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1 Department of Cell and Developmental Biology and the Neuroscience Program, University of Colorado Health Sciences Center, Aurora, CO 80045, USA
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(Received 15 December 2004;
accepted after revision 28 February 2005;
first published online 3 March 2005)
Corresponding author J. H. Caldwell: Campus Box 8315, Dept. of Cell/Devel Biology, UCHSC, PO Box 6511, Aurora, CO 80045, USA. Email: john.caldwell{at}uchsc.edu
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Introduction |
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-subunit and one or more auxiliary ß-subunits. Nine genes encode the -subunit in mammals, with the proteins designated NaV1.1–1.9. The majority of these subtypes are expressed in neurones, but NaV1.4 and NaV1.5 are expressed primarily in skeletal and cardiac muscle, respectively. Activation and inactivation properties of these channels are crucial for determining normal nerve function and muscle contraction. Perhaps the most dramatic illustration of the changes wrought by slight modifications to the activation and inactivation properties are the human diseases caused by mutations that produce myotonias or cardiac arrest (Head & Gardiner, 2003; Goldin, 2003). Mutations in the human sodium channels that disturb activation and/or inactivation are widely distributed over the primary sequence, indicating that many regions of the channel are capable of influencing activation and inactivation.
Auxiliary proteins to the -subunit can modify activation and inactivation of the channel. NaCh ß-subunits have dramatic effects on gating, especially inactivation (Isom, 2000). Calmodulin (CaM) is another protein that probably binds constitutively to the sodium channel. CaM regulates a vast array of different cellular processes, including ion channels (Saimi & Kung, 2002). Evidence for a functional role of CaM binding to NaChs is beginning to emerge. Yeast two-hybrid experiments performed in our laboratory and those of others (Mori et al. 2000) showed that the C-terminal region of NaChs interacts with CaM. This NaCh domain contains an IQ-binding motif, a Ca2+-independent CaM binding site that is highly conserved across all NaCh isoforms (Rhoads & Friedberg, 1997). The IQ domain in L-type calcium channels (CaChs) is known to be important for CaM-mediated inactivation of these channels (Qin et al. 1999; Zuhlke et al. 1999; Erickson et al. 2003; Liang et al. 2003).
A yeast two-hybrid screen using the C-terminal cytoplasmic tails of NaV1.4 and NaV1.5 yielded multiple full-length clones of CaM from a muscle library. Encouraged by the structural similarities of the C-terminal tail of NaChs to those of CaChs, we used whole-cell recordings to search for functional effects of CaM binding to NaV1.4 and NaV1.5. Recent work by Tan et al. (2002), Deschenes et al. (2002) and Herzog et al. (2003) has also shown functional effects of CaM on NaCh function. A perplexing aspect of these three studies is that there are many unexplained discrepancies between them. Our results agree with some aspects of the earlier reports, but other findings of ours are novel. Some of the differences between our results and those of other groups are likely to be due to the different cell lines (CHO versus HEK) used for heterologous expression. We found that coexpression of CaM with NaV1.4 and NaV1.5 affects the voltage dependence of either activation and/or steady-state inactivation. We used mutant CaMs and NaChs with point mutations in the IQ motif to identify regions of CaM and the NaCh required for modulation by CaM. Recordings from chimeric channels made between NaV1.4 and NaV1.5 also confirm the importance of the C-terminal domain for the effects of CaM on steady-state inactivation of the channel.
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Methods |
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The C-terminal cytoplasmic domains for both the skeletal (rSKM1 or NaV1.4) and cardiac (hH1 or NaV1.5) muscle sodium channels were cloned into pHybLex/Zeo (Invitrogen) for use as bait clones. Each loop was screened against a human-skeletal-muscle library in pGAD10 (Clontech) according to the manufacturer's instructions. Interaction of the bait and prey proteins was assayed by growth on yeast-peptone-dextrose rich medium (YPD)/–leu/–his/+zeo selective media. Autoactivation of the reporter gene was not observed on YPD/–his/+zeo, and autoactivation of the CaM cDNA clones was not observed on YPD/–leu/–his.
