Calmodulin Binds to the C Terminus of Sodium Channels Na_v1.4 and Na_v1.6 and Differentially Modulates Their Functional Properties

Abstract

Modulation of voltage-gated sodium channels (VGSC) can have a major impact on cell excitability. Analysis of calmodulin (CaM) binding to GST-fusion proteins containing the C-terminal domains of Na_v1.1-Na_v1.9 indicates that some of the tetrodotoxin-sensitive VGSC isoforms, including Na_V1.4 and Na_V1.6, are able to bind CaM in a calcium-independent manner. Here we demonstrate that association with CaM is important for functional expression of Na_V1.4 and Na_V1.6 VGSCs. Disrupting the interaction between CaM and the C terminus of Na_V1.4 and Na_V1.6 channels reduced current amplitude by 99 and 62%, respectively. Overexpression of CaM increased the current generated by Na_v1.4 and Na_v1.6 C-terminal mutant constructs that exhibited intermediate current densities and intermediate binding affinities for CaM, demonstrating that this effect on current density was directly dependent on the ability of the C terminus to bind CaM. In addition to the effects on current density, calmodulin also was able to modulate the inactivation kinetics of Na_v1.6, but not Na_v1.4, currents in a calcium-dependent manner. Our data demonstrate that CaM can regulate the properties of VGSCs via calcium-dependent and calcium-independent mechanisms and suggest that modulation of neuronal sodium channels may play a role in calcium-dependent neuronal plasticity.

Introduction

Voltage-gated sodium channels (VGSCs) are the main carriers of current underlying the fast upstroke of action potentials in excitable cells. Modulation of VGSCs can regulate the function of the brain (Catterall, 2000) and muscle (Weiss and Horn, 1986). The activity of many channel proteins is regulated by calmodulin (CaM) (Levitan, 1999; Saimi and Kung, 2002), a 16.7 kDa protein that is expressed in virtually all eukaryotic cells. CaM can induce changes in target proteins via its binding per se and in response to changes in calcium concentration (Chin and Means, 2000). For example, the activity of sodium channels in Paramecium tetraurelia is CaM-dependent (Ling et al., 1992; Saimi and Ling, 1995), and CaM plays a crucial role in the modulation of calcium channels (Lee et al., 1999; Peterson et al., 1999; Zuhlke et al., 1999).

Several lines of evidence suggest that VGSCs might be modulated by CaM in muscle and neurons. A large number of CaM binding proteins have an isoleucine-glutamine (IQ) CaM binding motif (Bahler and Rhoads, 2002). Rhoads and Friedberg (1997) identified IQ motifs in the C termini of Na_V1.1-1.3 and predicted that these VGSCs might bind CaM. Indeed, experiments that used a GST-fusion protein consisting of a 27-amino-acid fragment of the C terminus of Na_V1.2 showed that CaM binds directly to the Na_v1.2 IQ motif in a calcium-independent manner (Mori et al., 2000). Two recent studies suggest that CaM can affect the activity of Na_V1.4 (Deschenes et al., 2002) and Na_V1.5 (Tan et al., 2002) muscle VGSCs by binding to their IQ motifs.

IQ motifs have been identified in proteins that bind CaM permanently or transiently. Some proteins with IQ motifs bind CaM independently of calcium, whereas others selectively bind calcium/CaM or calcium-free CaM (Jurado et al., 1999; Bahler and Rhoads, 2002). The core of the IQ motif is often IQXXXRGXXXR, although the first residue is variable and can be ambiguous for isoleucine, leucine, or valine. The last residue reportedly can be either arginine or lysine, and the seventh residue is not well conserved. Thus a more generalized IQ motif might be [I,L,V]QXXXRXXXX[R,K] (Bahler and Rhoads, 2002). After aligning the C termini sequences of all known VGSC isoforms (see Fig. 1A), we observed that the key residues in the region of the C terminus sharing homology with a 23-residue-long IQ motif (Schultz et al., 2000) were conserved to varying degrees.

Figure 1.

CaM binds to VGSC both in vitro and in vivo. A, Alignment of IQ motifs within VGSC C termini. Identical and highly conserved residues are shaded in black and gray, respectively. Shown is a schematic of a GST-fusion protein with the 250-amino-acid-long C terminus of a sodium channel, indicating the position of the highly conserved IQ motif (Schultz et al., 2000). B, GST pull-down assay shows that CaM binds to the C termini of VGSC with different affinity. Fusion proteins of the individual isoforms, as indicated above each lane, were tested for their in vitro capability to pull down CaM in the presence (5 mm CaCl₂; top row) and absence (2 mm EDTA; bottom row) of Ca²⁺. Immunoblotting was performed with a monoclonal anti-CaM antibody. C, CaM associates with full-length Na_V1.4 in transfected HEK293 cells in vivo. Cell extracts from cells stably expressing rat Na_V1.4 that had been transfected either with EGFP cDNA (lanes 1, 2) or with CaM-EGFP cDNA (lanes 3, 4) were immunoblotted with an anti-CaM antibody. The immunoprecipitated proteins were visualized with a panspecific anti-VGSC antibody and show that CaM is required for a pull down of the Na_V1.4 channel.

In this study we asked whether CaM binding to Na_V1.6, a neuronal VGSC (Burgess et al., 1995; Schaller et al., 1995; Smith et al., 1998), had the same functional consequences as CaM binding to the skeletal muscle VGSC, Na_V1.4 (Trimmer et al., 1989). Our novel data show that CaM regulates the current density of both Na_V1.4 and Na_V1.6. In addition, we show that CaM regulates the kinetic properties of Na_V1.6 currents in a calcium-dependent manner. This work presents functional evidence for participation of VGSCs in the intricate CaM signaling network that is present within neurons (Marrion and Tavalin, 1998; Dolmetsch et al., 2001) and muscles (Hamilton et al., 2000).

Materials and Methods

Molecular biology. cDNAs corresponding to the C termini of all known voltage-gated sodium channels (Goldin et al., 2000) were generated by PCR. Rat brain or dorsal root ganglion (DRG) cDNA served as a template for Na_V1.1, 1.2, 1.3, 1.4, 1.5, 1.8, and 1.9. Na_V1.6 was amplified from mouse cDNA (Smith et al., 1998) and Na_V1.7 from a human cDNA construct (Klugbauer et al., 1995). PCR products were subcloned into EcoRI/BamHI sites of the pGEX3X vector (Amersham Biosciences, Piscataway, NJ) to generate glutathione S-transferase (GST) fusion constructs.

