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J. Biol. Chem., Vol. 279, Issue 43, 44303-44310, October 22, 2004

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The Sialic Acid Component of the ₁ Subunit Modulates Voltage-gated Sodium Channel Function^*

Daniel Johnson, Marty L. Montpetit, Patrick J. Stocker, and Eric S. Bennett

From the Department of Physiology & Biophysics and Program in Neuroscience, University of South Florida College of Medicine, Tampa, Florida 33612

Received for publication, August 4, 2004 , and in revised form, August 13, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Voltage-gated sodium channels (Na_v) are responsible for initiation and propagation of nerve, skeletal muscle, and cardiac action potentials. Na_v are composed of a pore-forming subunit and often one to several modulating subunits. Previous work showed that terminal sialic acid residues attached to subunits affect channel gating. Here we show that the fully sialylated ₁ subunit induces a uniform, hyperpolarizing shift in steady state and kinetic gating of the cardiac and two neuronal subunit isoforms. Under conditions of reduced sialylation, the ₁-induced gating effect was eliminated. Consistent with this, mutation of ₁ N-glycosylation sites abolished all effects of ₁ on channel gating. Data also suggest an interaction between the cis effect of sialic acids and the trans effect of ₁ sialic acids on channel gating. Thus, ₁ sialic acids had no effect gating on the of the heavily glycosylated skeletal muscle subunit. However, when glycosylation of the skeletal muscle subunit was reduced through chimeragenesis such that sialic acids did not impact gating, ₁ sialic acids caused a significant hyperpolarizing shift in channel gating. Together, the data indicate that ₁ N-linked sialic acids can modulate Na_v gating through an apparent saturating electrostatic mechanism. A model is proposed in which a spectrum of differentially sialylated Na_v can directly modulate channel gating, thereby impacting cardiac, skeletal muscle, and neuronal excitability.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The importance of voltage-gated sodium channels (Na_v)¹ in action potential initiation and propagation is well established. The orchestrated activation and inactivation gating of sodium channels 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 (1–7).

Na_v are complex transmembrane glycoproteins that are comprised of a large subunit that forms the pore through which ions can pass (8–10). Ten subunit isoforms have been cloned from excitable tissues, with orthologues present in a wide range of species (11). Although the subunit is sufficient to form functional channels when expressed alone, it is often associated with a ₁ subunit that modulates sodium channel activity; the exact manner by which ₁ alters channel function is still under investigation (12–15).

Isoforms of the subunit undergo extensive glycosylation. Estimates indicate that 15–40% of the total Na_v molecular weight is carbohydrate (16–18). Approximately 40–45% of the added carbohydrate residues are sialic acid moieties, resulting in the addition of an estimated 100 sialic acid residues per subunit molecule (16, 17). Recent studies have demonstrated that sialic acid is an important modulator of Na_v subunit gating. For example, enzymatically removing sialic acid or the entire glycosylation structure, from purified, transfected, or endogenous Na_v, shifts channel gating in the depolarized direction (19–22). Additionally, Na_v gate at more depolarized potentials when expressed in a mutant CHO cell line that is deficient in its ability to sialylate proteins (20, 23). Interestingly, the effects of sialylation on Na_v subunit gating appear to show some degree of isoform specificity. Gating of one human subunit isoform, Na_v1.4, was dependent on subunit sialic acids, whereas gating of a second isoform, Na_v1.5, was independent of sialic acid as expressed in the same experimental system. The functional sialic acids attached to Na_v1.4 were localized to the DIS5-S6 extracellular loop (23). Together, these studies demonstrate cis-regulation of Na_v gating by subunit sialic acids.

The ₁ subunit is predicted to have a single transmembrane spanning domain with an extracellular N-terminal end that contains four potential N-glycosylation sites and a single immunoglobulin-like fold (24). The small internal portion of the protein appears to be responsible for efficient association of ₁ with the Na_v subunit via ankyrin G (14, 25). The external domain of ₁ has been shown to be critical for correct modulation of sodium channel gating mediated by different Na_v isoforms (26–29). In general, ₁ causes a hyperpolarizing shift in the voltage dependence of inactivation. In several studies, activation gating was also shifted in the hyperpolarized direction by ₁ (3, 13, 25, 26, 28–31). Conversely, ₁ was shown to cause a depolarizing shift in voltage dependence of inactivation for Na_v expressed in HEK cell lines (12, 32, 33). This may be caused by competition between endogenous and heterologous ₁ in this system. Regardless, these differences in the impact of ₁ on channel gating strongly suggest that the cellular milieu has a dramatic effect on the ability of the ₁ subunit to modulate Na_v channel gating.