Cell culture
CHO-K1 (Chinese hamster ovary) cells (American Type Culture Collection) were used for expression of all of the NaCh and CaM constructs except those for Fig. 7, for which human embryonic kidney cells (HEK293) were used. CHO-K1 cells were grown in F12 (HAMS) medium (Sigma) fortified with 10% FCS and 1% penicillin/streptomycin in a humidified incubator at 37°C and 5% CO2. HEK293 cells were grown in F12 (DMEM) medium, 10% FCS, 1% penicillin/streptomycin and 1% non-essential amino acids (Invitrogen). The following DNAs were transiently transfected in various combinations as described in the Results section: NaV1.4 and NaV1.5 (pZemµ1–2 and pCDNA3.1-hH1, gift of S. R. Levinson, UCHSC), NaV1.4/CT1.5 and NaV1.5/CT1.4 (both in pCDNA3.1, gift of A. George, Vanderbilt University and E. Bennett, University of South Florida), I1727E and L1736R mutated NaV1.4, CaMWT and CaM1234 (gift of K. Beckingham, Rice University), CaM12 and CaM34 (gift of B. Peterson, Penn State University), and ß1-subunit (gift of S. Cannon, University Texas, Southwestern). CaMWT and CaM1234 were both cloned into the green fluorescent protein (GFP)-expression vector pEGFPN3 (Clontech). In cases where the DNA was not in a tagged vector, cells were cotransfected with pEGFPN3. Transfections were performed using Lipofectamine (Gibco/BRL) according to the manufacturer's instructions. For each transfection, 1–2 µg total DNA was used. Recordings were made 2–3 days after transfection.
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Mutation of NaV1.4
Mutations in the IQ domain were made using the QuikChange XL Site-Directed Mutagenesis Kit (Stratagene). The primers used were:
I1727e-5': AGG AGG TGT GTG CTA TCA AAG AGC AGA GGG CCT ACC GCC G
I1727e-3': CGG CGG TAG GCC CTC TGC TCT TTG ATA GCA CAC ACC TCC
L1736r-5': CCG CCA CCT GCG GCA GCG CTC CGT G
The underlined nucleotides show the mutation sites. XL10-Gold Ultracompetent cells were transformed with 2 µl of each reaction to produce the mutated DNA according to the manufacturer's instructions. Positive clones were sequenced by the UCHSC Cancer Center to verify that the mutation was incorporated.
RT-PCR. Primers were designed using GenBank sequences and the Perkin Elmer ABI Primer Express program. Each primer was searched against BLAST (http://www.ncbi.nlm.nih.gov/BLAST) to ensure that it did not match any known gene aside from that for which it was designed, including other family members. The primers for the CHO cells were designed to ensure 100% identity between rat and mouse for each subunit. All amplification products were the same size, and the primers had the same melting temperatures. RNA was made using a Qiagen Rneasy mini kit (Valencia, CA, USA). cDNA was made using Superscript II RT with oligo (dT)12–18 primers (Invitrogen, Carlsbad, CA, USA). For quantitative PCR, PCR was carried out in a GeneAmp Sequence Detection System 5700 (Perkin Elmer, Norwalk, CN, USA) using 40 cycles of 95 to 60°C PCR with a 10 min 95°C initial step. The fluorescent indicator Sybrgreen (Bio-Rad Laboratories, Hercules, CA, USA) was used to allow real-time light detection. Increased fluorescence resulting specifically from amplification of the target sequence was detected real time so that the number of PCR cycles and thus the number of molecules of each ß-subunit could be expressed as a percentage of hypoxanthine phosphoribosyltransferase (HPRT) mRNA. Each measurement was made in triplicate and averaged, with two individual replicate experiments used for statistical analysis. Primers used for amplification of ß-subunits are presented in the Supplemental material table.
Acquisition of data
Whole-cell patch-clamp recordings were made using standard techniques. Pipettes for recording were pulled from capillary micropipettes (Drummond Scientific Co.) using a 5-stage protocol on a micropipette puller (P-97 Sutter Instruments Co.), coated with blue ski wax, and fire-polished using a micro forge (MF-830 Narishige). Cells were voltage-clamped using an Axopatch 200A integrating amplifier (Axon Instruments). To minimize voltage-clamp errors, low-resistance electrodes (1.5–3 M
) were used with series resistance compensation correction > 95%. Voltage pulses were generated and membrane currents were measured using pCLAMP8 software (Axon Instruments). Currents were filtered at 5 kHz and digitized at 20 kHz. Leak current was subtracted using a P/8 protocol. With one exception, cells with currents > 1 nA were analysed to ensure that the records were not contaminated by endogenous currents. The exception was for IQ mutants that interfered with CaM binding; currents in these cells were often low and thus currents greater than 0.8 nA were included in the analysis. For all experiments, recording was started 5 min after gaining whole-cell access to ensure stabilization of currents.
For current–voltage data, cells were held at a holding potential of –120 mV. Voltage-clamp steps of 30 ms were applied from –90 to +100 mV in 10-mV increments. For steady-state inactivation data, a two-pulse protocol was used. A holding potential of –120 mV was used. Prepulse potentials of 100 ms were applied from –150 mV to +30 mV, followed by a test pulse of 30 ms to –10 mV.