All mutations were inserted into the respective sodium channel cDNA constructs by using the Quick Change XL (Stratagene, La Jolla, CA) mutagenesis kit, following the instructions of the manufacturer. Mutagenic primers were designed to introduce the appropriate base pair changes into the rat Na_V1.4 and the mouse Na_V1.6 sequences. Initially, both channels were made tetrodotoxin (TTX)-resistant by changing a pore tyrosine residue to a serine (Y401S in Na_V1.4 and Y371S in Na_V1.6) (Cummins et al., 2001) to allow currents to be isolated after expression in neurons and human embryonic kidney 293 (HEK293) cells. These two TTX-resistant channel constructs served as templates for all further mutagenesis. The plasmid Na_v1.4-RBG4 (Ukomadu et al., 1992) was used for the Na_v1.4 constructs. C-terminal truncation mutants of Na_V1.4 were made by introducing a stop codon at amino acid position 1741 (DEL1) after the IQ motif and at position 1719 (DEL2) before the IQ motif. Primers were designed to change Na_V1.4 amino acid residues ¹⁷²⁷IQ ¹⁷²⁸ to AQ, EQ, IA, IE, AA, and EE.

Construction of a mammalian expression vector encoding neuronal rat Na_v1.6 channel. These studies used a cDNA construct that encodes the mouse Na_V1.6 open reading frame. The complete open reading frame of mouse Na_V1.6, generously provided by Dr. A. Goldin (Smith et al., 1998), was excised from a pLCT1 oocyte expression vector first by digestion with AatII and then by blunting the ends with T4 DNA polymerase, followed by digestion with XhoI to release the Na_v1.6 insert. A mammalian expression vector pcDNA3.1 (Invitrogen, San Diego, CA), modified to render it a low-copy vector (Klugbauer et al., 1995), was digested with ApaI; the ends were blunted with T4 DNA polymerase and then digested with XhoI. In a subsequent ligation reaction the two DNA pieces were joined to form a 13 kb plasmid. Isolates that displayed the expected BamHI digestion pattern were verified by sequencing the entire Na_V1.6 insert. A TTX-resistant phenotype of Na_V1.6 (Na_V1.6r) was produced by converting tyrosine 371 to serine (Y371S) as previously described for the Na_v1.3 channel (Cummins et al., 2001). This point mutation was introduced into the channel sequence with the Quick Change XL mutagenesis kit (Stratagene), with two mutagenic primers designed according to the manufacturer's instructions. The mutant construct subsequently was verified by sequencing the whole Na_v1.6 insert. A similar approach was used to mutate the Na_V1.6 residues ¹⁹⁰⁰LQ ¹⁹⁰¹ to EQ, LE, and EE.

A CaM-EGFP (enhanced green fluorescent protein) fusion construct was generated by PCR. Forward and reverse primers containing an EcoRI and BamHI restriction site, respectively, were used to amplify a wild-type CaM DNA fragment from a rat brain cDNA library, which then was subcloned into a previously EcoRI/BamHI-digested pEGFP-N1 vector (Clontech, Palo Alto, CA). A dominant-negative calcium-insensitive CaM mutant was generated by mutating the first aspartates of each of the four calcium-binding EF hand motifs to alanines (Putkey et al., 1989; Peterson et al., 1999). All constructs were verified by sequencing.

Expression and purification of GST-fusion proteins. For expression of GST-fusion proteins the appropriate plasmids (pGEX-C terminus-Na_V 1.1-Na_V1.9) were transformed into Escherichia coli DH5α (Stratagene). Fusion proteins were affinity purified on glutathione-Sepharose beads, as previously described (Liu et al., 1999).

GST pull-down assay. To examine the binding of CaM to the C termini of all of the sodium channels as well as the mutants of the Na_V1.4 C terminus, in vitro, we incubated equal amounts of glutathione-Sepharose beads loaded with purified GST fusion proteins with 2 μg of bovine CaM in binding buffer AM-100 (Voit et al., 1997) at 4°C overnight. After being washed with binding buffer AM-100 four times, the bound proteins were released by boiling for 5 min in SDS-loading buffer and separated on a 12% SDS-PAGE. Subsequent immunoblot visualization was performed with monoclonal anti-CaM antibodies (Zymed, San Francisco, CA).

Coimmunoprecipitation. A HEK293 cell line that stably expressed rat Na_v1.4 was grown in 100 mm dishes to 80% confluence. Sister cultures then were transfected with 30 μg of CaM-EGFP cDNA or EGFP cDNA, using 30 μl of Lipofectamine 2000 (Invitrogen) transfection agent. After 24 hr the cells in each dish were lysed in 500 μl of cell lysis buffer. Fractions of the supernatants were saved for loading on the gel, and the rest of the cell extracts was incubated with 5 μl of polyclonal anti-GFP antibody (Clontech). Protein A-agarose (Invitrogen) then was used to precipitate the protein complexes at 4°C overnight. After extensive washes the proteins were eluted in SDS loading buffer, and CaM was visualized by immunoblotting with a panspecific anti-sodium channel antibody (Upstate Biotechnology, Lake Placid, NY) used at a 1:1000 dilution.

Electrophysiology. Standard whole-cell patch-clamp techniques were used as previously described (Cummins et al., 1998). All cells were held at -120 mV for 5 min before the measurement of peak sodium current amplitude. The currents of the TTX-resistant constructs were recorded in the presence of 250 nm TTX to minimize contamination from endogenous HEK293 sodium currents. The pipette solution contained (in mm): 140 CsF, 1 EGTA, 10 NaCl, and 10 HEPES, pH 7.3. The bathing solution contained (in mm): 140 NaCl, 3 KCl, 1 MgCl₂, 1 CaCl₂, and 10 HEPES, pH 7.3. HEK293 cells were transfected by using the calcium-phosphate precipitation technique as previously described (Cummins et al., 1998).

Culture of DRG neurons. DRG neurons were cultured as described previously (Caffrey et al., 1992). Briefly, the L4 and L5 DRG ganglia were harvested from adult Na_V1.8 null mice (Akopian et al., 1999). The DRG were treated with collagenase A (1 mg/ml) for 25 min and then collagenase D (1 mg/ml) and papain (30 U/ml) for 25 min, dissociated in DMEM and Ham's F-12 medium supplemented with 10% fetal bovine serum, and plated on glass coverslips. Na_v1.8 null neurons were kept under standard tissue culture conditions for 5-7 d before biolistic transfections. We previously showed that Na_v1.8 null DRG neurons do not express fast- or slow-inactivating TTX-resistant sodium currents (Cummins et al., 1999, 2001). Some Na_v1.8 null DRG neurons do express persistent TTX-resistant sodium currents (Cummins et al., 1999), but these currents are typically <1 nA after several days in culture and run down quickly in whole-cell recording configuration; therefore, these persistent TTX-resistant currents are not significant under the culture and recording conditions used in the present study.

