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Originally published In Press as doi:10.1074/jbc.M605060200 on July 17, 2006

J. Biol. Chem., Vol. 281, Issue 36, 25875-25881, September 8, 2006
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Isoform-specific Effects of the beta2 Subunit on Voltage-gated Sodium Channel Gating*

Daniel Johnson1 and Eric S. Bennett2

From the Department of Molecular Pharmacology & Physiology and Programs in Cardiovascular Sciences and Neuroscience, University of South Florida College of Medicine, Tampa, Florida 33612

Received for publication, May 26, 2006 , and in revised form, July 14, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Voltage-gated sodium channels (Nav) are complex glycoproteins comprised of an {alpha} subunit and often one to several beta subunits. We have shown that sialic acid residues linked to Nav {alpha} and beta1 subunits alter channel gating. To determine whether beta2-linked sialic acids similarly impact Nav gating, we co-expressed beta2 with Nav1.5 or Nav1.2 in Pro5 (complete sialylation) and in Lec2 (essentially no sialylation) cells. beta2 sialic acids caused a significant hyperpolarizing shift in Nav1.5 voltage-dependent gating, thus describing for the first time an effect of beta2 on Nav1.5 gating. In contrast, beta2 caused a sialic acid-independent depolarizing shift in Nav1.2 gating. A deglycosylated mutant, beta2-{Delta}N, had no effect on Nav1.5 gating, indicating further the impact of beta2 N-linked sialic acids on Nav1.5 gating. Conversely, beta2-{Delta}N modulated Nav1.2 gating virtually identically to beta2, confirming that beta2 N-linked sugars have no impact on Nav1.2 gating. Thus, beta2 modulates Nav gating through multiple mechanisms possibly determined by the associated {alpha} subunit. beta1 and beta2 were expressed together with Nav1.5 or Nav1.2 in Pro5 and Lec2 cells. Together beta1 and beta2 produced a significantly larger sialic acid-dependent hyperpolarizing shift in Nav1.5 gating. Under fully sialylating conditions, the Nav1.2·beta1·beta2 complex behaved like Nav1.2 alone. When sialylation was reduced, only the sialic acid-independent depolarizing effects of beta2 on Nav1.2 gating were apparent. Thus, the varied effects of beta1 and beta2 on Nav1.5 and Nav1.2 gating are apparently synergistic and highlight the complex manner, through subunit- and sugar-dependent mechanisms, by which Nav activity is modulated.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The importance of voltage-gated sodium channels (Nav)3 in action potential initiation and propagation is well established. Orchestrated Nav activation and inactivation is vital to normal neuronal signaling, skeletal muscle contraction, and regular heart rhythms. Even small syncopations from this normal gating rhythm may alter cellular excitability and whole animal physiology significantly, leading to such disorders as long QT syndrome and epilepsy (1–5).

Nav are complex transmembrane glycoproteins that are composed of a large {alpha} subunit that forms a pore through which ions can pass (6–8). Ten {alpha} subunit isoforms have been cloned from excitable tissues, with orthologues present in a wide range of species (9). Although the {alpha} subunit is sufficient to form functional channels when expressed alone, it is often associated with at least one auxiliary subunit, beta1–beta4, that modulates channel activity (10–13). The mechanism(s) by which the different auxiliary subunits act to modulate channel function is still under investigation (14–16).

Sodium channel {alpha} and beta subunits are typically extensively glycosylated, with up to 30% of the total mass of the channel estimated to be carbohydrate (17–19). Glycosylation structures are often capped with sialic acids (SA), which carry a negative charge at physiological pH. SA attached directly to Nav subunit N-glycosylation structures were shown to be important modulators of Nav gating (20–26). For example, the enzymatic removal of SA or the entire glycosylation structure, from purified, transfected, or endogenous Nav shifted channel gating in the depolarized direction (20, 23–26). Additionally, Nav gated at more depolarized potentials when expressed in a mutant Chinese hamster ovary cell line that is deficient in its ability to sialylate proteins (20, 21). Our recent work demonstrated that N-linked SA have an important role in beta1-dependent modulation of three different Nav {alpha} subunit isoforms (22).