Recording solutions
The external solution contained (mM): 132 NaCl, 4 KCl, 1.5 CaCl2, 1.5 MgCl2, 11 glucose and 10 Hepes (pH to 7.4 at room temperature using NaOH). The normal internal solution contained (mM): 115 CsF, 10 CsCl, 10 NaF, 10 Hepes and 5 EGTA (pH to 7.4 at room temperature using CsOH). The 10 µM free-Ca2+ internal solution contained 115 CsF, 10 CsCl, 10 NaF, 10 Hepes, 5 EGTA and 4.9 CaCl2. The intracellular Cl– solution contained (mM): 125 CsCl, 10 NaCl, 10 Hepes and 5 EGTA. Chemicals were purchased from Sigma. Inhibitors were purchased from Calbiochem. CaM-binding peptide (CBP), CaM-inhibitory peptide (CIP) and CaM-inhibitory peptide control (CIPc) stocks were dissolved in H2O. KN93 and KN92 stock solutions were dissolved in DMSO and diluted 1: 1000 prior to use. DMSO at this concentration had no effect on currents. All of the stock inhibitor solutions were stored at –20°C and diluted to their working concentrations immediately prior to use. The liquid junction potential between the internal solutions and the external bath was –8 mV, calculated using pCLAMP8.2. Reported membrane potentials were not corrected for this. All experiments were performed at room temperature.
Analysis of data
Analysis was performed using Clampfit8 (Axon Instruments), MatLab6.5 (The MathWorks, Inc.) and Origin6.1 (MicroCal Software, Inc., Northampton, MA, USA).
Time constants for inactivation were found by fitting a standard single exponential to the decay phase of the current using Clampfit8.
Conductance was calculated using the equation:
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Steady-state inactivation and conductance–voltage plots were fitted with a Boltzmann distribution to the equation:
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For producing graphs of the data, the means of the cells for each experimental condition were plotted using Origin6.1. For statistical analysis of the data, the curves for each individual cell were fitted with a Boltzmann distribution. The slope and V1/2 for each treatment group were then analysed using MatLab6.5 (Statistics are reported as means ± S.E.M. One-way ANOVA (analysis of variance) multiple comparison tests were used to test the significance of changes within and between treatment groups. This method compensates for the possibility of making at least one incorrect conclusion among pairs (greater than 5%) tested that can result when performing a series of t tests. Whole-cell current amplitudes, fast inactivation, and values of V1/2 and slope for the voltage dependence of activation and steady-state inactivation were compared between different treatment groups where appropriate. Differences were considered statistically significant for P < 0.05.
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Results |
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Application of CaM shifts the voltage dependence of activation and inactivation of NaV1.4
Currents were recorded from CHO cells transiently transfected with NaV1.4, with Ca2+-buffered intracellular recording solution (5 mM EGTA unless otherwise noted). The CaM inhibitors W5, mastoparan and CaM-inhibitory peptide (CIP, a 17 amino-acid peptide corresponding to the CaM-binding domain of myosin light-chain kinase), were applied to the cells through the pipette and had no effect on whole-cell currents measured (CIP data shown in Fig. 1A and B). Because interactions between CaM and its effector molecules are often mediated by Ca2+, we tested the effect of increasing intracellular Ca2+ on Na+ currents in these cells. Including Ca2+ (buffered to 10 µM) in the intracellular recording solution had no effect on currents measured (Fig. 1A and B).
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CaMWT and CaM1234 were expressed as fusion proteins with GFP at the amino-terminal end of CaM. For CaM1234, a glutamate required for Ca2+ binding was mutated to glutamine in all four Ca2+-binding EF hands, diminishing the Ca2+ affinity of CaM by several orders of magnitude (Mukherjea et al. 1996). Addition of the GFP tag to CaM does not change its cellular distribution or its functional properties when GFP-CaM is expressed in HEK293 cells (Erickson et al. 2001).
Coexpression of either CaMWT or CaM1234 with NaV1.4 did not significantly affect the current density (data not shown) or the time constant of rapid inactivation (Fig. 2A). However, coexpression of either CaMWT or CaM1234 with NaV1.4 did cause a hyperpolarizing shift in the peak of the I–V relationship (Fig. 2B). In addition, there was a hyperpolarizing shift of between 10 and 15 mV in the voltage dependence of activation (for both CaMWT and CaM1234) and inactivation (for CaMWT) of NaV1.4 current (Fig. 2C). The shift seen with CaMWT coexpression was similar to that observed with 50 µM CaM added to the intracellular recording solution (Fig. 1E and F), suggesting that endogenous CaM was indeed limiting in CHO cells with NaV1.4 overexpression. The hyperpolarizing shifts in both activation and steady-state inactivation by CaMWT (Fig. 2C) were statistically significant compared with NaV1.4 expressed alone. For reasons that are not clear, it is easier to obtain good whole-cell recordings with large sodium currents when the intracellular recording anion is fluoride (e.g. see Qu et al. 2000). For that reason most studies are done with high intracellular F–. Since Cl– is the physiological intracellular anion, we repeated these measurements with F– replaced by Cl– in the pipette. The voltage dependence of inactivation is shifted relative to that obtained with F–, but a hyperpolarizing shift still occurred when CaM was coexpressed (V1/2 = –52.0 ± 0.7 mV versus –44.5 ± 1.1 mV for control). Thus, the effect of CaM was independent of the intracellular anion.
