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Modulation of Voltage‐Gated Ion Channels by Sialylation

Andrew R. Ednie, Eric S. Bennett

10.1002/cphy.c110044

Source: Volume 2, Issue 2, April 2012

Published online: April 2012

Full Article on Wiley Online Library



Abstract

Control and modulation of electrical signaling is vital to normal physiology, particularly in neurons, cardiac myocytes, and skeletal muscle. The orchestrated activities of variable sets of ion channels and transporters, including voltage‐gated ion channels (VGICs), are responsible for initiation, conduction, and termination of the action potential (AP) in excitable cells. Slight changes in VGIC activity can lead to severe pathologies including arrhythmias, epilepsies, and paralyses, while normal excitability depends on the precise tuning of the AP waveform. VGICs are heavily posttranslationally modified, with upward of 30% of the mature channel mass consisting of N‐ and O‐glycans. These glycans are terminated typically by negatively charged sialic acid residues that modulate voltage‐dependent channel gating directly. The data indicate that sialic acids alter VGIC activity in isoform‐specific manners, dependent in part, on the number/location of channel sialic acids attached to the pore‐forming alpha and/or auxiliary subunits that often act through saturating electrostatic mechanisms. Additionally, cell‐specific regulation of sialylation can affect VGIC gating distinctly. Thus, channel sialylation is likely regulated through two mechanisms that together contribute to a dynamic spectrum of possible gating motifs: a subunit‐specific mechanism and regulated (aberrant) changes in the ability of the cell to glycosylate. Recent studies showed that neuronal and cardiac excitability is modulated through regulated changes in voltage‐gated Na+ channel sialylation, suggesting that both mechanisms of differential VGIC sialylation contribute to electrical signaling in the brain and heart. Together, the data provide insight into an important and novel paradigm involved in the control and modulation of electrical signaling. © 2012 American Physiological Society. Compr Physiol 2:1269‐1301, 2012.

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Figure 1. Figure 1.

External [Ca2+] shifts voltage‐gated ion channel (VGIC) activation voltage. Decreased [Cao2+] results in a 10‐ to 30‐mV shift in squid giant axon Na+ current activation voltage. Adapted, with permission, from B Frankenhaeuser and AL Hodgkin. J Physiol 137: 218‐244, 1957 59.

Reprinted with permission.
Figure 2. Figure 2.

External negative surface charge impacts membrane potential sensed by VGIC. Illustration of the putative effects of external negative charges on the membrane electric field and potential. Surface potential theory predicts that an increase in external divalent cations nullifies the impact of the surface charge on the membrane potential. Figure adapted, with permission, from B Hille. Ion channels of Excitable Membranes. 3rd edition 84.

Reprinted, with permission, from Sinauer Associates.
Figure 3. Figure 3.

Extracellular glycosylation is a sequential process that occurs in the endoplasmic reticulum (ER) and Golgi and involves the activity of hundreds of glycogenes. Illustration of the process of protein N‐glycosylation as it occurs in the ER and Golgi. Figure adapted, with permission, from A Helenius and M Aebi. Science 291: 2364‐2369, 2001 79.

Reprinted, with permission, from AAAS.
Figure 4. Figure 4.

Voltage‐dependent gating of the skeletal muscle Nav was shifted nearly identically under each of three independent conditions of reduced sialylation. Conductance‐voltage (G‐V) relationships for Nav1.4 expressed under conditions of full sialylation (+SA) and three independent conditions of reduced sialylation [−SA; neuraminidase‐treated, to remove sialic acids (top), in the essentially nonsialylating Lec2 cell line (middle), and two deletion mutants in which four and five N‐glycosylation sites were removed through mutagenesis (bottom)]. G‐V relationships here and generally, were determined by measuring the peak conductance during a brief depolarizing test pulse from a negative resting potential. Adapted, with permission, from E Bennett et al. J Gen Physiol 109: 327‐343, 1997 17.
Figure 5. Figure 5.