The beta2 subunit is predicted to have a single transmembrane-spanning domain, a small intracellular domain, and a large extracellular N-terminal end that contains three potential N-glycosylation sites and an immunoglobulin-like fold that shows similarity to the neural cell adhesion molecule, contactin (11, 27). The external portion of beta2 is responsible for efficient association with the {alpha} subunit via disulfide bonds (28, 29). beta2 is expressed in the central nervous system and in cardiac tissue (11, 30–32). In general, beta2 caused a depolarizing shift in the voltage dependence of activation and/or inactivation of Nav1.2 and Nav1.8 but was reported to not affect the channel properties of Nav1.3 or Nav1.5 (14, 15, 32, 33). The loss of beta2 resulted in hyperpolarizing shifts in the voltage dependence of inactivation as well as significant decreases in sodium current density in acutely dissociated hippocampal neurons isolated from mice lacking functional beta2 (34). Thus, beta2 is an important modulator of several Nav isoforms expressed in cardiac and neuronal tissues.

Here, we wished to determine whether beta2 SA are involved in modulating Nav gating. We also wished to determine whether the SA-dependent effects of beta2, if any, were additive with those recently observed for beta1 sialic acids (22). To this end, we expressed two different Nav {alpha} subunit isoforms, Nav1.2 and Nav1.5 (central nervous system and cardiac isoforms, respectively) in the presence or absence of beta2 in two Chinese hamster ovary cell lines that differ in their ability to sialylate proteins (35–37). The Pro5 cell line allows normal Chinese hamster ovary cell sialylation, whereas Lec2 cells, which are deficient in the CMP-sialic acid transporter, produce proteins that are essentially nonsialylated and thus can act as a model for an inherited disorder resulting in glycoproteins with deficient levels of sialic acids attached, carbohydrate-deficient glycoprotein syndrome (CDGS), type IIf (38).

Our data indicate that beta2 modulates Nav gating through SA-dependent and SA-independent mechanisms; the exact mechanism is determined by the {alpha} subunit with which it associates. In addition, we show that the SA-dependent effects of beta1 on a specific {alpha} subunit are apparently additive with the SA-dependent and SA-independent effects of beta2 on that {alpha} subunit.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Clones—Nav1.2, Nav1.5, and green fluorescent protein have been described previously (22). The hbeta1 and hbeta2 cDNAs were subcloned into the bicistronic vectors, pIRES-DsRed2 and pIRES2-EGFP, respectively. This was done to verify the expression of each beta subunit through fluorescence. The beta2 subunit was mutated similarly to that described previously for beta1 subunit mutagenesis (22). Specifically, each asparagine that initiates a potential N-glycosylation site (Asn42, Asn66, and Asn74) was changed to a glutamine residue to create the deglycosylated mutant, beta2-{Delta}N.

Mammalian Cell Culture and Transfection—Pro5 and Lec2 cells were grown as previously described (21, 22). Transfections involving only {alpha} and beta1 were carried out as previously described (22). Transfections involving {alpha} and beta2 were performed using 2.2 µg of DNA (91% {alpha} sodium channel, 9% hbeta2 vector cDNAs). Triple transfections of {alpha}, beta1, and beta2 were performed using a total of 2.5 µg of DNA (80% {alpha} sodium channel, 12% hbeta1, and 8% hbeta2 vector cDNAs). The experiments were performed 72 h post-transfection.

Whole Cell Recording and Data Analysis—INa were recorded at room temperature (~22 °C) using established whole cell patch clamp techniques, pulse protocols, data analyses, and solutions as previously described (20–22). Although series resistance was compensated 95–98% for all data, the smaller current produced using the low sodium solutions further minimized any remaining series resistance error, resulting in <1 mV error. All of the data shown are recorded at least 5 min after attaining whole cell configuration to ensure complete dialysis of the intracellular solution. All of the solutions were filtered using Gelman 0.2-µm filters immediately prior to use.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
beta2 Has Opposing Effects on Gating of two Nav {alpha} Subunit Isoforms—To determine whether modulation of Nav1.2 and/or Nav1.5 by beta2 is dependent on the presence of beta2 sialic acids, Nav1.2 and Nav1.5 were individually co-expressed with beta2 in the fully sialylating Pro5 and essentially nonsialylating Lec2 cell lines.