At least three of the four N-linked glycosylation sites present in the N terminus of ₁ are thought to be glycosylated in the mature protein (24, 34). Could sialic acids attached to ₁ alter subunit gating through a novel trans-regulatory mechanism? Although the ₁ gene product expressed in heart, skeletal muscle, and brain is identical, the level of post-translational modification appears to vary among cell types (35). In addition, expression of ₁ is tightly regulated over the course of development. Depending on the tissue, expression is first observed in the first 4 days after birth and increases to maximal, maintained levels after 2–4 weeks (35–39). Thus, expression of ₁, an important modulator of Na_v gating, is developmentally regulated. Also, ₁ is heavily and differentially glycosylated. If ₁ glycosylation alters subunit gating, then channel gating might be modulated differently by various levels of ₁ sialic acid among excitable tissues and from one developmental stage to another.

Given that subunit sialic acids impact channel gating, we wished to test the hypothesis that sialic acids attached to the ₁ subunit are involved in modulating channel function. To this end, we expressed four different Na_v subunit isoforms in the presence or absence of ₁ in a CHO cell line (Pro5) that essentially fully sialylates proteins, and in a mutant daughter cell line (Lec2) that is unable to sialylate proteins. Our data indicate a novel mechanism by which ₁ can modulate Na_v gating in a saturating, sialic acid-dependent manner and that ₁ sialic acids account for all effects of ₁ on sodium channel gating. In addition, the data indicate for the first time that within a single membrane, transmembrane protein function is modulated by sialic acid residues attached to a second membrane protein.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Vector Construction and Mutagenesis
The cDNA containing the rNa_v1.2 open reading frame (ORF) inserted into pRC-CMV (Invitrogen) was a kind gift of Dr. Alan Goldin. The hNa_v1.7 cDNA ORF was inserted into pcDNA3.1. Expression vectors containing hNa_v1.4 and hNa_v1.5 were as previously described (23). h₁ was subcloned into the bicistronic vector, pIRES2-EGFP (Clontech), to ensure expression of ₁ through visual inspection. The quadruple h₁ mutant (h_1-N) was created using the GeneEditor (Promega) site-directed mutagenesis kit with h₁ subcloned into pBluescript vector (Stratagene) as a template. Each asparagine residue initiating an external N-linked consensus sequence, NX(S/T), was mutated to a serine residue through sequential mutagenesis. The constructs were sequenced to confirm successful mutagenesis and sequence fidelity. h_1-N was then subcloned into pIRES2-EGFP for co-expression experiments. The h₁ and h_1-N ORFs minus their stop codons were amplified using PCR with the following oligonucleotides 5'-TCCGGCCACCTGGACGCCCG-3' and 5'-GCGCAGCACGCGCCGCGCAG-3'. PCR products were subcloned into pcDNA3.1/V5-His TOPO TA expression vector (Invitrogen). Both ORFs were subsequently subcloned into pEGFP-N1 (Clontech) to generate C-terminal, GFP-tagged h₁ and h_1-N constructs.

Mammalian Cell Culture and Transfection
Pro5 and Lec2 cells were grown as described previously (23). For transfections, the cells were seeded onto 35-mm dishes 24 h prior to exposure to 1 ml of Opti-mem medium containing 8 µl of Lipofectamine (Invitrogen) and 2.0–2.5 µg of DNA (85–92% sodium channel vector and 8–15% pGreen Lantern Fluorescent Protein (GFP; Invitrogen) or the ₁ subunit vector) and then incubated for 24 h. The transfection mix was replaced with the appropriate growth medium, and the cells were incubated for a further 48 h before use.

Electrophysiology and Data Analysis
I_Na were recorded at room temperature (22 °C) using established whole cell patch clamp techniques, pulse protocols, data analyses, and solutions as previously described (20, 23). For the Ca²⁺ perfusion studies, the seals were formed in the bath solution containing 2.0 mM Ca²⁺. The cells were first perfused with the 2.0 mM Ca²⁺ bath solution and then with the 0.2 mM Ca²⁺ bath solution to determine directly the shift in V_a with a 10-fold decrease in external Ca²⁺ concentration. 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 a <1-mV error. All of the data shown are recorded at least 5 min after attaining whole cell configuration to assure complete dialysis of the intracellular solution. All of the solutions were filtered using Gelman 0.2-µm filters immediately prior to use.