Gating of less‐sialylated Nav are not as sensitive to changes in [Cao2+]. Conductance‐voltage (G‐V) curves for Nav1.4 expressed under conditions of full sialylation (top) and reduced sialylation (bottom) at 2.0 (filled circles) and 0.2 (open circles) mmol/L Ca2+. Note the much larger hyperpolarizing shifts in G‐V relationship under reduced [Cao2+] for the fully sialylated channels. Figure adapted, with permission, from E Bennett et al. J Gen Physiol 109: 327‐343, 1997 17.
Figure 6. Figure 6.

Functional channel sialic acids are localized to the DIS5‐S6 loop. Conductance‐voltage (G‐V) curves for Nav1.4 (hSkM1, top left), Nav1.5 (hH1, top right), and chimeras in which the DIS5‐S6 loop of Nav1.5 replaced the Nav1.4 loop (hSkM1P1, bottom left) or vice versa (hH1P1, bottom right) as expressed in fully sialylating Pro5 (circles) and nonsialylating Lec2 (squares) cells. Figure adapted, with permission, from ES Bennett. J Physiol 538: 675‐690, 2002 18.
Figure 7. Figure 7.

The fully sialylated β1‐subunit modulates gating of three Nav α subunits. Conductance‐voltage (G‐V) curves for four Nav α‐subunits ± the β1‐subunit under conditions of full and reduced sialylation. Filled symbols: α‐subunit alone. Open symbols: α + β1. Circles: in Pro5 cells (+SA). Squares: in Lec2 cells (−SA). Solid lines: α‐subunit alone. Dashed lines: α + β1. A, Nav1.4. B, Nav1.5. C, Nav1.7. D, Nav1.2. Figure adapted, with permission, from D Johnson et al. J Biol Chem 279: 44303‐44310, 2004 106.
Figure 8. Figure 8.

The β1‐subunit had no effect on Nav α‐subunit gating following removal of β1 N‐glycosylation sites. Conductance‐voltage (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 coexpressed with β1 (filled squares with dashed lines; n = 9‐12 for each) or with β1‐ΔN, a mutant β1 in which all four putative N‐linked glycosylation Asparagine residues were mutated to Serine residues (filled triangles with dotted lines; n = 4‐6 for each). Removal of β1 N‐linked sugars prevented fully the β1 sialic acid‐induced hyperpolarizing shifts in channel gating. Filled triangles with dotted line, β1‐ΔN. All other symbols and lines are as described in Figure 7. Figure and legend adapted, with permission, from D Johnson et al. J Biol Chem 279: 44303‐44310, 2004 106.
Figure 9. Figure 9.

The impact of channel sialic acids on Nav gating is apparently a saturating phenomenon. Voltage‐dependent steady state and kinetic gating for hSkM1P1 ± β1 ± SA. Circles, hSkM1P1 alone; squares, hSkM1P1 ± β1. Filled symbols, in Pro5 cells; open symbols, in Lec2 cells. n = 8‐11 for each condition. 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) Conductance‐voltage (G‐V) relationship. (B) Steady‐state channel availability curve (SSI). Peak currents were measured during a short, maximally depolarizing pulse that was preceded by a 500 ms prepulse to increasingly depolarized potentials. (C) Fast inactivation time constants. Fractional current that recovered from fast inactivation during recovery prepulses of variable duration was used to determine time constants. (D) Time constants for recovery from fast inactivation. Figure and legend adapted, with permission, from D Johnson et al. J Biol Chem 279: 44303‐44310, 2004 106.
Figure 10. Figure 10.