As shown in Fig. 1, co-expression of beta2 with Nav1.5 in the fully sialylating Pro5 cell line caused a generalized hyperpolarizing shift in voltage-dependent gating. Specifically, steady state activation (Fig. 1A) and the kinetics of fast inactivation (Fig. 1C) and recovery from fast inactivation (Fig. 1D) all were shifted similarly (~8 mV) to the left along the voltage axis. Note that beta2 had no significant effect on steady state inactivation (Fig. 1B). When beta2 and Nav1.5 were co-expressed in the essentially nonsialylating Lec2 cell line, beta2 had no measurable effect on Nav1.5 gating. That is, all steady state and kinetic gating characteristics measured for the nonsialylated Nav1.5·beta2 complex were nearly identical to gating characteristics of Nav1.5 alone with or without attached sialic acids (Fig. 1). These data suggest that all gating effects of beta2 on Nav1.5 can be assigned to beta2 sialic acids and represent, to our knowledge, the first time that a gating effect of beta2 on Nav1.5 was observed.

Similar questions were asked for the effect of beta2 on Nav1.2 gating, with very different results. beta2 caused a uniform depolarizing shift (~7 mV) in all four measured gating characteristics (Fig. 2). Gating of the Nav1.2·beta2 complex was nearly identical as expressed in Pro5 or in Lec2 cells, indicating that the beta2-induced depolarizing shift in Nav1.2 gating was not dependent on beta2 sialic acids.

Thus, beta2 had opposing effects on the gating of two different Nav {alpha} subunits. There were only modest and inconsistent effects of beta2 on the slope factors for steady state activation or inactivation relationships (Ka and Ki, respectively), indicating that the beta2 has little to no impact on the effective gating valence for Nav1.2 or Nav1.5. In addition, we did not observe a significant effect on peak conductance values in any condition except when Nav1.5 was expressed with beta2 alone. Gating parameters measured in this study are detailed in Fig. 6 and in Table 1.


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  		TABLE 1
The measured gating parameters for {alpha} ± beta1 ± beta2 ± sialic acid

The data are the mean parameter values ± S.E. The parameters were determined as described previously (22). See Fig. 6 for the definitions of the parameters. For {tau}h, the data were measured at –40 mV for Nav1.5 and at –30 mV for Nav1.2. Significance was tested using a two-tailed Student's t test. The parameter measured for the fully sialylated Nav complex was compared with the parameter for the fully sialylated {alpha} subunit alone.

 
N-linked Sialic Acids Attached to beta2 Fully Account for Effects of beta2 on Nav1.5 Gating but Have No Impact on Nav1.2 Gating—To determine whether the SA-dependent effects of beta2 on Nav1.5 gating were limited to N-linked sialic acids, a deglycosylated mutant beta2 was constructed in which each asparagine residue that initiates an N-glycosylation consensus sequence was mutated to a glutamine residue (beta2-{Delta}N). We co-expressed beta2-{Delta}N with Nav1.5 or Nav1.2 in the fully sialylating Pro5 cells and compared channel gating to the {alpha} subunit alone and to the {alpha}·beta2 channel complex (Fig. 3). Note that the deglycosylated beta2-{Delta}N had no effect on Nav1.5 gating (3A). The data shown in Fig. 3B show that beta2-{Delta}N and wild type beta2 modulate Nav1.2 gating nearly identically and thereby suggest that beta2-{Delta}N likely still interacts with Nav {alpha} subunits. Together, these data support the conclusion that N-linked sialic acids fully account for the gating effects of beta2 on Nav1.5, whereas beta2 N-linked sugars are not involved in the effects of beta2 on Nav1.2 gating.