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The essential underlying question in these studies was whether changes in CaM conformation elicited by molecules or proteins that bind to CaM would affect the biophysical properties of the channel. A change in Ca2+ concentration is the most obvious modulator of CaM conformation. Increased Ca2+ in the cells expressing NaV1.4 and CaMWT caused a depolarizing shift in steady-state inactivation but not activation (the depolarizing shift in activation in Fig. 2D was not statistically significant). We reasoned that other factors that would produce a conformational change in CaM might also alter the voltage-dependent properties of the channel. Two CaM-binding peptides, 75 pM CIP and 25 µM CBP (a 29-amino-acid peptide corresponding to the CaM-binding domain of CaM-kinase II (CKII)) also caused significant depolarizing shifts of inactivation in the CaM-cotransfected cells (Fig. 2D and F). One explanation for this depolarizing shift is that CIP and CBP compete with the IQ domain for binding to CaM and could cause CaM to dissociate from the channel producing voltage dependence of inactivation similar to that of the channel expressed without CaM. The depolarizing shifts of 7–10 mV seen with Ca2+, CIP and CBP compared with CaMWT alone were significant for the voltage dependence of steady-state inactivation (Fig. 2F), but despite a similar trend, the shifts were not significant for activation (Fig. 2E). It seems likely that binding of Ca2+ produces a different conformational change in CaM than the binding of the peptides. Nevertheless, both types of ligands produced a depolarizing voltage shift of inactivation when CaMWT was coexpressed. The inclusion of 10 µM Ca2+, 25 µM CBP or 75 pM CIP in the pipette had no effect on either activation or inactivation when the Ca2+-insensitive mutant CaM1234 was coexpressed with NaV1.4 (Fig. 2E and F). We conclude that Ca2+ and CaM-binding peptides modulate the effects of CaM and that activation and inactivation are not equally regulated by CaM.
CaM often affects target proteins through effector molecules such as phosphatases or kinases. NaV1.4 is not affected by cAMP-dependent protein kinase, despite the presence of consensus sequences for phosphorylation in the -subunit (Yang & Barchi, 1990; Smith & Goldin, 1992). NaChs in rat cerebellar granule cells are regulated by CKII (Carlier et al. 2000). Therefore, it was possible that CaM was acting on NaV1.4 through phosphorylation of the channel by CKII. To test this hypothesis, we applied KN93, a specific inhibitor of CKII that competes for the binding site of CaM on the kinase. KN93 had no effect on the hyperpolarizing shifts in both activation and inactivation seen with CaMWT coexpression (Fig. 2E and F). This suggests that the voltage-dependent shifts caused by CaM were not due to phosphorylation of NaV1.4 by CKII.
The yeast two-hybrid interaction and the failure of a kinase inhibitor to modify the effects of CaM coexpression indicate that the hyperpolarizing shifts in the V1/2 of activation and inactivation of the Na+ current when CaM is coexpressed are a result of CaM binding directly to the channel. This interaction is likely to be Ca2+ independent, since the Ca2+ concentration in these cells was held in the low nanomolar range by including EGTA in the intracellular recording solution. Further tests, described below, were designed to identify the regions of CaM and NaV1.4 that are required for this interaction.
Effects of mutations in the N and C lobes of CaM
The N- and C-terminal halves of CaM have different effects on ion channels. For example, an intact C lobe is required for Ca2+-dependent inactivation of the L-type CaCh (Peterson et al. 1999) while an intact N lobe is required for the effects of CaM on SK Ca2+-activated K+ channels (Keen et al. 1999). Yue and colleagues (Liang et al. 2003) have recently proposed a unifying model for CaM's effects on CaChs: the N lobe detects global Ca2+ and the C lobe responds to high local Ca2+. In addition, the N lobe of CaM is important for voltage-dependent inactivation and the C lobe for Ca2+-dependent inactivation. The structural basis for these effects must rely on differing affinities for Ca2+ of the two lobes of CaM with different conformational changes of the protein (Tjandra et al. 1995; Finn et al. 1995; Kuboniwa et al. 1995; Zhang et al. 1995). By perturbing states of Ca2+ binding between the lobes, we can alter specific conformational changes of CaM that could affect its ability to bind to NaV1.4. Therefore, NaV1.4 was coexpressed with either CaM12 or CaM34, which have reduced affinity for Ca2+ in either the N (CaM12) or C (CaM34) lobes of CaM (an aspartate to alanine mutation in each pair of EF hands in either the N or C lobe of CaM) (Putkey et al. 1989; Peterson et al. 1999; DeMaria et al. 2001). These CaM mutants allowed us to test the relative importance of the individual lobes of CaM for the shifts in the voltage dependence of activation and inactivation.