Biolistic transfection of Na_v1.8 null DRG neurons. The Helios Gene Gun System (Bio-Rad Laboratories, Hercules, CA) was used for biolistic transfection of neurons. Na_v1.6r or Na_v1.4r DNA (10 μg) was mixed with 5 μg of GFP DNA in 50 μm spermidine and coprecipitated onto 1.6 μm gold particles with CaCl₂. The DNA gold suspension was washed twice in 100% ethanol and resuspended in 0.05% polyvinylpyrrolidone in ethanol; it was used for coating the inner wall of 10 inches of Tefzel tubing (Bio-Rad Laboratories). The tubing was dried by using ultrapure nitrogen and was cut into ∼20 cartridges for the Helios Gene Gun. This process resulted in a density of 1 mg of gold particles per shot and 0.75 μg of total DNA per cartridge.

Immediately before biolistic transfection, the culture medium was removed from the Petri dish. The gene gun was held ∼2 cm above the cells, and a pressure of ∼120 psi was used to discharge the gold particles. A 70 μm nylon mesh (Small PartsMiami Lakes, FL) was placed just in front of the Helios Gene Gun barrel liner to achieve a more uniform distribution of gold particles (Wellmann et al., 1999). Within 24 hr the cells usually showed expression of GFP, indicating a successful biolistic transfection. Electrophysiological studies were conducted 18-48 hr after transfection, and most of the cells that expressed GFP also expressed fast-inactivating TTX-resistant sodium currents. Because these currents are not observed in untransfected Na_v1.8 null neurons or Na_v1.8 null neurons transfected with GFP alone, this confirmed that most of the cells that expressed GFP also had been cotransfected successfully with the Na_v1.6 TTX-resistant channel.

Statistical analysis. One-way ANOVAs were used to test for differences between the experimental groups. The difference between pairs of individual data sets was tested by Fisher's least significant difference (LSD) multiple comparison test, and p values < 0.05 were considered to be significant. Where appropriate, data were compared with Student's t test. Data are expressed as the mean ± SEM.

Results

Several members of the VGSC family are able to bind CaM directly

Many proteins, including voltage- and ligand-gated channels, are regulated by CaM (Levitan, 1999; Zhu et al., 2001). Some proteins bind to CaM in a constitutive manner, whereas others are able to do so if calcium ions are present in a sufficiently high concentration. To determine to which category the VGSCs belong, we examined CaM binding in the presence and the absence of Ca²⁺ions. In analogy to the experiments conducted with calcium channels (Peterson et al., 1999), we generated cDNAs of GST-fusion proteins of the complete C terminus of all of the known members of the VGSC family (Goldin et al., 2000), Na_V1.1 through Na_V1.9. We used the whole C terminus rather than shorter peptide fragments because studies have shown that residues outside the immediate CaM binding region can have significant effects on binding affinity (Peersen et al., 1997). After expression and purification we incubated the GST-fusion proteins overnight with 2 μg of bovine CaM in the presence and absence of calcium ions. Because binding occurred under these in vitro conditions, we could exclude the possibility that any other factors might have mediated the observed interaction. Immunoblotting with an anti-CaM antibody revealed isoform-specific differences in CaM binding (Fig. 1B). Under both high and low calcium conditions the fusion proteins of Na_V1.2, Na_V1.4, and Na_V1.6 exhibited very strong binding to calmodulin, suggesting that, like Na_v1.2 (Mori et al., 2000), Na_V1.4 and Na_V1.6 also might be considered as high-affinity targets for CaM. The C termini of Na_V1.1 and Na_V1.3 showed weaker binding but also were able to bind CaM in the presence and absence of Ca²⁺ ions. The C terminus of Na_v1.7 exhibited weak binding to CaM only in the low calcium condition. The three TTX-resistant sodium channels (Na_V1.5, Na_V1.8, and Na_V1.9) did not show detectable binding of CaM to their C termini in our assay. This assay indicates that, whereas the TTX-sensitive VGSC isoforms (with the noted exception of Na_v1.7) bind CaM to their C termini in the presence and absence of Ca²⁺ ions, the TTX-resistant isoforms probably exhibit a much lower affinity for CaM.

CaM binds to Na_V1.4 in vivo

CaM binding to VGSCs has not been shown to occur in mammalian cells yet, raising the possibility that CaM may not bind to full-length channels. To address this issue, we performed a coimmunoprecipitation with the skeletal muscle sodium channel Na_V1.4, which showed high-affinity binding to CaM in the GST pull-down assay and has been well characterized (Ukomadu et al., 1992). We transfected a HEK293 cell line that stably expressed the rat Na_V1.4 channel with a plasmid encoding a CaM-EGFP fusion protein. It has been shown previously that EGFP does not interfere with normal CaM-target interactions (Erickson et al., 2001). A pull-down assay for the sodium channel protein complex, using a polyclonal anti-GFP antibody and a panspecific anti-sodium channel antibody for visualization, showed that CaM-EGFP was part of the sodium channel complex (Fig. 1C). This demonstrates that CaM truly can bind to the full-length Na_V1.4 protein in an in vivo environment. Electrophysiological examination of the currents produced by Na_V1.4 in this experiment revealed no change of any of its properties in comparison to the sister cultures expressing Na_V1.4 alone (data not shown). However, this cannot be interpreted as evidence that CaM does not alter the functional properties of Na_v1.4 channels. The data in Figure 1B suggest that Na_v1.4, like Na_v1.2, binds CaM with a high affinity. Because CaM is thought to be expressed in all eukaryotic cells, it is possible that the endogenous levels of CaM in HEK293 cells are sufficient to saturate Na_v1.4 channels; therefore, the expression of additional CaM has no further effect.

CaM directly modulates Na_V1.4 function

To investigate further the functional implications of the CaM-VGSC complex, we constructed two different C-terminal deletion mutants (Fig. 2A) and compared their sodium currents with those produced by the full-length Na_V1.4. In construct DEL1 we deleted the amino acids from the end of the C-terminal up to, but not including, the IQ motif. In construct DEL2 we deleted an additional 23 amino acids in the C terminus that comprise the IQ motif. Channel constructs were transiently expressed in HEK293 cells to determine whether the C-terminal region containing the IQ motif was functionally relevant. All of our electrophysiological measurements were conducted by using Na_V1.4 channels mutated to be TTX-resistant by changing the tyrosine at position 401 to serine (Y401S) and by recording in the presence of TTX to help differentiate our recombinant channel currents from the endogenous TTX-sensitive currents that are often present at low, but detectable, levels in HEK293 cells (Cummins et al., 1993). In this and subsequent experiments the term “wild-type Na_v1.4” will be used to refer to the TTX-resistant variant Na_v1.4-Y401S.