The Effects of beta1 and beta2 on Nav Gating Are Apparently Additive—Sodium channel {alpha} subunits often associate with more than one auxiliary subunit. For example, Nav1.2, beta1, and beta2 are expressed at the same time in granule cells of the cerebellum (for reviews see Refs. 9 and 39). Previous work from our laboratory demonstrated that beta1 induced similar, uniform, SA-dependent hyperpolarizing shifts in Nav1.2 and Nav1.5 gating (22). To determine whether the SA-dependent effects of beta1 were apparently additive with the {alpha} subunit-specific, SA-dependent and SA-independent effects of beta2, {alpha} (Nav1.2 or Nav1.5) ± beta1 ± beta2 subunits were expressed in Pro5 and in Lec2 cells.


Figure 1
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  		FIGURE 1.
beta2 causes a sialic acid-dependent hyperpolarizing shift in Nav1. 5 gating. Filled symbols, Pro5 cells (+SA); open symbols, Lec2 cells (–SA); circles, Nav1.5 alone; squares, Nav1.5 + beta2. n = 8–13 for each condition tested (Table 1). A, conductance-voltage relationships for Nav1.5 ± beta2 ± SA. The data are the mean normalized peak conductance ± S.E. at a membrane potential, and the curves are single Boltzmann fits to the data. Inset, typical whole Pro5 cell Nav1.5 + beta2 current traces measured during 10-ms step depolarizations. B, steady state inactivation (hinf) curves for Nav1.5 ± beta2 ± SA. The data are the mean normalized peak currents ± S.E. measured during a maximally depolarizing test pulse following a 500-ms prepulse to the plotted potentials. Inset, typical whole Pro5 cell currents measured during a 5-ms test pulse to +60 mV following a 500-ms prepulse to increasing depolarizations. C, time constants of fast inactivation ({tau}h) for Nav1.5 ± beta2 ± SA. Data are the mean ± S.E. Lines are non-theoretical point-to-point. Inset, representative whole Pro5 cell Na+ current traces measured during a –30-mV test pulse. Note the rate at which the current attenuates (inactivates) is faster in the presence of beta2 (under fully sialylating conditions). Peak current amplitudes were normalized, resulting in a 1.55-fold amplification of the Nav 1.5 current trace. D, time constants of recovery from fast inactivation ({tau}rec) for Nav1.5 ± beta2 ± SA. The data are the {tau}rec ± S.E. measured for each condition at three recovery potentials. The lines are nontheoretical point-to-point. Inset, typical plot of the fractional recovery measured following –120-mV recovery pulses of various durations for Nav1.5 ± beta2 expressed in Pro5 cells. The data are the means ± S.E. fractional current (I/I0) measured during a second depolarizing test pulse following the plotted interval at –120 mV from the original depolarizing test pulse. The lines are exponential fits to the data from which the {tau}rec were determined.

 
Co-expression of Nav1.5 with beta1 and beta2 in Pro5 cells caused a generalized hyperpolarizing shift (~15 mV) in Nav1.5 gating significantly greater than that induced by beta1 or beta2 alone (Fig. 4, A, C, and D). However, under fully sialylating conditions, steady state inactivation curves (Fig. 4B) were shifted negatively to potentials similar to those observed for the Nav1.5·beta1 complex (by ~9 mV), consistent with the previous data indicating that beta2 had no measurable effect on Nav1.5 channel availability (Fig. 1B). Gating of all channel complexes studied (i.e. Nav1.5 alone, Nav1.5·beta1, Nav1.5·beta2, and Nav1.5·beta1·beta2) behaved similarly under conditions of reduced sialylation. That is, neither beta1 nor beta2 modulated Nav1.5 gating in the absence of sialic acids regardless of the combination of subunits. Thus, the gating effects of beta1 and beta2 on Nav1.5 are SA-dependent and apparently additive and independent. The measured gating parameters for Nav1.5 ± beta1 ± beta2 ± SA are represented graphically in Fig. 6 and listed in Table 1. Note the apparent additive effects of beta1 and beta2 sialic acids on these values. Further, note that under conditions of reduced sialylation, there are no significant differences in gating parameter values among the channel complexes studied.