The biophysical properties of NaV1.4 did not change when the channel was coexpressed with CaM12 compared with the channel expressed alone. In contrast to the shift in the voltage dependence of currents seen with CaMWT coexpression with NaV1.4, CaM12 coexpression had no effect on the voltage dependence of activation or inactivation (Fig. 3A and filled bars in C). This mutation of the Ca2+ binding sites in the N lobe of CaM produced an inactive CaM when coexpressed with the channel. Increased Ca2+ and CIP had no further effect on the voltage dependence of activation or inactivation when CaM12 was expressed (Fig. 3D and E). The loss of Ca2+ binding in the N lobe of CaM abolished not only the voltage shifts in activation and inactivation, but also the modulatory effects of Ca2+ and CIP, providing further evidence that the effects of Ca2+ and CIP (Fig. 2D–F) depend on the association of a Ca2+-sensitive CaM with the channel. Since CaM12 had no effects on measured channel properties, we used anti-CaM antibodies for immunolabelling of transfected cells (cotransfected with GFP) to ensure that the mutant CaM was being expressed. Labelling of GFP and CaM cotransfected cells was 2- to 5-fold greater than non-transfected cells and control GFP- transfected cells. This increase in CaM labelling was similar to that observed for wild type and the other CaM mutants (data not shown). In summary, these effects of mutations in the N lobe were simple, i.e. a complete loss of action by CaM.
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Mutations of two amino acids in the IQ domain
The IQ-binding motif is a known consensus sequence for CaM binding in a variety of different proteins (Rhoads & Friedberg, 1997) and is present in all known NaChs in the C-terminal domain. Mutations in this domain in CaChs alter or abolish CaM binding and its functional effects on the channel (Peterson et al. 1999; Zuhlke et al. 1999). CaM has been shown to bind NaV1.2 in this region by yeast two-hybrid and gel mobility-shift assays (Mori et al. 2000). To define the region of the C-terminal required for binding between NaV1.4 and CaM, two mutations (expressed separately) in the IQ domain (IQRAYRRHLLQRSKV; I1727E and L1736R) were made at residues known to participate in CaM binding in CaChs and NaV1.5 (Tan et al. 2002).
When the channel was expressed by itself, these mutations did not change channel properties compared with native NaV1.4 (data not shown). However, when coexpressed with CaMWT or CaM1234, the I1727E mutation blocked all effects of CaMWT (Fig. 4A and B) and CaM1234 (data not shown). Although currents tended to be smaller for the mutated channel, there was no significant difference in the current density between native NaV1.4 and the I1727E mutant channel coexpressed with CaMWT (data not shown). Thus, the mutation does not affect surface expression even though the channel behaves as if CaM is not associated with it. The addition of Ca2+ or CIP in the recording solution also had no effect on voltage dependence (filled bars in Fig. 4E and F). Therefore, this mutation in the IQ domain of NaV1.4 abolished the shifts in voltage dependence caused by CaMWT and by modulators of CaM. This is consistent with a model in which the proximal end of the IQ domain (which includes the IQ) is required for CaM binding to the sodium channel (see Discussion).
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One mutation (I1727E) blocked all effects of CaM, as though CaM was no longer bound to or was able to interact with the channel. The other mutation (L1736R) preserved some effects, presumably due to association of apoCaM with the channel, while abolishing others, as though it interferes with a conformational change or shift in the binding of CaM to the channel in the presence of increased Ca2+ or CIP. These results suggest that the amino-terminal end of the IQ domain is essential for apoCaM binding. The carboxyl end of the IQ domain is more important for effects caused by increased Ca2+ in the cell.