Pulse Protocols
Conductance-Voltage (G-V) Relationship—The cell was held at –100 or –120 mV. The voltages were stepped to various depolarized potentials (ranging from –100 to +70 mV in 10 mV increments) for 10 ms and then returned to the holding potential. Consecutive pulses were stepped every 1.5 s, and the data were leak-subtracted using the P/4 method, stepping negatively from the holding potential. At each test potential, steady state whole cell conductance was determined by measuring the peak current at that potential and dividing by the driving force (i.e. difference between the membrane potential and the observed reversal potential). The maximum conductance generated by each cell was used to normalize the data for each cell to its maximum conductance by fitting the data to a single Boltzmann distribution (equation 1, solving for maximum conductance). The average V_a± S.E. values listed in Table I were determined from these single Boltzmann distributions. The normalized data were then averaged with those from other cells, and the resultant average conductance-voltage curve was fit via least squares using the following Boltzmann relation.

View this table: [in this window] [in a new window]   TABLE I The measured gating parameters for ± 1 ± sialic acid The data are the mean parameter values ± S.E. For h, the data were measured at -40 mV for Nav1.4, Nav1.5, and hSkM1P1 and at -30 mV for Nav1.2 and Nav1.7. To determine the effects of 1 sialic acids, significance was tested using a two-tailed Student's t test, with each condition compared with the parameter measured for the fully sialylated subunit alone.

(Eq. 1)

where V is the membrane potential, V_a is the voltage of half-activation, and K_a is the slope factor.

Measurement of Inactivation Time Constants—Inactivation time constants were determined by fitting the current traces used to measure G-V relationships. Attenuating currents from 90 to 10% of the peak values were fit to a single exponential function to determine the time course of fast inactivation.

Steady State Inactivation Curves (h_inf)—Voltage dependence of steady state inactivation was determined by first prepulsing the membrane for 500 ms from the holding potential, then stepping to +60 mV for 5 ms, and then returning to the holding potential. The prepulse voltages ranged from –130 mV to +10 mV in 10-mV increments. The currents from each cell were normalized to the maximum current measured by fitting each single cell data to a single Boltzmann distribution (Equation 2, solving for maximum current), from which the mean V_i± S.E. values listed in Table I were determined. The normalized data for many cells were then averaged and fit to Equation 2, from which the average h_inf curves describing steady state inactivation for the channel population were calculated.

(Eq. 2)

Recovery from Inactivation—The cells were stepped to +60 mV for 10 ms from the holding potential and then returned to the recovery potential for varying duration ranging from 1 to 20 ms in 1-ms increments. Following this recovery pulse, the potential was again stepped to +60 mV for 10 ms. The peak current measured during the two +60-mV depolarizations were compared, and the fractional peak current remaining during the second depolarization was plotted as a function of the recovery pulse duration. This represents the fraction of channels that recovered from inactivation during the recovery interval. Time constants of recovery were determined by fitting the data to single exponential functions.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
₁ Modulates Gating of Three of Four Na_v Subunits—The external domain of ₁ is critical for correct modulation of sodium current mediated by different Na_v subunit isoforms (26–29). Reports indicate effects of ₁ ranging from increased fast inactivation rate to hyperpolarizing and even small depolarizing shifts in the voltage dependence of steady state channel gating (3, 12, 13, 31, 32, 33). These varied effects apparently depend on the subunit and the cellular expression system used to study ₁ function. To minimize the variation observed among cellular expression systems, we co-expressed ₁ with one of four different subunits in CHO cells to compare directly subunit isoform-specific effects of ₁ on sodium current. Fig. 1 shows the average Na_v conductance voltage relationships (G-V) recorded from cells expressing one of four Na_v subunits: the adult skeletal muscle isoform (Na_v1.4), the cardiac isoform (Na_v1.5), a peripheral nerve isoform (Na_v1.7), and a brain isoform (Na_v1.2), in the presence or absence of ₁. Note that Na_v1.4 activation is unaffected by ₁, whereas the G-V curves for Na_v1.2, Na_v1.5, and Na_v1.7 are shifted in the hyperpolarized direction by 8–9 mV when ₁ is present. Fig. 2 shows that the voltage dependence of steady state channel availability (h_inf) is similarly affected by ₁, with the voltages of half-inactivation (V_i) for Na_v1.2, Na_v1.5, and Na_v1.7 shifted 6–9 mV in the hyperpolarized direction in the presence of ₁, whereas V_i for Na_v1.4 is unaffected by ₁.