Steady state and kinetic gating of Nav1.2 co-expressed with beta1 and beta2 in Pro5 cells were similar to gating of Nav1.2 alone (Fig. 5). In the presence of full sialylation, the opposing effects of the two beta subunits on Nav1.2 gating essentially balanced one another, resulting in an apparent trimeric channel complex that gates similarly to that of the {alpha} subunit alone. When sialylation was reduced, the beta1 subunit no longer impacted Nav1.2 gating, whereas the effect of beta2 was left intact. Thus, the nonsialylated Nav1.2·beta1·beta2 channel complex gated at more depolarized potentials. The calculated gating parameter values for Nav1.2 ± beta1 ± beta2 ± SA are shown in Fig. 6 and listed in Table 1. Note the consistent (among gating parameters) spectrum of effects that are dependent on the combination of beta subunits and sialylation conditions.

The data shown in Figs. 4, 5, 6 demonstrate that modulation of different Nav {alpha} subunit isoforms by multiple beta subunits in conditions of varied post-translational modification is a complex process that results in multiple functional phenotypes depending on the combination of subunits expressed by the cell and the degree to which each subunit is glycosylated/sialylated.


Figure 2
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  		FIGURE 2.
beta2 causes a uniform sialic acid-independent depolarizing shift in Nav1. 2 gating. All of the data were acquired as described in the legend to Fig. 1. Filled symbols, Pro5 cells (+SA); open symbols, Lec2 cells (–SA); circles, Nav1.2; squares, Nav1.2 + beta2. n = 9–12 for each condition tested (Table 1). A, conductance-voltage relationships for Nav1.2 ± beta2 ± SA. B, steady state availability (hinf) curves for Nav1.2 ± beta2 ± SA. C, time constants of fast inactivation ({tau}h) for Nav1.2 ± beta2 ± SA. D, {tau}rec values for Nav1.2 ± beta2 ± SA.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
beta2 Has Multiple Mechanisms of Action That Are Dependent on the {alpha} Subunit with Which It Associates—Previous work has demonstrated that beta1 has essentially uniform SA-dependent effects on the gating of several Nav isoforms, including the adult brain (Nav1.2) and cardiac (Nav1.5) isoforms (22). Here we show that beta2, unlike beta1, has opposing effects on the gating of Nav1.2 and Nav1.5. Two different mechanisms can explain these opposing isoform-specific effects, an SA-dependent and an SA-independent mechanism.

Co-expression of beta2 induced a depolarizing shift in Nav1.2 gating (Fig. 2). This effect is in general agreement with the observations of Chen and co-workers (34), who demonstrated that the voltage dependence of inactivation for INa of hippocampal neurons isolated from mice lacking beta2 was shifted in the negative direction compared with mice expressing beta2 (34). Studies of Nav1.2·beta2 expressed in HEK cells demonstrated that beta2 could positively shift the voltage dependence of activation and inactivation (14). In addition, here we show that beta2 slows the rate of inactivation and increases the rate of recovery from fast inactivation. That is, we find that beta2 caused all measured voltage-dependent gating parameters for Nav1.2 to shift in the depolarized direction essentially along the voltage axis.

When sialylation or N-glycosylation was prevented, beta2 still modulated Nav1.2 gating nearly identically to its effect under fully sialylated conditions (Figs. 2 and 3). Thus, the effects of beta2 on the gating of Nav1.2 appear to be entirely SA-independent (in this cellular system). These effects are opposite to those we previously described for beta1 modulation of Nav1.2 in this system. We showed that beta1 shifted the gating parameters of Nav1.2 in the hyperpolarized direction and was entirely dependent on beta1 sialic acids (22). Together these studies demonstrate the specific and alternative modes of action of two different auxiliary subunits on gating of a specific Nav {alpha} subunit.