Effects of CaM coexpression on the cardiac channel, NaV1.5
In the yeast two-hybrid assay, CaM interacted with the C-terminal domains of both NaV1.4 and NaV1.5. We therefore tested whether CaM affected channel properties in NaV1.5 as well. The time constants of fast inactivation were not significantly different for NaV1.5 coexpressed with CaMWT or CaM1234 compared with NaV1.5 alone (Table 1). Coexpression of CaMWT with NaV1.5 caused a shift in the I–V curve toward negative potentials, accompanied by a significant shift in the voltage dependence of activation (Fig. 5A and B). There was no significant shift in the voltage dependence of inactivation (Fig. 5B). CaM1234 coexpression had no significant effect on either activation (unlike its effect on NaV1.4) or inactivation. In contrast to NaV1.4, including 10 µM Ca2+, 75 pM CIP or 25 µM CBP in the pipette had no effect on activation or inactivation of NaV1.5 currents coexpressed with CaMWT (Fig. 5C, E and F). Increased Ca2+ had a slight depolarizing effect on the voltage dependence of inactivation, but this shift was not significant (Fig. 5F).
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A comparison of the results seen with CaMWT coexpressed with either NaV1.4 (Fig. 2) or NaV1.5 (Fig. 5) suggests that CaMWT acts on these channel isoforms through different pathways. CaMWT affected the voltage-dependent properties of both channels. However, CaM modulators caused depolarizing shifts on NaV1.4 currents, but had no effect on NaV1.5 currents, while KN93 only affected NaV1.5 currents.
Chimeric sodium channels formed by swapping the C-terminal domains
Studies using chimeric channels between NaV1.2 and NaV1.5 (Mantegazza et al. 2001) showed that the cytoplasmic C-terminus is important in determining the voltage dependence of steady-state inactivation but has no effect on activation. Cytoplasmic loops in the amino terminal half of the protein are important in distinguishing the activation properties of NaV1.4 and NaV1.5 from one another (Bennett, 2001). Since the C-terminal region of NaV1.4 was required for CaM's effects on the channel and coexpression of CaM had different effects on NaV1.4 and NaV1.5, chimeric channels of these two NaCh isoforms were used to test the possibility that some of the differences in the effects of CaM between NaV1.4 and NaV1.5 were based solely on the C-terminal tail. Recordings were made with NaV1.4/CT1.5 (NaV1.4 parent channel with NaV1.5 C terminal) and NaV1.5/CT1.4 (NaV1.5 parent channel with NaV1.4 C terminal; see inset in Fig. 6) coexpressed with CaMWT. The time constants of fast inactivation for the chimeric channels shifted to values of the C-terminal donor (Table 1), consistent with Deschenes et al. (2001) who showed that the donor of the C-terminal domain determined the time constant of fast inactivation for these channel isoforms. Coexpression of CaMWT had no further effect on fast inactivation.
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For the other chimera (NaV1.5/CT1.4), the voltage dependence of activation and steady-state inactivation of NaV1.5/CT1.4 were also shifted with coexpression of CaMWT (Fig. 6B). Ca2+ and CIP included in the recording solution did not cause a depolarizing shift in activation, similar to the lack of effect on NaV1.5 currents (Fig. 6C). However, Ca2+ and CIP caused a significant shift in the V1/2 of steady-state inactivation, similar to NaV1.4 (compare striped bars to filled bars in Fig. 6D). Thus, this chimera with a NaV1.4 carboxyl tail had inactivation properties similar to the native NaV1.4.
Dependence upon the expression system for CaM effects
One of the disturbing aspects of studies of the functional effects of CaM upon voltage-gated sodium channels is that the results are inconsistent from one laboratory to another, despite efforts to duplicate the recording solutions and cell lines used. Three previous studies used HEK293 cells (Deschenes et al. 2002; Tan et al. 2002; Herzog et al. 2003) and, in some cases, identical solutions and still reported results different from each other. We used CHO cells for the experiments described above. To determine if the use of a different cell line contributed to the effects we observed, we also recorded from HEK cells and tested the effect of coexpression of CaM and Nav1.4 upon the voltage dependence of activation and inactivation. In contrast to the shift in voltage dependence seen in CHO cells (Fig. 2C), we found no evidence of a shift in HEK cells (Fig. 7A).