View larger version (28K): [in this window] [in a new window]   FIG. 1. 1 sialic acids cause a hyperpolarizing shift in the peak conductance-voltage relationships for three subunits. Conductance-voltage (G-V) relationships for four voltage-gated sodium channel subunits ± 1 as expressed in two CHO cell lines with varying abilities to sialylate proteins. Expression in Pro5 cells allows normal CHO cell sialylation, whereas expression in Lec2 cells prevents sialylation. The data are the mean normalized peak conductance (G) ± S.E. at a membrane potential, and the curves are fits of the data to single Boltzmann relationships. n = 9–13 for each condition tested. Significant differences among conditions tested throughout are listed in Table I. Subunit schematics demarcate the location and number of N-glycosylation consensus sequences along each subunit. Circles with solid lines, subunit alone; squares with dashed lines, + 1. Filled symbols, in Pro5 cells; open symbols, in Lec2 cells. A, Nav1.4. B, Nav 1.5. C, Nav1.7. D, Nav1.2.

View larger version (21K): [in this window] [in a new window]   FIG. 2. 1 sialic acids cause a similar hyperpolarizing shift in channel availability curves. Steady state channel availability (hinf) curves for the four subunits ± 1 ± sialic acid. The data are the mean normalized peak current () ± S.E. measured during a maximally depolarizing test pulse following a 500-ms prepulse to the plotted potentials. The symbols, lines, sample numbers, and panels are as described in the legend to Fig. 1.

View larger version (22K): [in this window] [in a new window]   FIG. 3. 1 sialic acids increase the rate of fast inactivation. The data are the means ± S.E. time constants for fast inactivation (h) as a function of membrane potential. Lines are nontheoretical, point-to-point. The symbols, lines, sample numbers, and panels are as described in the legend to Fig. 1. Inset to C, representative normalized whole cell Na+ current traces measured at –20 mV for Nav1.7. Note that the rate at which the current attenuates (inactivates) is much faster in the presence of 1 sialic acids, consistent with the observed shift in h along the voltage axis. The scale shown is for Na 1.7 + 1 + SA current traces, to which the other current traces were normalized.

View larger version (21K): [in this window] [in a new window]   FIG. 4. 1 sialic acids slow the rate to recovery from fast inactivation causing a similar negative shift in the voltage dependence of recovery rate. The data are the time constants for recovery from fast inactivation (rec) ± S.E. measured for each construct at three recovery potentials. The lines are nontheoretical point-to-point. The symbols, lines, sample numbers, and panels are as described in the legend to Fig. 1. Inset to C, typical plot of the fractional recovery measured using a standard twin pulse protocol to determine the rate of recovery from fast inactivation at a –120-mV recovery potential for Nav1.7 ± 1 ± SA. The data are the means ± S.E. fractional current 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 of the data from which the rec were determined.

₁ Sialic Acids Can Modulate the Gating of an Na_v1.4 Mutant with Reduced Glycosylation—Here we observed that Na_v1.4 gating is not affected by the presence of ₁. Na_v1.4 is predicted to have more N-glycosylation than Na_v1.2 or Na_v1.7, and we showed previously that Na_v1.4 is more heavily glycosylated than Na_v1.5 in this system (23), consistent with work performed in other laboratories (44). The majority of Na_v1.4 N-glycosylation sites are present in the DIS5-S6 extracellular linker, and previous work demonstrated that the sialic acid effects on Na_v1.4 gating are localized to this domain (23). Given that ₁ can modulate gating of channels with putatively less glycosylation than Na_v1.4 (e.g. Na_v1.2, Na_v1.5, and Na_v1.7), we examined the effects of ₁ on the Na_v1.4 DIS5-S6 loop chimera, hSkM1P1, which is less glycosylated than wild type Na_v1.4 (23, 27). As shown in Fig. 5, gating of hSkM1P1 alone is not dependent on sialic acid. However, co-expression of ₁ imposes a sialic acid-dependent hyperpolarizing shift in hSkM1P1 gating similar in magnitude and direction to the effects of ₁ sialic acids on gating of Na_v1.2, Na_v1.5, and Na_v1.7. This, together with the lack of effect of ₁ on Na_v1.4 gating observed here, would suggest that there is a potential saturating limit to the amount (location) of ₁ sialic acids that can affect subunit channel gating. More precisely, apparently combinations of the sialic acids associated with subunit IS5-S6 and with ₁ determine the overall impact of these sugars on channel gating, and this effect is saturating.