Figure 3
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  		FIGURE 3.
Deglycosylated beta2-{Delta}N no longer alters Nav1. 5 gating but still imposes a depolarizing shift in Nav1.2 gating. Conductance-voltage relationships for Nav1.5 or Nav1.2 ± beta2 or ± beta2-{Delta}N. The data are the mean conductance values ± S.E. at a membrane potential measured as expressed in fully sialylating Pro5 cells. The curves are single Boltzmann distribution fits to the data. A, deglycosylated beta2-{Delta}N has no effect on Nav1.5 gating. Circles, Nav1.5 (n = 8); squares, Nav1.5 +beta2 (n = 9); open triangles, Nav1.5 +beta2-{Delta}N (n = 4). B, the effect of deglycosylated beta2-{Delta}N on Nav1.2 gating mimics effect of beta2 on gating. Circles, Nav1.2 (n = 9); squares, Nav1.2 + beta2 (n = 12); open triangles, Nav1.2 + beta2-{Delta}N (n = 8).

 
In contrast, beta2 caused a generalized hyperpolarizing shift in the voltage dependence of Nav1.5 gating but had no significant effect on steady state inactivation (Fig. 1). This effect was entirely SA-dependent, because beta2 could not modulate Nav1.5 gating in the absence of sialic acids.

Although the effect on steady state inactivation was not statistically significant, it may be a real, albeit small, SA-dependent shift in half-inactivation voltage. The reasons for this possibility include: 1) all of the other parameters measured are shifted to more negative potentials by sialic acids and 2) the mean value of the half-inactivation voltage for Nav1.5+beta2 does shift by ~4 mV in the negative direction.

Next, we sought to question whether N-linked sialic acids were fully responsible for this novel effect of beta2 on Nav1.5. As shown in Fig. 3, a mutant beta2 in which all potential N-glycosylation sites were removed, beta2-{Delta}N, had no effect on Nav1.5 gating, indicating that N-linked sialic acids attached to beta2 were fully responsible for the effect of beta2 on Nav1.5 gating. beta2-{Delta}N imposed the same depolarizing effect on Nav1.2 gating as observed for wild type beta2, indicating that beta2 N-linked sugars had no effect on Nav1.2 gating and that deglycosylation did not prevent association of beta2 with the {alpha} subunits.


Figure 4
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  		FIGURE 4.
The sialic acid-dependent effects ofbeta1 andbeta2 on Nav1. 5 gating are apparently additive. All of the data were acquired as described in the legend to Fig. 1. Filled symbols, Pro5 cells (+SA); open symbols, Lec2 cells (–SA); circles, Nav1.5; squares, Nav1.5 + beta2; diamonds, Nav1.5 + beta1; triangles, Nav1.5 + beta1 + beta2. n = 9–12 for each condition tested (Table 1). A, conductance-voltage relationships for Nav1.5 ± beta1 ± beta2 ± SA. Note the additional hyperpolarizing shift in the conductance-voltage curve when Nav1.5 is expressed with both beta1 and beta2 in Pro5 cells. B, channel availability curves for Nav1.5 ± beta1 ± beta2 ± SA. Note only the presence of beta1 sialic acids are sufficient to shift the curve. C, {tau}h for Nav1.5 ± beta1 ± beta2 ± SA. Note inactivation is fastest when both beta1 and beta2 sialic acids are present. D, {tau}rec values for Nav1.5 ± beta1 ± beta2 ± SA. Note recovery is slowest in the presence of both beta1 and beta2 sialic acids.

 
To our knowledge this is the first report describing a significant functional effect of beta2 on Nav1.5 gating. beta2 is expressed in the heart, and recent work has shown that Nav1.5 and beta2 colocalize to the intercalated disks of ventricular myocytes (32, 40). In addition, Nav1.5 is also expressed in both fetal and adult brain and could potentially be co-expressed with beta2 (41). Thus, control of beta2 expression and sialylation may provide a means for modulating Nav1.5 gating in the heart and brain.

Another important and novel finding is that beta2 differentially modulates the gating of two Nav {alpha} subunit isoforms. That is, modulation of Nav gating by beta2 appears to be determined by the {alpha} subunit with which it associates. Nav1.5 is modulated only in the presence of beta2 sialic acids, whereas modulation of Nav1.2 by beta2 is SA-independent. This suggests that beta2 has at least two different mechanisms of action: one that involves sialic acids and one that does not.