Figure 2.

The IQ motif is required for VGSC currents. A, Schematic of C-terminal deletion constructs: wild-type rat Na_V1.4 (WT), C-terminal deletion mutant (DEL1) in which a stop codon has been inserted immediately after the IQ motif, and second deletion mutant (DEL2) in which the 23-residue-long IQ motif was removed also. B, The corresponding current recordings from HEK293 cells transfected with these three constructs are shown. Whereas the peak current amplitude of DEL1 (5.7 ± 0.9 nA; n = 13) is similar to that of wild-type Na_V 1.4 (4.9 ± 0.6 nA; n = 45), DEL2 exhibits little or no current (0.32 ± 0.04 nA; n = 18). C, Protein expression levels of deletion constructs. A Western blot of wild-type Na_V1.4, DEL1, and DEL2 in transiently transfected HEK293 cells with anti-VGSC panspecific antibody shows equal amounts of protein in each lane.

The currents produced by construct DEL1 (Fig. 2B) had activation and inactivation kinetics and peak amplitudes (n = 15) that were identical to those of the wild-type Na_v1.4 channel (n = 44), indicating that the distal portion of the C terminus is not vital for normal channel function in HEK293 cells. In contrast, the DEL2 construct produced little or no sodium current (n = 18) when tested with the same voltage-clamp protocol as the wild-type channel and construct DEL1, showing that the region containing the IQ motif is critical for the generation of inward current by Na_V1.4 (Fig. 2B). To determine whether the DEL2 construct produced protein levels comparable to wild-type Na_V1.4 and DEL1, we performed a Western blot with protein extracted from cells that were transfected, with the same amounts of DNA for all three constructs (Fig. 2C). Similar amounts of protein were produced by all three constructs, demonstrating that, although the IQ motif is necessary for Na_V1.4 current, it is not required for protein expression.

Prompted by this drastic effect of the DEL2 deletion on Na_V1.4 current density, we wanted to refine further our analysis of the importance of the IQ motif. Mutations that change the hydrophobicity and charge of the core residues isoleucine and glutamine of the CaM binding motif have been used previously to study the interaction of CaM with its targets (Peterson et al., 1999; Zuhlke et al., 2000; Deschenes et al., 2002). We designed Na_v1.4 constructs in which the corresponding amino acids were mutated to either alanines or glutamic acids and tested their electrophysiological properties. The isoleucine and the glutamine of the IQ motif (Fig. 1A) first were changed individually to either an alanine or a glutamic acid; then both residues were mutated together (Fig. 3A). Changing the isoleucine at position 1727 to the similar alanine (referred to as AQ) induced no significant changes in current properties when compared with the wild-type (IQ) channel. However, introducing a charged glutamic acid (EQ) at position 1727 reduced the peak sodium current by >90% (Fig. 3D). The remaining current is similar in amplitude to the endogenous TTX-resistant sodium current often observed in untransfected HEK293 cells (Cummins et al., 1999). In contrast, altering the second residue, glutamine 1728, to either alanine (IA) or glutamic acid (IE) reduced sodium currents by ∼50% (Fig. 3B). Interestingly, neither the voltage dependence of activation and steady-state inactivation nor any of the time constants that were measured were different for the IE or for any other mutant that we compared with wild-type Na_V1.4 (IQ) (Fig. 3C). The double mutations, I1727A/Q1728A (AA) and I1727E/Q1728E (EE), reduced sodium currents to background levels (Fig. 3B,D), similar to the current reduction observed with construct DEL2 (Fig. 2B). This shows that the current reduction is not attributable to the addition of negative charges into the C terminus alone and suggests that it is attributable to disruption of the IQ motif.

Figure 3.

CaM binding is required for VGSC function. A, The portion of the C-terminal containing the CaM binding IQ motif is shown for wild-type Na_v1.4 (IQ) and the alanine and glutamic acid substitution mutants (boldface letters). B, Representative families of voltage-clamp recordings from HEK293 cells expressing wild-type Na_V1.4 (IQ), IE, and EE constructs are shown in the left column. The IE mutation caused a 60% reduction in peak current amplitude (2.0 ± 0.3 nA; n = 20) as compared with wild-type channels (4.9 ± 0.6 nA; n = 45), and the EE mutation nearly eliminated currents (0.39 ± 0.09 nA; n = 15). Increasing the available intracellular CaM by coexpressing the channel constructs with CaM-EGFP (right column) increased the peak current amplitude of the IE construct (3.8 ± 0.4 nA; n = 26) but had no effect on IQ amplitude (3.8 ± 0.5 nA; n = 18) or on the amplitude of the EE double mutant (0.33 ± 0.07 nA; n = 10). Cells were held at -120 mV, and currents were elicited by 40 msec test pulses to potentials ranging from -80 to -40 mV. C, Mutation of IQ does not induce changes in voltage dependence of activation or steady-state inactivation. Comparison of a mutant that produced intermediate current amplitudes (IE; open symbols) with wild-type Na_V1.4 (filled symbols) revealed an identical voltage dependence of activation and inactivation. D, Shown are peak current amplitudes of Na_V1.4 constructs in which the core residues of the CaM binding IQ motif were replaced. The number of cells recorded from for each group is indicated above the bars. ANOVA plus pairwise comparisons (Fisher's LSD multiple comparison test) revealed that the AA, EQ, and EE current amplitudes were significantly lower than wild-type (IQ) current amplitudes, but not different from the amplitude of endogenous TTX-resistant currents in untransfected HEK293 cells (0.18 ± 0.03 nA). The AQ current amplitudes were not significantly different from wild-type (IQ) current amplitudes. Two mutants, IA and IE, produced intermediate current amplitudes that were significantly different (Fisher's LSD multiple comparison test, p > 0.05) from both wild-type Na_v1.4 (IQ) and endogenous HEK293 current amplitudes. Overexpression of CaM significantly increased the current in cells expressing intermediate mutants (IA+Cam, and IE+CaM), but not wild-type (IQ+CaM) or EE (EE+CaM) channels. E, Changing I and Q alone affects CaM binding in a GST pull-down assay. The same mutations as above were introduced into the fusion proteins of the C terminus of Na_V1.4. The EQ, EE, and AA mutations drastically reduced CaM binding, but the IE mutation showed an intermediate affinity.