Although many differences between CHO and HEK cells could account for this difference, we tested one of the most obvious possibilities, namely that ß-subunit expression differs between these two cell lines. ß-Subunits are not required for channel formation but do affect kinetics, voltage dependence and surface expression. Four ß-subunit genes have been described. We used quantitative PCR to measure the relative abundance of the mRNA for each of these genes in CHO and HEK cells. Mouse and human brain RNA were used as controls (data not shown). The primers were chosen in coding regions that are strictly conserved for each subtype in mouse, rat and human. mRNA levels were normalized to the copies of HPRT (hypoxanthine phosphoribosyltransferase) mRNA in every case (Fig. 7B and C). Three conclusions can be drawn. First, ß4 expression is non-existent in CHO cells. Second, ß1 expression is absent in HEK cells. Finally, assuming that the primers have the same amplification efficiency, the overall levels of mRNA for ß-subunits are much lower in HEK cells than in CHO cells (roughly a 10-fold difference). To ensure that the lack of expression of ß4 in CHO cells and of ß1 in HEK cells was not due to a poor choice of PCR primers, a second set of PCR primers to different coding regions was generated and used for amplification. The results were the same: ß1 mRNA is missing in HEK cells and ß4 mRNA is missing in CHO cells (data from both sets of primers are included in Fig. 7B and C). Thus, some of the differences between the results presented here using CHO cells and those of other laboratories using HEK cells could be due to different levels of ß-subunits in the cell types (see Discussion). We tested the possibility that the absence of ß1 in HEK cells was responsible for the lack of a CaM effect. Coexpression of ß1 with Nav1.4 and CaM did not produce a shift in voltage dependence of inactivation (data not shown). Thus, ß1 cannot explain the failure to observe an effect in HEK cells. The involvement of ß4 has not been tested.
Effects of a CaM-binding peptide on Na++ currents in C2C12 cells
Endogenous muscle sodium channels are expressed by the muscle-derived C2C12 cell line. Inclusion of CIP (75 pM) in the pipette caused a significant depolarizing shift (7.4 mV, n = 10, P < 0.01) in the voltage dependence of inactivation (Fig. 8). There was no effect on the I–V relationship or the voltage dependence of activation. The magnitude of this effect is close to that observed for Nav1.4 (8.3 mV, see Table 1). Since CIP had no effect on inactivation in CHO cells when CaM1234 was expressed with Nav1.4 (Fig. 2F), the shift in C2C12 cells implies that it acts via CaM bound to the endogenous sodium channel.
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Discussion |
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These results are consistent with the following simple model (Fig. 9). The C lobe of CaM binds to a region near to or including the IQ portion of this motif at low resting levels of Ca2+. Mori et al. (2003) used circular dichroism to show that apoCaM binds to the C-terminal of Nav1.2, suggesting that the binding of apoCaM is a general feature of NaChs. Our assumption is that the Ca2+-free form of CaM is similar to the crystal structure of myosin light chain bound to IQ domains (Terrak et al. 2003). The canonical sequence (IQxxxRGxxxR) of the IQ domain of both skeletal and cardiac muscle NaChs is missing the conserved glycine (replaced by an R) and the conserved arginine at the end (replaced by a Q). The muscle IQ sequences are very similar to an IQ sequence of myosin that causes myosin light chain to adopt an extended conformation, with the C lobe binding to the IQ domain and the N lobe in a position that is away from and not bound to the IQ domain (Terrak et al. 2003). Therefore, we propose that CaM is in an extended conformation in which the N lobe of CaM binds to a different domain (the N lobe binding domain, NLBD). The NLBD could be another part of the carboxyl terminal of the sodium channel, another cytoplasmic domain of the NaCh, or another protein associated with the NaCh. Increased Ca2+ or CaM-binding peptides cause a conformational change in CaM and a shift in CaM binding that is likely to be different for Ca2+ versus the CaM-binding peptides. When Ca2+ is elevated, this CaM-binding shift could be a movement of the N lobe from the NLBD to the distal IQ (containing the leucine that we mutated to arginine, which blocked the Ca2+ effect). It is unlikely that the N lobe can bind to the IQ domain when CaM-binding peptides are applied because they would interfere with this interaction. This would imply that it is the release from the NLBD that produces shifts in voltage dependence of inactivation. This conformational change in the channel/CaM interaction results in a depolarizing shift in the V1/2 of inactivation of NaV1.4. The binding of the C lobe of CaM to the IQ domain is Ca2+ independent since the CaM34 mutant retains the ability to shift the V1/2 curve. However, depolarizing shifts seen with increased Ca2+ or CIP were greatest with CaM34, indicating that these effects are primarily due to Ca2+ binding to the N lobe of CaM.
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We would have predicted that coexpression of CaMWT (in low Ca2+), CaM1234 and CaM12 would have produced equal shifts in the voltage dependence, but each one had a different effect. ApoCaMWT shifted both activation and inactivation, CaM1234 shifted only activation, and CaM12 shifted neither activation nor inactivation. The differences between apoCaMWT, CaM1234 and CaM12 cannot be simply explained since they should ideally all bind if apoCaMWT is able to bind to the channel. Since NMR studies have shown considerable flexibility of CaM in solution (Chou et al. 2001) we speculate that the mutations in the N lobe and/or C lobe to lower Ca2+ affinity produce CaMs that are not structurally equivalent to apoCaMWT or to each other.