Co-expression of beta1 and beta2 Provides Multiple Mechanisms for Modulating Nav Function—We report here that the beta2 subunit has varied effects on two Nav {alpha} subunits, causing an SA-dependent hyperpolarizing shift in Nav1.5 gating and an SA-independent depolarizing effect on Nav1.2 gating. Previously, we showed that beta1 shifted all tested gating parameters for Nav1.5 and Nav1.2 in the hyperpolarized direction in an SA-dependent manner (22). Nav {alpha} subunits might be associated with both auxiliary subunits. If so, what is the impact on Nav gating? Are the SA-dependent effects of beta1 and beta2 on Nav1.5 independent? Are the SA-dependent and -independent effects additive?


Figure 5
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  		FIGURE 5.
The beta1 sialic acid-dependent and the beta2 sialic acid-independent effects on Nav1. 2 gating are also apparently additive. All of the data were acquired as described in the legend to Fig. 1. Filled symbols, Pro5 cells (+SA); open symbols, Lec2 cells (–SA); circles, Nav1.2; squares, Nav1.2 + beta2; diamonds, Nav1.2 + beta1; triangles, Nav1.2 + beta1 + beta2. n = 8–13 for each condition tested (Table 1). A, conductance-voltage relationships for Nav1.2 ± beta1 ± beta2 ± SA. Note in the presence of sialic acids, beta1 shifts the conductance-voltage curve for Nav1.2 leftwards; in the absence of SA the depolarizing effects of beta2 dominate. B, channel availability curves for Nav1.2 ± beta1 ± beta2 ± SA. Note that in the presence of sialic acids beta1 shifts the hinf curve for Nav1.2 leftwards; in the absence of SA the depolarizing effects ofbeta2 dominate. C, {tau}h for Nav1.2 ± beta1 ± beta2 ± SA. Note the presence of beta1 SA negates the effects of beta2 on inactivation kinetics. D, {tau}rec values for Nav1.2 ± beta1 ± beta2 ± SA. Note beta1 negates the beta2-induced faster recovery, but only when SA are present.

 
As shown in Figs. 5 and 6 and in Table 1, co-expression of beta1 and beta2 with Nav1.2 indicated that the gating effects of the beta subunits are apparently additive and independent. That is, the depolarizing influence of beta2 essentially negated the hyperpolarizing influence of beta1 under fully sialylating conditions. Thus, the measured gating behavior of Nav1.2 alone was nearly identical to the fully sialylated Nav1.2·beta1·beta2 complex. However, under conditions of reduced sialylation, only SA-independent beta2 effects were observed. The data indicate that the effects of beta1 and beta2 on Nav1.2 are independent and, indeed, apparently additive.

Interestingly, the same was true for beta subunit effects on Nav1.5, even though each beta subunit conferred SA-dependent effects. That is, channel gating (except steady state inactivation) shifted to even greater hyperpolarized potentials (shifted ~15 mV) when beta1 and beta2 were co-expressed with Nav1.5 under fully sialylated conditions than when only one beta subunit was present (shifted ~8–9 mV). When sialylation was reduced, Nav1.5 gating was unaffected by expression of the beta subunits either alone or in combination (Figs. 4 and 6 and Table 1). Thus, these data suggest the SA-dependent effects of both beta subunits on Nav1.5 gating are apparently additive and likely independent.