Model predicting the putative saturating effects of α and β1 sialic acids on Nav gating. (A) Examples of possible interactions of β1 sialic acids with Nav1.4 (left) and the glycosylation‐deficient chimera, hSkM1P1 (right). The data suggest that β1 sialic acids cannot contribute further to the gating of Nav1.4 but do contribute to the gating of the other α‐subunits through an apparent electrostatic mechanism. Thus, the left panel shows ineffectual β1 sialic acids as distant from Nav1.4, whereas the right panel illustrates that fewer α‐subunit functional DIS5‐S6 sialic acids may allow β1 sialic acids to interact more intimately with the α‐subunit and contribute to channel gating. (B) A theoretical concentration response curve comparing relative contributions to Va by functional sialic acids associated with various αβ1 combinations. A typical sigmoidal curve is shown, with the various αβ1‐subunit combinations aligned along the curve; the location of each is not precise but is consistent, relatively, with the data shown in Figures 7‐9. For example, Nav1.4 must contain essentially saturating levels of functional sialic acids in this experimental system. Thus, Nav1.4 alone and Nav1.4 ± β1 are pictured along the maximum of the curve. In a second example, Nav1.5 alone cannot contain many DIS5‐S6 functional sialic acids, and therefore Nav1.5 expressed alone must lie somewhere along the minimum portions of the curve. When β1 is coexpressed with Nav1.5, the levels of functional sialic acids increase, causing a hyperpolarization in Va, and hence, Nav1.5 + β1 is shown somewhere along the rising slope of the curve. Although the data presented in Johnson et al. J Biol Chem, 2004, showed uniform effects of β1 sialic acids on measured voltage‐dependent Nav gating parameters, there is no reason, a priori, for this to occur. The impact of β1 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 β1 sialic acids with a specific α‐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 β1 sialic acids on Va and Vi. “P1” represents hSkM1P1. Figure and legend adapted, with permission, from D Johnson et al. J Biol Chem 279: 44303‐44310, 2004 106.
Figure 11. Figure 11.

The sialic acid‐dependent effects of β1 and β2 on Nav1.5 gating are additive. Filled symbols, Pro5 cells (+SA); open symbols, Lec2 cells (−SA); gold circles/lines, Nav1.5; red squares/lines, Nav1.5 + β2; cyan diamonds/lines, Nav1.5 + β1; purple triangles/lines, Nav1.5 + β1 + β2. n = 9‐12 for each condition tested. (A) Conductance‐voltage (G‐V) relationships. (B) Channel availability (SSI) curves. (C) Fast inactivation time constant. (D) Time constants of the recovery from fast inactivation. Figure and legend adapted, with permission, from D Johnson and ES Bennett. J Biol Chem 281: 25875‐25881, 2006 105.
Figure 12. Figure 12.

Sialic acids modulate Kv1.1 gating through apparent electrostatic mechanisms. Bar graphs illustrating the impact of changes in [Cao2+] on activation gating of Kv1.1 under three conditions of glycosylation (full, in Pro5; reduced sialylation, in Lec2; terminal galactose and sialic acid residues missing, in Lec8).

Figure adapted, with permission, from WB Thornhill et al. J Biol Chem 271: 19093‐19098, 1996 194. Reprinted with permission.
Figure 13. Figure 13.

Kv1.2 gating can be modulated by the addition or removal of N‐glycans. Conductance‐voltage (G‐V) curves for wild‐type Kv1.2 (contains one N‐glycosylation site) and two mutant Kv1.2 constructs in which N‐glycosylation sites were added (Kv1.2Tri‐N) or removed (Kv1.2N207Q) through mutagenesis.

Figure adapted, with permission, from I Watanabe et al. Brain Res 1144: 1‐18, 2007 211. Reprinted with permission.
Figure 14. Figure 14.

Cardiac transient outward K+ current, Ito, is sensitive to desialylation. (A) Whole cell IK traces from adult ventricular myocytes ± neuraminidase treatment. (B) Peak Ito density ± neuraminidase. (C) Conductance‐voltage (G‐V) relationships for Ito ± neuraminidase. (D) Steady‐state channel availability (SSI) curves for Ito ± neuraminidase. Figure adapted, with permission, from CA Ufret‐Vincenty et al. Am J Physiol Cell Physiol 281: C464‐C474, 2001b 200.
Figure 15. Figure 15.