Based on our assumption that CaM is in an extended conformation, we anticipated an effect of peptides that are CaM binding sites in CaM kinase II and myosin light chain kinase. Nevertheless, the close similarity to the effects of elevated Ca2+ was remarkable. The effects of these peptides raise the possibility that CaM might coordinate a complex of the NaCh with other CaM-binding proteins such as CKII. This would place CKII in a position that could allow rapid control of phosphorylation of the channel.
Two point mutations in the IQ domain of the NaV1.4 sodium channel altered the effects of CaM. A mutation of I to E at the IQ of this domain (I1727) abolished all effects of CaM. This is a highly conserved amino acid (hence the name of the motif) and this region has been shown to be essential for binding of CaM to the IQ motif (Deschenes et al. 2002; Herzog et al. 2003). The second point mutation occurred in a distal part of the IQ motif and at a position that tends to be hydrophobic but is not strictly conserved (L1736). The L to R mutation retained the shift in voltage dependence with CaM coexpression, but the responses to Ca2+ and the CaM-binding peptide CIP were abolished. The conformations of CaM and of the NaCh at the IQ domain and the surrounding domains are not known, but we speculate on the CaM conformation based on recent crystal structure results with myosin light chain (MLC), a molecule that binds to repeated IQ domains in the myosin heavy chain and has a structure similar to CaM (Terrak et al. 2003). An IQ motif with the L substituted by a positively charged amino acid (R or K) is also associated with MLC binding in an extended conformation (Terrak et al. 2003), suggesting that the mutation L1736R could prevent the N lobe from binding to the IQ domain when Ca2+ is elevated (see Fig. 9).
Comparisons between the native and chimeric channels made by switching the C-termini of NaV1.4 and NaV1.5 (Fig. 6) showed that the effects of CaM on steady-state inactivation were determined primarily by the C-terminal donor, while activation properties of the channels were more complex. For example, activation of the NaV1.4/CT1.5 chimera was shifted by Ca2+ and CIP but neither native channel showed this shift. Activation of NaV1.5 was selectively affected by KN93, further strengthening the argument that activation and inactivation are separable processes and that CaM modulates these channel properties through different pathways. These results highlight the fact that activation and inactivation are not confined solely to the S4 loop (activation) and the inactivation loop between domains III and IV (Goldin, 2003). Our data suggest that the C-terminal region of the channel is the cytoplasmic domain necessary for the interaction of CaM with the NaCh and for its effects on steady-state inactivation. However, CaM affects both the voltage dependence of activation and steady-state inactivation (Fig. 2), despite the fact that it is binding primarily in the carboxyl tail. This suggests that it may also bind other cytoplasmic domains or modify the interaction of the C-terminal with the other cytoplasmic regions.
Three recent papers have reported functional effects of CaM coexpressed with NaChs (Deschenes et al. 2002; Tan et al. 2002; Herzog et al. 20
, n = 12); Nav1.4 + CaM (
, n = 7). B and C, relative abundance of mRNA of each of the four NaCh ß-subunits, expressed as the number of copies relative to 1000 copies of HPRT (an internal control) in HEK and CHO cells, respectively. Note the logarithmic scale.
, n = 13) or CIP (
, n = 6) included in the pipette. Inset shows one example of current traces used to generate the I–V curve. B, voltage dependence of steady-state activation and inactivation. Steady-state inactivation was measured by a two-pulse protocol with 100 ms conditioning pulses from –150 to +20 mV followed by a 30 ms test pulse to –10 mV. The data were fitted using a Boltzmann distribution. There was no shift in either activation or inactivation with 10 µM Ca2+ (
, n = 11), 30 µM CaM (
, n = 4), or 50 µM CaM (
, n = 13) or CaM1234 (
). For the L1736R mutation, V1/2 of activation with CaMWT was significantly shifted compared with L1736R alone, but not different from native NaV1.4 coexpressed with CaMWT. Ca2+ and CIP caused no significant shift compared with L1736R + CaMWT. F, shifts in the V1/2 of steady-state inactivation for I1727E and L1736R coexpressed with CaMWT, relative to NaV1.4. Data for coexpression of native NaV1.4 coexpressed with CaMWT were replotted from 
, n = 5). KN92 was unchanged from CaMWT alone. E, shifts in the V1/2 of activation for NaV1.5 relative to CaMWT coexpression. Coexpression with CaMWT caused a significant hyperpolarizing shift. Ca2+, CBP and CIP had no significant effect compared with CaMWT alone. 10 µM KN93 caused a further hyperpolarizing shift compared with CaMWT (
). F, shifts in the V1/2 of steady-state inactivation for NaV1.5 relative to CaMWT coexpression. Coexpression with CaMWT had no significant effect on NaV1.5. CaM modulators had no significant effect compared with CaMWT alone. KN93 (10 µM) caused a hyperpolarizing shift compared with CaMWT coexpression alone (
).