Figure 6
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  		FIGURE 6.
Measured voltage-dependent gating parameters support the diverse and apparently additive effects of beta1 and beta2 on Nav1. 2 versus Nav1.5 gating. The measured parameter values ± S.E. are plotted as bar graphs for Nav1.5 (left graphs) or Nav1.2 (right graphs) ± beta1 ± beta2 ± SA. The parameter values were determined as described previously (22). The parameters are: Va, half-activation voltage (in mV); Vi, half-inactivation voltage (in mV); {tau}h, time constant of fast inactivation (in ms); {tau}rec, time constant for recovery from fast inactivation (in ms). The bars are demarcated as follows: in Pro5 (+SA; solid bars) and Lec2 (–SA; open bars) cells. {alpha} alone (no cross-hatching); +beta1 (x cross-hatching); +beta2 (\ cross-hatching); +beta1 + beta2 (x cross-hatching). Solid horizontal lines represent the mean parameter values measured for Nav1.5 + SA and Nav1.2 + SA. Significance was tested using a two-tailed Student's t test, comparing the test condition to the values measured for the fully sialylated {alpha} subunit alone (p ≤ 0.05); significant differences are demarcated with an asterisk. Sialic acid dependence is demarcated with a + and represents a significant difference in gating parameter between the fully sialylated (in Pro5 cells) and nonsialylated (in Lec2 cells) channel complex. (p ≤ 0.05).

 
Could Less Sialylated Nav Partially Account for Symptoms Exhibited by Those Suffering from Carbohydrate-deficient Glycoprotein Syndrome?—Patients with CDGS present with severe developmental delay, neurological abnormalities, and failure to thrive. Surviving adults suffer from progressive muscle atrophy (42). Currently, there are 20 documented forms of CDGS, each caused by dysfunction of one gene product involved in N-linked (16 of 20) or O-linked protein glycosylation (43, 44). In addition to CDGS, there are other disorders whose pathology is linked to aberrant glycosylation such as several forms of congenital muscular dystrophy (43–47). Nearly all CDGS impact multiple organ systems, with prominent effects on neuronal, muscular, and cardiovascular systems. Although the exact mutated glycosylation structure varies from one CDGS to another, all CDGS victims suffer from reduced protein sialylation. Because many of the observed phenotypes suggest sluggish or reduced neuromuscular and/or cardiovascular activity, it is intriguing to consider that the lowered SA levels attached to the glycoproteins of the patient may be responsible for changes in ion channel activity, which might contribute to symptoms. For example, consistent with our data here and described previously (22, 23), perhaps cardiac Nav function in CDGS patients is compromised because of the reduced levels of sialic acids attached to the {alpha}, beta1, and beta2 subunits expressed in the heart. Such studies will be the focus of future investigations.

Summary—Taken together these data suggest an increasingly complex mechanism by which voltage-gated sodium channels, and thereby cardiac, skeletal muscle, and neuronal action potentials, can be regulated. The SA attached to a specific Nav {alpha} subunit can differentially impact channel gating (21–23). Co-expression of the beta1 subunit uniformly shifts Nav1.2 and Nav1.5 gating, but only in the presence of SA. Co-expression of beta2 alone causes an SA-dependent shift in Nav1.5 gating but an SA-independent shift in Nav1.2 gating. The data indicate that the varied SA-dependent and SA-independent effects of beta subunits on two different Nav {alpha} subunits are apparently independent and additive. The results indicate for the first time that Nav activity can be modulated by altering the functional Nav subunit composition coupled with varying levels of subunit glycosylation/sialylation. The implications from this study are potentially far-reaching given the diversity and ubiquitous presence of ion channel glycosylation and the vital role of ion channels in cellular function.


   FOOTNOTES
 
* This work was supported in part by NIAMS, National Institutes of Health Grant R-01AR45169 (to E. S. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 Present address: Faculty of Life Sciences, University of Manchester, 2nd Floor, Core Technology Facility, 46 Grafton St., Manchester, M13 9NT, UK. Back

2 To whom correspondence should be addressed: Dept. of Molecular Pharmacology and Physiology, University of South Florida, College of Medicine, MDC 8, Tampa, FL 33612. Tel.: 813-974-1545; Fax: 813-974-3079; E-mail: esbennet{at}hsc.usf.edu.

3 The abbreviations used are: Nav, voltage-gated sodium channel(s); SA, sialic acid(s); CDGS, carbohydrate-deficient glycoprotein syndrome. Back


   ACKNOWLEDGMENTS
 
We acknowledge the technical expertise of Jean Harper.



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 EXPERIMENTAL PROCEDURES
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 DISCUSSION
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