View larger version (27K): [in this window] [in a new window]   FIG. 5. 1 sialic acids can modulate the gating of an Nav1.4 mutant with reduced glycosylation. Gating of hSkM1P1 is sensitive to 1 sialic acids. Voltage-dependent steady state and kinetic gating for hSkM1P1 ± 1± SA is shown. Circles, hSkM1P1 expressed alone; squares, hSkM1P1 + 1. Filled symbols, in Pro5 cells; open symbols, in Lec2 cells. n = 8–11 for each condition (see Table I). A schematic of hSkM1P1 structure illustrates that the chimera consists of Nav1.4 with the less glycosylated Nav1.5 DIS5-S6 loop replacing the Nav1.4 DIS5-S6. A, G-V relationship. B, steady state channel availability. C, fast inactivation time constants. D, time constants for recovery from fast inactivation.

View larger version (13K): [in this window] [in a new window]   FIG. 6. The effect of 1 sialic acids on gating is consistent with an electrostatic mechanism. 1 sialic acids apparently contribute to an external negative surface potential that affects Nav gating. Shown is a bar graph of the observed hyperpolarizing shifts in Va for hSkM1P1 ± 1± SA with a 10-fold decrease in external Ca2+ concentration used to differentially screen external negative surface charges. The Va shifts by about –15 mV when extracellular Ca+2 is changed from 2.0 to 0.2 mM for hSkM1P1 alone ±1 SA and for hSkM1P1 + - SA (in Lec2 cells). However, the Ca2+-dependent shift in Va under conditions of fully sialylated 1 is a much larger, significant (p < 0.02), –25 mV. n = 3–4 for each condition.

1-N

1-N

1-N

View larger version (21K): [in this window] [in a new window]   FIG. 7. 1 N-linked sialic acids account fully for 1 modulation of NaV gating. G-V relationships for hSkM1P1 (A), Nav1.5 (B), Nav1.7 (C), and Nav1.2 (D) under fully sialylated conditions alone (filled circles with solid lines; n = 9–13 for each) or co-expressed with 1 (filled squares with dashed lines; n = 9–12 for each) or with 1-N a mutant 1 in all which four putative N-linked glycosylation asparagine residues were mutated to serine residues (filled triangles with dotted lines; n = 4–6 for each). Removal of 1 N-linked sugars prevents fully the 1 sialic acid induced hyperpolarizing shifts in channel gating. The data are the means ± S.E. peak conductance at a membrane potential. The lines are fits of the data to single Boltzmann relationships. Filled triangles with dotted line, 1-N. All other symbols, lines, and sample numbers are as previously described. Schematics of the wild type 1, with its four putative N-glycosylation structures attached, and 1-N, with all four carbohydrate structures missing, are pictured in A.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Previous work from our laboratory demonstrated that the gating of Na_v subunits expressed in CHO cells is altered by changes in sialylation levels (20, 23). We recently described how Na_v1.5 gating parameters were unaffected by subunit sialic acids, whereas Na_v1.4 gated at more depolarized potentials when expressed in essentially nonsialylating Lec2 cells versus fully sialylating Pro5 cells (23). Here, we extended this study to question the role of sialic acids in the gating of two neuronal subunits, Na_v1.2 and Na_v1.7. As shown in Figs. 1, 2, 3, 4, much like that observed for Na_v1.5 previously, reduced sialylation of Na_v1.7 had no effect on gating. Na_v1.2 gating was, in general, not significantly altered by subunit sialic acids, although a consistent, small, depolarizing shift in gating was observed for Na_v1.2 expressed in Lec2 cells compared with gating in the fully sialylating Pro5 cells.

The ₁ subunit is predicted to have four N-linked glycosylation sites, at least three of which are glycosylated (24, 34). We tested the hypothesis that ₁ sialic acids modulate Na_v gating by co-expressing ₁ with the four subunits in the fully sialylating Pro5 cells and compared channel function with those observed in the nonsialylating Lec2 cells. As shown in Figs. 1, 2, 3, 4, all of the effects of ₁ on subunit gating measured were removed under conditions of reduced ₁ sialylation. That is, ₁ induced hyperpolarizing shifts in Na_v1.2, Na_v1.5, and Na_v1.7 gating only under fully sialylating conditions. ₁ expressed in the nonsialylating Lec2 cells had no effect on subunit gating. Thus, ₁ sialic acids can account for all of the effects of ₁ on Na_v gating observed here.