To determine whether the observed amplitude changes were correlated with altered binding of CaM to the channel, we introduced the glutamic acid mutations EQ, IE, and EE into the GST-fusion protein of the Na_V1.4 C terminus and tested their ability to bind CaM (Fig. 3E). Changing IQ to IE reduced binding, and changing IQ to EQ or EE seemed to prevent binding of CaM to the C terminus completely. This correspondence between CaM binding of the different IQ mutants and peak current amplitudes provides evidence for a direct link between CaM binding and the regulation of current amplitude and suggests that the current reduction was not attributable to CaM-independent interactions.

If the hypothesis that the reduced current levels observed with the IE and IA mutant channels were the result of a lower affinity of CaM for the C terminus of these channels is correct, then increasing the intracellular CaM concentration should increase the current produced by the IE and IA mutant Na_v1.4 channels. In contrast, increasing CaM concentration would not be predicted to increase the current produced by the EE mutant, because this double mutation appeared to eliminate CaM binding to the C terminus of Na_v1.4. Therefore, we tested whether over-expression of CaM was able to rescue the loss of current observed with the IQ mutants. We coexpressed the CaM-EGFP construct with wild-type Na_V1.4, the IE and IA mutant constructs that produced intermediate current amplitudes, and the EE construct, which produced little or no current (Fig. 3B). Higher intracellular CaM concentrations had no significant influence on current amplitude (Fig. 3D) or voltage dependence of the wild-type channel (data not shown). Although we did not observe an increase in current with the EE mutant, which eliminated CaM binding in the GST-fusion protein assay, overexpression of CaM boosted the current of the intermediate IE and IA constructs to wild-type current levels (Fig. 3B,D). These data confirm that CaM can regulate Na_V1.4 sodium current density directly by binding to the C terminus.

CaM also modulates Na_V1.6 current amplitude

We next asked whether neuronal VGSC isoforms might be regulated by CaM in the same manner as Na_v1.4. The initial GST pull-down assay indicated that Na_V1.6, an isoform that is very abundant in brain (Burgess et al., 1995; Tzoumaka et al., 2000), binds CaM with an affinity comparable to that of Na_V1.4 and Na_v1.2. The IQ motif of Na_V1.6 is slightly different from that of the other VGSCs (Fig. 1A) in that Na_V1.6 has a leucine instead of an isoleucine at the position corresponding to I1727 of Na_V1.4. However, this residue of the IQ motif has been shown to be variable in conventional myosins, and isoleucine, leucine, or valine generally is considered equivalent at this position (Bahler and Rhoads, 2002). In addition, the I1727L mutation had no effect on the functional properties of Na_v1.4 (data not shown). To test the functional consequences of CaM binding to the IQ motif of Na_V1.6, we mutated two of the core residues of the IQ motif in Na_V1.6, residues L1900 and Q1901, to glutamates. We compared the properties of wild-type Na_V1.6 channels (LQ) with Na_V1.6-Q1901E (LE) and Na_V1.6-L1900E-Q1901E (EE) mutant channels. Because Na_V1.6 channels express poorly in HEK cells (0.31 ± 0.05 nA; n = 30), we used a TTX-resistant variant of Na_V1.6 (Na_V1.6-Y371S) expressed in DRG neurons from Na_V1.8 null mice (Akopian et al., 1999). After 5 d in culture these neurons lack TTX-resistant currents and therefore provide a very suitable expression system for expression of sodium channels that have been mutated to be TTX-resistant (Cummins et al., 2001).

At 24 hr after biolistic transfection large (peak amplitude, 40.8 ± 4.0 nA; n = 31) Na_V1.6 sodium currents (Fig. 4A) were recorded in the presence of 250 nm TTX. The currents produced by the LE construct were 74% smaller than wild-type (LQ) currents (Fig. 4B). In contrast to the effect of the IQ mutations on Na_V1.4, the Na_v1.6 construct with the EE double mutation reduced Na_V1.6 current amplitudes down to only ∼37% of control (Fig. 4A,B). ANOVA plus pairwise comparisons (Fisher's LSD multiple comparison test) revealed that the mutant current amplitudes were significantly smaller than wild-type current amplitude but that the difference between the current amplitudes produced by LE and EE mutants was not significant. To determine whether the difference in the effect of the EE double mutation on Na_V1.4 and Na_V1.6 current amplitude was the result of the different expression backgrounds (HEK293 cells vs DRG neurons), we expressed Na_V1.4 and Na_V1.4-EE channels in Na_V1.8 null DRG neurons. Because we were using TTX-resistant constructs, we again were able to isolate the transfected currents by using 250 nm TTX. Although large TTX-resistant sodium currents were recorded from neurons transfected with Na_V1.4 channels, virtually no current was observed in neurons transfected with Na_V1.4-EE channels (Fig. 4B). It is not clear what accounts for the difference in the effect of the EE double mutation on Na_V1.4 and Na_V1.6 current amplitude, but it could reflect additional binding sites for CaM on Na_V1.6 channels, as has been reported for some L-type calcium channels (Pitt et al., 2001).

Figure 4.

The IQ motif of Na_v1.6 channels is an important determinant of current amplitude. A, Representative families of voltage-clamp recordings from Na_v1.8 null DRG neurons TTX-resistant Na_V1.6 (left) and Na_V1.6-EE (right) channels are shown. Cells were held at -120 mV, and currents were elicited by 40 msec test pulses to potentials ranging from -80 to -40 mV in 5 mV increments. B, Shown are peak current amplitudes of Na_V1.6 and Na_v1.4 constructs in which the core residues of the CaM binding IQ motif were replaced. The number of neurons recorded from for each group is indicated above the bars. ANOVA plus pairwise comparisons (Fisher's LSD multiple comparison test) revealed that the Na_v1.6-LE and Na_v1.6-EE mutant channels produced lower current amplitudes than wild-type (LQ) Na_v1.6 channels (black bars) and that overexpression of CaM increased the current amplitude produced by Na_v1.6-LE mutant channels, but not wild-type channels (open bars). The EE double mutant reduced the amplitude of currents produced by Na_v1.4 channels expressed in DRG neurons by >99% (shaded bars).

In analogy to the study of Na_V1.4, we wanted to determine whether the modulation of Na_v1.6 current amplitude by the IQ motif mutations depended on the reduced binding of CaM to the channel. Therefore, we tested whether overexpression of CaM could rescue the reduced current of the LE construct. The LE current amplitude was increased by 118% (Student's t test, p < 0.001) when more CaM was available to the channel. As with Na_V1.4, this change in current density was not associated with changes in the voltage dependence of activation or inactivation.