Adult dorsal root ganglia (DRG) neuronal Nav is less sialylated and less sensitive to neuraminidase treatment than the neonatal Nav. Comparison of INa in neonatal and adult DRG neurons. (A) Current‐voltage (I‐V) relationships. (B) Conductance‐voltage (G‐V) relationships. (C) Steady‐state channel availability (SSI) curves.

Adapted, with permission, from L Tyrrell et al. J Neuroscience 21: 24 9629‐9637, 2001 198. Reprinted with permission.
Figure 16. Figure 16.

Neonatal ventricular Nav gate at more depolarized potentials. (A) Conductance‐voltage (G‐V) relationships. Inset: whole cell Na+ current (INa) from an isolated adult ventricular myocyte. (B) Steady‐state inactivation (SSI). Inset: INa measured during a series of +40 mV test pulses, each following a prepulse to a larger depolarization. (C) Time constant of fast inactivation (τh). Inset: INa during a −50 mV test pulse. Solid traces: neonatal atrial (NA), adult atrial (AA), and adult ventricular (NV) myocytes. Dotted trace: neonatal ventricular (NV) myocyte. (D) Voltage dependence of the recovery time constants. Inset: plotted recovery data and fits for all four conditions at a −130 mV recovery potential. Solid and dotted curves as described in (C). n = 6‐17. Figure and legend adapted, with permission, from PJ Stocker and ES Bennett. J Gen Physiol 127: 253‐265, 2006 180.
Figure 17. Figure 17.

Removal of Nav1.5 glycosylation and sialylation can account fully for gating difference in Nav1.5 among neonatal and adult atrial and ventricular Nav. Conductance‐voltage (G‐V) (A), and steady‐state inactivation (SSI) (B) curves, fast inactivation (C), and recovery (D) kinetics for all channel types following treatment with PNGase‐F (open symbols, triangles, NA; inverted triangles, AA; circles, NV; squares, AV), and for Nav from neonatal ventricles under control/untreated (solid circles) and neuraminidase‐treated (dotted, open circles) conditions. Figure and legend adapted, with permission, from PJ Stocker and ES Bennett. J Gen Physiol 127: 253‐265, 2006 180.
Figure 18. Figure 18.

Gel‐shift analyses suggest that Nav1.5 sialylation levels are regulated between cardiac chambers and during ventricular development. (A‐C) Immunoblots of Nav α subunits from neonatal and adult atria and ventricles ± glycosidase treatments. (A) Untreated lysates. (B) Lysates ± PNGase‐F treatment. (C) Lysates ± neuraminidase treatment. (D) Bar graph of average measured Nav α‐subunit Mr ± PNGase‐F ± neuraminidase. Significance was determined for the untreated data using a two‐tailed Student's t test comparing the Mr for the α‐subunit from neonatal ventricles to the Mr for the α‐subunits from neonatal atria and adult atria and ventricles. *, significant (P < 0.01). Figure and legend adapted, with permission, from PJ Stocker and ES Bennett. J Gen Physiol 127: 253‐265, 2006 180.
Figure 19. Figure 19.

Suggested mechanisms by which differential sialylation modulates voltage‐gated ion channel (VGIC) gating. Simplified model depicts subunit‐ and cell‐specific differential sialylation relevant to Now gating. Each mechanism potentially can produce a modest spectrum of channel gating motifs [shown for illustrative purposes only as different conductance‐voltage (G‐V) relationships]. Top left: subunit‐specific differential sialylation. Each α‐ (and β‐) subunit will have an inherent “glycosylation signature” that predetermines the level/location of glycosylation that can be attached to channel. Top right: cell‐specific differential sialylation. The relative activity of one to several of the 20 sialyltransferases (STs) will determine the amount and location of sialic acids added to one α‐ (and/or β‐) subunit. When combined together (bottom), perhaps hundreds of gating motifs could be realized. For example, by assuming that each of the Nav α‐subunits might be sialylated differently by the activity of each of the 20 known STs, 200 different gating motifs might be possible. This model is likely oversimplified, as it does not account for the even greater number of gating motifs that might be realized through regulated expression of the Nav β‐subunits, the activity of multiple STs in various combinations, or for the varied expression/activity of other glycogenes. Thus, with the regulated expression of specific α‐ and β‐subunits combined with the regulated expression and activity of specific STs, Nav gating might be modulated across a large and finely tuned spectrum of voltages. Figure and legend adapted, with permission, from PJ Stocker and ES Bennett. J Gen Physiol 127: 253‐265, 2006 180.
Figure 20. Figure 20.