In addition to N-linked glycosylation, membrane proteins often have sugars attached through different linkages. For example, O-linked glycosylation (attached to serine or threonine residues) is prevalent. We sought to determine whether the ₁ sialic acid-dependent effects on subunit gating were limited to N-linked sialic acids by mutating all four asparagine residues that initiate each N-glycosylation consensus sequence. The mutant ₁, _1-N, contains no N-linked sugars but would maintain other post-translational modifications, including O-linked sugars. As shown in Fig. 7, fully sialylated _1-N had no effect on the gating of any of the four subunits previously shown to be affected by ₁ sialic acids. Thus, the data confirm that ₁ N-linked sialic acids are responsible fully for the observed shifts in subunit gating.

₁ Sialic Acids Modulate Na_v Channel Gating through an Apparent Saturating Electrostatic Mechanism—We showed previously that subunit sialic acids impose effects on gating through an apparent electrostatic mechanism. It is well established that Na_v gating is sensitive to changes in external Ca²⁺ concentrations, requiring a larger depolarization as external Ca²⁺ levels increase. A surface potential theory is often assigned to this phenomenon. Thus, external negative surface charges produce a surface potential that alters the voltage sensed by the channel gating mechanism (49). As Ca²⁺ levels increase, these charges may be screened, minimizing their impact on gating. The voltage sensed by the gating mechanism of the channel becomes more negative, moving away from the voltage of activation, and thus a larger depolarization is required to activate the channel. If sialic acids contribute to such a negative surface potential, then sensitivity to screening by external Ca²⁺ should increase with the level of functional sialic acids. If ₁ sialic acids contribute to a negative surface potential, then gating of channels co-expressed with fully sialylated ₁ should be the most Ca²⁺ sensitive as shown in Fig. 6. The shift in V_a with changing external Ca²⁺ levels for hSkM1P1 alone ± SA and for hSkM1P1 + ₁ expressed in Lec2 cells were similar and significantly smaller than the shift observed for the fully sialylated hSkM1P1 + ₁. Thus, ₁ sialic acids alter subunit gating through an electrostatic mechanism, apparently contributing to an external negative surface potential.

Figs. 1, 2, 3, 4 showed that gating of the heavily glycosylated Na_v1.4 was affected by subunit sialic acids (cis effect) but not further altered by ₁ sialic acids. Conversely, gating of each of the putatively lesser glycosylated subunits was not dependent on sialic acids but was dependent on ₁ sialic acids (trans effect). These data suggest that in this system there is a saturating limit to the contribution of sialic acids to channel gating, with Na_v1.4 sialic acids likely achieving saturation. Fig. 5 provides further supporting evidence, showing that gating of the less glycosylated Na_v1.4 chimera, hSkM1P1, is no longer sensitive to sialic acids but is sensitive to ₁ sialic acids. These data suggest that with hSkM1P1, Na_v1.4 functional sialylation is reduced to levels below saturation such that ₁ sialic acids might contribute to channel gating. Thus, it appears that the combined effects of cis subunit DIS5-S6 and trans ₁ subunit functional sialic acids on channel gating are saturating (Fig. 8).