Time course of inactivation varies between wild-type Na_V1.6 (LQ) and EE mutant currents

Although the activation time constant τ_m did not differ between the LQ and EE Na_V1.6 channel constructs, there was a significant difference (Student's t test, p < 0.05) in the inactivation time constant τ_h between LQ and the EE constructs (Fig. 5). The EE double mutation slowed Na_V1.6 inactivation by 48% at -30 mV and 51% at +20 mV. The rate of fast inactivation was slower for the EE mutant channels at all voltages from -30 to +40 mV. Because shifts in the voltage dependence of channel gating can contribute to changes in inactivation kinetics, we compared the voltage dependence of activation and steady-state inactivation for Na_V1.6 LQ and EE constructs. The midpoints for activation (determined with a Boltzmann function) were not significantly different for LQ (-25.4 ± 1.0 mV; n = 7) and EE (-22.9 ± 2.4 mV; n = 8) Na_v1.6 currents. The midpoints for steady-state inactivation were also similar for LQ (-64.3 ± 2.1 mV; n = 10) and EE (-65.5 ± 2.0 mV; n = 9) Na_v1.6 currents. Thus shifts in the voltage dependence of channel gating do not seem to contribute to the differences in inactivation kinetics.

Figure 5.

The EE mutation in the IQ motif alters the inactivation kinetics of Na_v1.6 channels. A, Comparison of representative traces from Na_v1.8 null DRG neurons transfected with wild-type and EE mutant Na_v1.6 channels. Currents were elicited with test pulses to -30 mV (left) and +20 mV (right). The EE mutant channels exhibited slower inactivation. B, Inactivation kinetics as a function of voltage. The macroscopic decay time constant is greater for Na_v1.6 EE currents (open circles; n = 8) than for Na_v1.6 wild-type currents (filled squares; n = 16) at all voltages ranging from -25 to +40 mV (Student's t test, p < 0.05). Time constants were estimated from single exponential fits to the decay phase of currents elicited by 40 msec step depolarizations to the indicated potential.

Calcium/calmodulin switches Na_V1.6 from a fast into a slow mode

In the GST pull-down assay we showed that CaM binds to Na_V1.6 in the presence and absence of calcium ions. However, CaM is considered the prototype of a family of intracellular calcium sensor proteins that confer calcium sensitivity to their targets (Chin and Means, 2000; Burgoyne and Weiss, 2001). Because reducing the binding of CaM to Na_V1.6 changes its inactivation kinetics, we asked whether this reflects a physiologically relevant CaM-dependent mechanism that also could be regulated by changes in intracellular calcium concentration. All of the experiments described above were conducted with low intracellular calcium in which the pipette solution contained 1.1 mm EGTA and no added calcium. Similar results were obtained with 20 mm BAPTA and no added calcium in the pipette. To examine Na_V1.6 current properties at a high [Ca²⁺]_i, we added 1.1 mm calcium to the pipette solution that contained 1.1 mm EGTA, which raised the free calcium level to ∼10 μm. We also examined the effect of a CaM mutant in which the aspartates in the first position of the four calcium-binding EF hand motifs had been replaced by an alanine to render it calcium-insensitive. This mutant usually is referred to as a dominant-negative form of CaM and is abbreviated as CaM₁₂₃₄ (Peterson et al., 1999; Pitt et al., 2001).

We first examined the effect of coexpression of wild-type CaM with Na_V1.6 on Na_V1.6 current characteristics. Coexpression of wild-type CaM with Na_V1.6 did not alter the properties of currents recorded with low [Ca²⁺]_i. Raising [Ca²⁺]_i in the presence of overexpressed CaM did not alter the voltage dependence of activation (Fig. 6A) or steady-state inactivation (Fig. 6B). However, with high [Ca²⁺]_i the currents recorded from cells transfected with Na_V1.6 and CaM exhibited slower time constants of inactivation (Fig. 6C,D). On the other hand, when Na_V1.6 channels were coexpressed with CaM₁₂₃₄, the inactivation kinetics were fast in the presence of both high and low [Ca²⁺]_i (Fig. 6D). Thus high [Ca²⁺]_i with wild-type CaM, but not with the calcium-insensitive CaM₁₂₃₄, had the same effect on the rate of inactivation as did the EE double mutation. This indicates that calcium-free CaM can enhance the rate of Na_v1.6 inactivation.

Figure 6.

Calcium/CaM modulates Na_v1.6 inactivation kinetics, but not voltage-dependent properties. A, Normalized peak current-voltage relationship for TTX-resistant Na_v1.6 channels recorded with low (filled squares; n = 5) and high (open squares; n = 6) intracellular calcium. The currents were elicited by 40 msec test pulses to various potentials from -80 to -40 mV. Cells were held at -120 mV. B, Comparison of TTX-resistant Na_v1.6 steady-state inactivation recorded with low (filled squares; n = 5) and high (open squares; n = 6) intracellular calcium. Steady-state inactivation was estimated by measuring the peak current amplitude elicited by 20 msec test pulses to -10 mV after 500 msec prepulses to potentials over the range of -130 to -10 mV. Current is plotted as a fraction of the maximum peak current. C, Representative currents from whole-cell recordings of Na_v1.8 null DRG neurons expressing TTX-resistant Na_v1.6 channels in high or low intracellular calcium. Currents were elicited by a step depolarization to -10 mV from a holding potential of -120 mV and were scaled for comparison. The current recorded in high calcium displays slower kinetics. D, Inactivation kinetics as a function of voltage for Na_v1.6 currents. The macroscopic decay time constant is greater for TTX-resistant Na_v1.6 currents recorded with high intracellular calcium (open squares; n = 6) than with low intracellular calcium (filled squares; n = 16) or for TTX-resistant Na_v1.6 currents coexpressed with calcium-deficient CaM₁₂₃₄ with either high (open triangles; n = 5) or low (filled triangles; n = 5) intracellular calcium. Time constants were estimated from single exponential fits to the decay phase of currents elicited by 40 msec step depolarizations to the indicated potential. All recordings were made in the presence of 250 nm TTX from Na_v1.8 null DRG neurons transfected with TTX-resistant Na_v1.6 channels. Significant differences were determined using Student's t test with the significance value set at p < 0.05.

For comparison, we also tested the calcium sensitivity of Na_V1.4 channels. Increasing [Ca²⁺]_i had no effect on the voltage dependence of activation (Fig. 7A) or steady-state inactivation (Fig. 7B) of Na_v1.4 channels expressed in HEK293 cells. The inactivation kinetics of Na_V1.4 currents expressed in either HEK293 cells (Fig. 7C) or Na_V1.8 null DRG neurons (data not shown) also were not altered by changes in [Ca²⁺]_i. This indicates that the calcium-dependent calmodulin regulation of VGSC inactivation kinetics is isoform-specific.

Figure 7.