Mutant polysialyltransferase modulates activity of Drosophila Nav. (A) Tetrodotoxin (TTX) sensitivity of the Drosophila excitatory junction potentials (EJP) is reduced in mutant DSiaT (S23), suggesting reduced Para (Drosophila Nav) activity in the mutant. (B) The TTX sensitivity of the EJP is rescued by the DSiaT rescue mutant.

Figure adapted, with permission, from E Repnikova et al. J Neuroscience 30: 186466‐6476, 2010 162. Reprinted with permission.
Figure 21. Figure 21.

Hippocampal Nav gating is modulated by sialylation. Nucleated patch (A and B) and whole cell recording (C and D) measurements of isolated hippocampal neuronal Nav conductance‐voltage (G‐V) (A and C) and SSI (B and D) ± neuraminidase treatment. In (C) and (D), G‐V and SSI curves measured following treatment with neuraminidase + inhibitor are essentially the same as the untreated curves.

Figure adapted, with permission, from D Isaev et al. J Neuroscience 27: 4311587‐11594, 2007 96. Reprinted with permission.
Figure 22. Figure 22.

Hippocampal excitability is modulated by apparent changes in hippocampal protein sialylation. (A) Field recordings ± neuraminidase ± neuraminidase blocker following multiple injections of higher [Ko+] used to induce epileptiform activity in vivo. (B) Summary data of seizure occurrence as a function of injection number. (C) Duration of ictal‐like activity. (D) Maximum population spike frequency.

Figure adapted, with permission, from D Isaev et al. J Neuroscience 27: 4311587‐11594, 2007 96. Reprinted with permission.
Figure 23. Figure 23.

Atrial action potential (AP) waveform is modulated by expression of the polysialyltransferase, ST8Sia2. APs were measured from isolated control and ST8sia2(−/−) neonatal atrial and ventricular myocytes. Top left: typical AP waveforms measured from control (in black) and ST8sia2(−/−) (in red) atrial myocytes. (A‐C) The mean ± SEM AP waveform parameters (relevant portions of AP marked with arrows). Black, control atrium (n = 7); red, ST8sia2 (−/−) atrium (n = 9); dark blue, control ventricle (n = 7); blue, ST8sia2(−/−) ventricle (n = 5). (A) Time to AP peak. (B) AP duration at 50% repolarization (APD50). (C) AP duration at 90% repolarization (APD90). Significance tested using a two‐tailed t test and comparing ST8sia2(−/−) to control parameters. *, significant (P<0.005); #, not significant (P>0.1). Figure and legend adapted, with permission, from ML Montpetit et al. Proc Natl Acad Sci, U S A 106: 16517‐16522, 2009 142.
Figure 24. Figure 24.

Atrial Nav gating is modulated by ST8Sia2 expression. ST8sia2(−/−) and control myocyte Nav gating. Left panels (A, C, E, and G): atrium, ST8sia2(−/−) (red; n = 10); control (black; n = 4). Right panels (B, D, F, and H): ventricle, ST8sia2(−/−) (blue; n = 4); control (dark blue; n = 6). (A and B) Steady‐state activation. Insets: half‐activation voltage. (C and D) Steady‐state channel availability (SSI). Dashed line: ST8sia2(−/−) data shifted along voltage axis by −7.3 mV (to mimic measured shift in half‐activation voltage). Insets: half‐inactivation voltage. (E and F) Fast inactivation time constants. Dashed line: The ST8sia2(−/−) data shifted along voltage axis by −7.3 mV. Insets: typical whole cell Na+ current traces elicited by stepping to a −40 mV test potential. (G and H) Time constants of recovery from fast inactivation at a −130 mV recovery potential. Significance tested using a two‐tailed t test and comparing ST8sia2(−/−) to control parameters. *, significant (P ≤ 0.005); #, not significant (P > 0.1). Figure and legend adapted, with permission, from ML Montpetit et al. Proc Natl Acad Sci, U S A 106: 16517‐16522, 2009 142.
Figure 25. Figure 25.