View larger version (33K): [in this window] [in a new window]   FIG. 8. Model predicting the putative saturating effects of and 1 sialic acids on Nav gating. A, examples of possible interactions of 1 sialic acids with Nav1.4 (left panel) and the glycosylation-deficient chimera, hSkM1P1 (right panel). The data suggest that 1 sialic acids cannot contribute further to the gating of Nav1.4 but do contribute to the gating of the other subunits through an apparent electrostatic mechanism. Thus, the left panel shows ineffectual 1 sialic acids as distant from Nav1.4, whereas the right panel illustrates that fewer subunit functional DIS5-S6 sialic acids may allow 1 sialic acids to interact more intimately with the subunit and contribute to channel gating. B, a theoretical concentration response curve comparing relative contributions to Va by functional sialic acids associated with various 1 combinations. A typical sigmoidal curve is shown, with the various 1 subunit combinations studied here aligned along the curve; the location of each is not precise but is consistent, relatively, with the data shown here. For example, Nav1.4 must contain essentially saturating levels of functional sialic acids in this experimental system. Thus, Nav1.4 alone and Nav1.4 + 1 are pictured along the maximum of the curve. In a second example, Nav1.7 alone cannot contain many DIS5-S6 functional sialic acids, and therefore Nav1.7 expressed alone must lie somewhere along the minimum portions of the curve. When 1 is co-expressed with Nav1.7, the levels of functional sialic acids increase, causing a hyperpolarization in Va, and hence, Nav 1.7 + 1 is shown somewhere along the rising slope of the curve. Although the data presented here show uniform effects of 1 sialic acids on measured voltage-dependent Nav gating parameters, there is no reason, a priori, for this to occur. The impact of 1 sialic acids on specific Nav gating parameters will depend on several factors including the extent of coupling among channel kinetic states and/or any inherent voltage dependence of the transition from one state to another. Thus, for example, the interaction of 1 sialic acids with a specific subunit may place the channel at a different position along the concentration response curves for shifts in Va versus shifts in Vi. This would likely be observed as differences in the impact of 1 sialic acids on Va and Vi.

Thus, the cell would have two complementary methods to modulate sodium current through differential sialylation. Changing the level of sialylation through regulation of sialyl-transferase activity might alter the function of some channels chronically. For example, sodium currents in adult rat cortical and dorsal root ganglion neurons are less sensitive to sialic acid than sodium currents from neonatal neurons (22, 40). A developmental decrease in Na_v glycosylation was observed in one study (22). Recent work from our laboratory indicates that adult ventricular myocyte Na_v are more heavily sialylated and gate at more hyperpolarized potentials than do neonate ventricular myocyte Na_v.² Thus, there are tissue-specific changes in sialylation over the course of development.

Second, control of ₁ expression (and the specific subunit expressed) causes acute changes in functional sialic acid levels with the additional ₁ sialic acids potentially altering the gating of the subunits with which they associate. Thus, gating of a specific subunit could be modulated by varying the expression of ₁ over time in combination with manipulating the extent of and ₁ post-translational modification, producing a spectrum of differentially sialylated Na_v. As a result, the excitability of the cell would be affected in a controlled manner that is potentially more efficient than directly manipulating the channel primary structure. Such negative shifts in gating imposed by ₁ sialic acids would shift the voltage range of the window current, causing persistent channel activity to occur at more hyperpolarized potentials. There are several reports showing that point mutations of SCN1A (Na_v1.1) and SCN5A (Na_v1.5) cause shifts in the window current voltage range that may be responsible for such maladies as epilepsy and arrhythmias (long QT syndrome) (1, 4, 5). In addition, ₁ sialic acids slow the rate of recovery from fast inactivation, which would directly affect action potential relative refractory periods. That is, if ₁ sialic acids induce slower recovery rates, then at a short time following initialization of a Na_v kinetic cycle, the percentage of inactivated channels will increase. This will directly affect the rate at which subsequent depolarizations might lead to activation of Na_v sufficient to cause an action potential to fire. Thus, we report novel findings that are relevant to the modulation of voltage-gated sodium channel activity that will directly impact how one's heart, muscle, and brain function.

Trans-regulation of Membrane Proteins by Carbohydrate Structures Attached to a Second Protein: a Unique Process?—In addition, a more global phenomenon can be described for the first time. Our data indicate that ₁ sialic acids directly alter subunit function, consistent with a mechanism by which transmembrane protein function can be modulated solely and directly by the sugars attached to a second protein. Because nearly all transmembrane proteins are glycosylated, such a phenomenon may be involved in processes other than the modulation of Na_v gating, with significant potential for sugar residues to impose direct functional effects on many transmembrane proteins.

	FOOTNOTES

 
^* This work was supported in part by a grant from the NIAMS, National Institutes of Health. 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.

To whom correspondence should be addressed: Dept. of Physiology & Biophysics, 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.

¹ The abbreviations used are: Na_v, voltage-gated sodium channel; CHO, Chinese hamster ovary; ORF, open reading frame; GFP, green fluorescent protein; SA, sialic acid residue.

² P. J. Stocker and E. S. Bennett, unpublished data.

	ACKNOWLEDGMENTS

 
We thank Dr. Alan Goldin for generously providing the Na_v1.2 cDNA and acknowledge the technical expertise of Jean Harper. We also thank Dr. Jahanshah Amin for careful reading of the manuscript.

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