Calcium/CaM does not modulate Na_v1.4 inactivation kinetics or voltage-dependent properties. A, Normalized peak current-voltage relationship for Na_v1.4 channels recorded with low (filled squares; n = 8) and high (open squares; n = 8) intracellular calcium. The currents were elicited by 40 msec test pulses to various potentials from -80 to -40 mV. Cells were held at -120 mV. B, Comparison of Na_v1.4 steady-state inactivation recorded with low (filled squares; n = 8) and high (open squares; n = 8) intracellular calcium. Steady-state inactivation was estimated by measuring the peak current amplitude elicited by 20 msec test pulses to -10 mV after 500 msec prepulses to potentials over the range of -130 to -10 mV. Current is plotted as a fraction of the maximum peak current. C, Inactivation kinetics as a function of voltage for Na_v1.4 currents. The macroscopic decay time constant is similar for Na_v1.4 currents recorded with high intracellular calcium (open squares; n = 8) and with low intracellular calcium (filled squares; n = 8). Time constants were estimated from single exponential fits to the decay phase of currents elicited by 40 msec step depolarizations to the indicated potential. All recordings were made in the presence of 250 nm TTX from HEK293 cells transfected with TTX-resistant Na_v1.4 channels. Significant differences were determined using Student's t test with the significance value set at p < 0.05.

Recently, Deschenes et al. (2002) reported that calcium/CaM shifted steady-state inactivation of Na_v1.4 currents in the negative direction by ∼5 mV. Because we did not observe any shift in steady-state inactivation under our conditions, we performed additional sets of experiments that used conditions similar to those used by Deschenes and colleagues. The first set of experiments used the intracellular and bath solutions (Fig. 8) described by Deschenes et al. (2002) with no calcium buffering capacity in the pipette solution. No difference was observed in the voltage dependence of activation (Fig. 8A) or steady-state inactivation (Fig. 8B) between cells transfected with either Na_v1.4 plus CaM or Na_v1.4 plus EGFP. In the second set of experiments that used these solutions, the intracellular free calcium was buffered to ∼10 μm with the addition of 1.1 mm EGTA and 1.1 mm CaCl₂ to the pipette solution. In this set of experiments we recorded from HEK293 cells that stably expressed Na_v1.4 channels with or without the addition of 10 μm CaM (Calbiochem) to the pipette solution. At 10 min after establishing the whole-cell recording configuration, we measured the voltage dependence of activation (Fig. 8C) and steady-state inactivation (Fig. 8D). Calcium/calmodulin did not alter the voltage dependence of Na_v1.4 currents under these conditions. Therefore, we conclude that calcium/calmodulin does not alter the voltage-dependent or kinetic properties of Na_v1.4 currents.

Figure 8.

Calcium/CaM does not shift the voltage dependence of Na_v1.4 activation or steady-state inactivation. In these experiments the intracellular solution contained (in mm): 35 NaCl, 105 CsCl, and 10 Cs-HEPES, pH 7.3, and the extracellular solution contained (in mm): 150 NaCl, 2 KCl, 2 CaCl₂, 1 MgCl₂, 10 glucose, and 10 Na-HEPES, pH 7.4, as described by Deschenes et al. (2002). A, Normalized peak current-voltage relationship for Na_v1.4 channels recorded with no intracellular calcium buffering. HEK293 cells were cotransfected with Na_v1.4 channels and either EGFP (filled circles; n = 5) or CaM (open circles; n = 5). The currents were elicited by 40 msec test pulses to various potentials from -80 to -40 mV. Cells were held at -120 mV. B, Comparison of Na_v1.4 steady-state inactivation recorded with no intracellular calcium buffering. HEK293 cells were cotransfected with Na_v1.4 channels and either EGFP (filled circles; n = 5) or CaM-EGFP (open circles; n = 5). Steady-state inactivation was estimated by measuring the peak current amplitude elicited by 20 msec test pulses to -10 mV after 500 msec prepulses to potentials over the range of -130 to -10 mV. Current is plotted as a fraction of the maximum peak current. C, Normalized peak current-voltage relationship for Na_v1.4 channels recorded from HEK293 cells stably expressing Na_v1.4 channels with (open triangles; n = 8) or without (filled triangles; n = 6) 10 μm CaM in the pipette solution. The currents were activated as described in A. D, Comparison of Na_v1.4 steady-state inactivation recorded from HEK293 cells stably expressing Na_v1.4 channels with (open triangles; n = 8) or without (filled triangles; n = 6) 10 μm CaM in the pipette solution. The currents were elicited as described in B.

Discussion

In this study we describe the modulation of two VGSC isoforms, Na_V1.4 and Na_V1.6, by CaM and intracellular calcium. We show that functional expression of Na_v1.4 current is critically dependent on the ability of Na_v1.4 to bind CaM to its C terminus, indicating that this is a novel mechanism for the regulation of sodium current amplitude. Mutations in the IQ motif of Na_V1.6 channels also reduced Na_V1.6 current amplitude, although to a smaller extent than Na_V1.4 channels. We did not find any evidence that CaM modulated the voltage dependence of activation or inactivation of either Na_v1.4 or Na_v1.6 channels. However, we found that changes in the intracellular calcium ion concentration altered the inactivation kinetics of Na_V1.6 currents via a CaM-dependent mechanism. These data show that CaM can regulate the properties of VGSCs in isoform-specific ways and via both calcium-dependent and calcium-independent mechanisms.

The regulation of Na_v1.4 and Na_v1.6 current density by CaM was surprising, but not unprecedented. CaM appears to be an important regulator of several different ionic currents; recently, Saimi and Kung (2002) proposed that CaM could be considered a channel subunit in itself. The high voltage-activated sodium channels of Paramecium are critically dependent on CaM for activation (Saimi and Ling, 1995). CaM has been shown to interact with the C terminus of SK4 potassium channels, and this interaction controls channel assembly and surface expression of SK4 channels (Joiner et al., 2001). Functional expression of KCNQ2/Q3 potassium channels also requires CaM binding, although in this case CaM is not required for targeting of KCNQ2 to the cell membrane despite the fact that KCNQ2 seems to bind CaM constitutively in a calcium-independent manner (Wen and Levitan, 2002).

