms of LQTS.

One interesting detail that emerges from our experiments is the requirement for Ca²⁺ in this interaction (Fig. 4, A and B). Whether Ca²⁺ plays a dynamic signaling role is unclear since the in vitro interaction between the Na_v1.5 CT and III–IV linker was maintained and apparently unaffected over the physiological range of [Ca²⁺]_i. The Ca²⁺ dependence cannot be attributed to the ostensible requirement for CaM interaction with the Na_v1.5 CT to promote efficient interaction between the Na_v1.5 CT and III–IV linker since our data suggest that CaM binds the C terminus in Ca²⁺-independent manner. Moreover, the role for Ca²⁺ in this interaction must be interpreted with caution for two reasons. First, although the absence of Ca²⁺ markedly impaired the interaction between the Na_v1.5 CT and III–IV linker in the in vitro assay, the requirements in the context of the entire channel and the other determinants that may contribute to the inactivation process may be different. For example, it is possible that Ca²⁺ substitutes in the in vitro assay for a voltage-dependent process available only to the intact channel. Nevertheless, the Ca²⁺-dependent conformational changes detected in the intrinsic Tyr fluorescence assays and the Ca²⁺-dependent shift in mobility of the Na_v1.2 CT·CaM complex bearing the epileptogenic R1902C mutation might hint at subtle yet important changes that underlie the small but physiologically significant differences that promote stabilization of inactivation rather than sustained current that leads to LQTS or epilepsy. Second, ascribing any result from a biochemical assay to the etiology of a sustained current that measures only 0.5% of the total peak current is challenging; changes that lead to the small yet clinical significant functional difference might be beyond the sensitivity of these biochemical assays. Nevertheless, the correlation between functional impairment and decreased interaction between the Na_v1.5 CT and III–IV linker (this study and Ref. 33) and the Ca²⁺ sensitivity promoted by the epileptogenic R1902C mutation (Fig. 3) are intriguing.

The displacement of CaM from the Na_v1.5 CT by III–IV linker is another interesting aspect of this interaction and offers potentially important functional insights. First, this observation suggests that CaM competes with III–IV linker for binding to the C terminus, raising the possibility that the binding sites overlap. Alternatively, III–IV linker could promote CaM dissociation as an allosteric modifier. Second, since this displacement occurs more strongly in the presence of Ca²⁺, the Na_v1.5 CT falls into a special category of CaM-binding proteins that have a lower affinity for Ca²⁺/CaM than for apoCaM (38). The prototypical member of this class, neuromodulin, is a neuronal membrane-associated protein that sequesters apoCaM through binding to an IQ motif and releases Ca²⁺/CaM when [Ca²⁺]_i rises. The properties of the IQ motif in the Na_v1.5 CT appear to be similar to those of IQ motifs found in other members of this class (such as IQGAP) that can bind apoCaM and Ca²⁺/CaM.

These data therefore point to a role for CaM in Ca²⁺ modulation of Na⁺ channel function and contrast with the model proposed by Wingo et al. (23), in which Ca²⁺ shifts availability through binding to a paired EF hand motif within the Na_v1.5 CT. Further, our data are inconsistent with that alternative proposal, as we failed to detect Ca²⁺-induced changes in intrinsic Trp fluorescence. Moreover, in comparison with the data obtained by Wingo et al., we observed an 15-nm blue shift in _max, consistent with decreased solvent accessibility of the single tryptophan (39). The larger Stokes shift observed by Wingo et al. may therefore reflect partial unfolding of their preparation. Furthermore, the methionine (Met¹⁷⁹³) immediately after the Z-position in the EF hand model presented by Wingo et al. is highly unusual, suggesting that Ca²⁺ coordination by this motif is unlikely. In a comprehensive data base of EF hand sequences (structbio.vanderbilt.edu/cabp_database/index.html), methionine is not found in that position.

The model presented for CaM function in Na_v1.5 regulation may not apply to Na_v1.2 since isoform-specific differences in CaM function have been described (18, 19). It is difficult to confirm these differences, however, since each of the three published studies examining CaM regulation of Na⁺ channel function (17–19) present some results that conflict with at least one of the other studies using the same channel isoform. Thus, studies such as the one presented here offer new approaches to define prospectively the functional parameters for testing. In this regard, it is intriguing that the R1902C mutation in Na_v1.2, which is linked to autism (22), was the only mutant that caused a Ca²⁺-dependent shift in mobility of the Na_v1·CaM complex upon gel filtration chromatography. The possibility that this mutation endows a larger conformational change, similar to that observed for CaM in complex with Ca_v1.2, and confers on Na_v1.2 a new sensitivity to Ca²⁺ signaling may provide hints about the pathogenesis of this mutation and the molecular defects in at least one form of autism.

	FOOTNOTES

 
^* This work was supported by grants from the National Institutes of Health (HL-71165 to G. S. P. and HL-56810 and HL-67849 to R. S. K.) and the Irma T. Hirschl Trust (to G. S. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Both authors contributed equally to this work.

^|| To whom correspondence should be addressed: Dept. of Pharmacology, Columbia University College of Physicians and Surgeons, 630 W. 168th St., PH 7W 318, New York, NY 10032. Tel.: 212-305-1009; Fax: 212-305-8780; E-mail: gp2004{at}columbia.edu.

¹ The abbreviations used are: SK, small conductance K⁺; CaM, calmodulin; FPLC, fast protein liquid chromatography; MOPS, 4-morpholinepropanesulfonic acid; GST, glutathione S-transferase; CT, C terminus; CTs, C termini; WT, wild-type; LQTS3, long QT syndrome subtype 3; BrS, Brugada syndrome; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl.

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