Although CaM is expressed at relatively high concentrations (1-100 μm) in many cell types, CaM binding proteins may outnumber CaM by as much as 2:1. Persechini and Cronk (1999) estimated that, although under normal conditions high-affinity (K_D ≤ 10 nm) CaM binding proteins are able to bind CaM, low-affinity (K_D ≥ 100 nm) CaM binding proteins are able to bind CaM only if the local concentration of free CaM can be increased significantly. Mori et al. (2000) reported that affinity of the IQ motif of Na_v1.2 for CaM was 5 nm; therefore, Na_v1.2 would be considered a high-affinity CaM binding protein. Our data suggest that Na_v1.4 and Na_v1.6 also might be high-affinity CaM binding proteins (Fig. 1B) and might bind CaM constitutively. Indeed, although eliminating CaM binding to the IQ motifofNa_v1.4orNa_v1.6 channels had major effects on current density, the overexpression of CaM did not seem to alter the properties of wild-type Na_v1.4 or Na_v1.6 currents, suggesting that the channels might be saturated with CaM. The fact that overexpression of CaM was able to increase the current amplitude in cells expressing Na_v1.4 or Na_v1.6 channels with IQ mutations that reduced, but that did not eliminate, current and CaM binding provides compelling evidence for a direct regulation of sodium current amplitude by CaM. Interestingly, a recent study indicated that a mutation identified in the C terminus of the human Na_v1.2 channel (R1902C) from an individual with autism might reduce the binding affinity of Na_v1.2 for calcium/calmodulin (Weiss et al., 2003).

Our GST-fusion protein assays indicated that the C termini of Na_v1.1, Na_v1.3, and Na_v1.7 bound CaM with a lower affinity than the other TTX-sensitive isoforms. Although the IQ motifs of the various sodium channel isoforms are fairly well conserved, there are differences in this region (for example, Na_v1.3 has an asparagine at a position at which the other isoforms have an alanine; Fig. 1A), and these differences could alter CaM binding. However, it is likely that other regions in the C termini of the different isoforms also influence CaM binding. It will be interesting to determine whether CaM also can regulate the current density produced by the TTX-sensitive VGSC isoforms other than Na_v1.4 and Na_v1.6. None of the three known TTX-resistant isoforms (Na_v1.5, Na_v1.8, and Na_v1.9) showed detectable CaM binding in our assay. Two previous studies have suggested that Na_v1.5 might bind CaM. Tan et al. (2002) reported that a 20-amino-acid peptide containing the IQ motif of Na_v1.5 was able to bind CaM, and Deschenes et al. (2002) reported that a truncated form of the C terminus of Na_v1.5 that contained 53 amino acids was able to bind CaM. Our experiments were conducted by using the complete C terminus of Na_v1.1-Na_v1.9 for the in vitro binding studies, and, because regions outside the IQ motif can influence CaM binding (Peersen et al., 1997), conformational changes in the full-length C terminus might account for the lack of CaM binding to Na_v1.5 in our assay. Interestingly, Tan et al. (2002) reported that the 20 AA peptide containing the Na_v1.5 IQ motif showed a lower affinity for CaM than the 290-309 CaM antagonist peptide derived from CaM kinase II. Because the 290-309 peptide has an affinity for CaM of 52 nm (Payne et al., 1988), this indicates that the affinity of the Na_v1.5 IQ motif for CaM is >10-fold lower than that of the Na_v1.2 IQ motif. Based on these data, Na_v1.5 would be considered a low-affinity binding protein and would not be expected to bind CaM constitutively. Although the study by Tan et al. (2002) suggested that calcium/CaM might decrease the amplitude of Na_v1.5 currents by ∼5-15% by regulating an intermediate form of slow inactivation, Deschenes et al. (2002) concluded that Na_v1.5 currents are modulated only indirectly by CaM via a CaM-dependent kinase.

We did not see any effect of CaM overexpression on the voltage-dependent properties of Na_v1.4 or Na_v1.6 currents. In contrast, Deschenes et al. (2002) reported that overexpression of CaM in HEK293 cells produced a -6 mV shift in the voltage dependence of steady-state inactivation of Na_v1.4 currents (Deschenes et al., 2002). Whereas we used 1.1 mm EGTA to buffer intracellular free calcium in our control experiments, Deschenes et al. (2002) did not include any calcium buffers in their intracellular solution for their control conditions. However, we also did not observe any effect of overexpression of CaM on Na_v1.4 currents when we used the solutions described by Deschenes and colleagues without intracellular calcium buffering. In unbuffered solutions the free calcium levels can be quite variable and even can be ≥10 μm (Pallotta et al., 1992). Therefore, we also examined the effect of adding CaM to the Deschenes et al. (2002) pipette solution when free calcium was buffered to ∼10 μm with EGTA. Under these conditions the additional CaM had no effect on the voltage dependence of Na_v1.4 currents. We did not find any evidence that the properties of Na_v1.4 were regulated by CaM in a calcium-dependent manner in any of the experiments that we conducted.

Our data showed that the functional properties of Na_v1.6 currents are regulated by calcium/CaM, which slowed inactivation by ∼50%. However, because the Na_v1.6-EE currents also exhibited slowed inactivation, our data indicate that calcium-free CaM enhances the rate of inactivation, and, when calcium binds to CaM, calcium/CaM disrupts this enhancement, thereby effectively slowing inactivation. By disrupting the binding of CaM to the IQ motif, the EE mutation effectively would prevent the calcium-free CaM enhancement of inactivation. Although in this proposed mechanism calcium-free CaM is the active molecule, which is the case for other actions of CaM (Jurado et al., 1999), this still represents a calcium-dependent modulation of sodium current properties. This is the first description of a calcium-dependent CaM modulation of a neuronal sodium channel isoform. CaM has been shown to confer calcium sensitivity to several other transmembrane channel proteins (Saimi and Kung, 1994; Levitan, 1999). The fact that we are now able to observe a similar regulatory mechanism for Na_v1.6, a neuronal VGSC that is present along axons (Caldwell et al., 2000) and extends to axon terminals (Black et al., 2002), has potentially interesting implications for the control of neuronal excitability and synaptic transmission by calcium/CaM. The calcium/CaM-dependent slowing of Na_v1.6 inactivation kinetics could prolong action potential duration, and in nerve terminals this type of modulation might enhance neurotransmitter release.

Conclusions

Our data show that CaM binding to the C terminus of VGSCs can be critical for obtaining functional sodium current expression and may provide a mechanism for regulating the amplitude of sodium currents in a concentration-dependent manner. Although our experiments with GST-fusion proteins indicate that CaM binding to the C terminus of VGSCs is not dependent on calcium concentration, calcium and CaM are known to play important roles in excitation contraction coupling in muscle (Hamilton et al., 2000) and in controlling the excitability of neurons (Sola et al., 2001), and the interaction of the CaM-VGSC complex with other proteins in neurons and muscle cells may be modulated by calcium. In addition, although we did not observe any calcium/CaM-dependent regulation of Na_v1.4 currents, the inactivation kinetics of Na_v1.6 currents were modulated by calcium/CaM. The array of mechanisms that involve finely tuned Ca²⁺ concentrations just below the cell membrane is growing, and our data add a new mechanism, mediated by CaM, to the ways in which VGSCs can be regulated.