MLP−/− ventricular myocytes are susceptible to early afterdepolarizations. MLP−/− mice show extended QT intervals (A), increased action potential durations (B), and increased susceptibility to early after depolarizations (C and D).

Figure adapted, with permission, from CA Ufret‐Vincenty et al. J Biol Chem 276: 28197‐28203, 2001 199. Reprinted with permission.
Figure 26. Figure 26.

Gating of MLP−/− ventricular Nav is less sensitive to desialylation than the wild‐type channel. INa for desialylated control myocytes mimicked untreated and neuraminidase‐treated MLP−/− myocyte INa. (A) Current‐voltage (I‐V) relationships. (B) Conductance‐voltage (G‐V) relationships. (D) SSI curves. (E) Fast inactivation times constants.

Figure adapted, with permission, from CA Ufret‐Vincenty et al. J Biol Chem 276: 28197‐28203, 2001 199. Reprinted with permission.


Figure 1.

External [Ca2+] shifts voltage‐gated ion channel (VGIC) activation voltage. Decreased [Cao2+] results in a 10‐ to 30‐mV shift in squid giant axon Na+ current activation voltage. Adapted, with permission, from B Frankenhaeuser and AL Hodgkin. J Physiol 137: 218‐244, 1957 59.

Reprinted with permission.


Figure 2.

External negative surface charge impacts membrane potential sensed by VGIC. Illustration of the putative effects of external negative charges on the membrane electric field and potential. Surface potential theory predicts that an increase in external divalent cations nullifies the impact of the surface charge on the membrane potential. Figure adapted, with permission, from B Hille. Ion channels of Excitable Membranes. 3rd edition 84.

Reprinted, with permission, from Sinauer Associates.


Figure 3.

Extracellular glycosylation is a sequential process that occurs in the endoplasmic reticulum (ER) and Golgi and involves the activity of hundreds of glycogenes. Illustration of the process of protein N‐glycosylation as it occurs in the ER and Golgi. Figure adapted, with permission, from A Helenius and M Aebi. Science 291: 2364‐2369, 2001 79.

Reprinted, with permission, from AAAS.


Figure 4.

Voltage‐dependent gating of the skeletal muscle Nav was shifted nearly identically under each of three independent conditions of reduced sialylation. Conductance‐voltage (G‐V) relationships for Nav1.4 expressed under conditions of full sialylation (+SA) and three independent conditions of reduced sialylation [−SA; neuraminidase‐treated, to remove sialic acids (top), in the essentially nonsialylating Lec2 cell line (middle), and two deletion mutants in which four and five N‐glycosylation sites were removed through mutagenesis (bottom)]. G‐V relationships here and generally, were determined by measuring the peak conductance during a brief depolarizing test pulse from a negative resting potential. Adapted, with permission, from E Bennett et al. J Gen Physiol 109: 327‐343, 1997 17.



Figure 5.

Gating of less‐sialylated Nav are not as sensitive to changes in [Cao2+]. Conductance‐voltage (G‐V) curves for Nav1.4 expressed under conditions of full sialylation (top) and reduced sialylation (bottom) at 2.0 (filled circles) and 0.2 (open circles) mmol/L Ca2+. Note the much larger hyperpolarizing shifts in G‐V relationship under reduced [Cao2+] for the fully sialylated channels. Figure adapted, with permission, from E Bennett et al. J Gen Physiol 109: 327‐343, 1997 17.



Figure 6.

Functional channel sialic acids are localized to the DIS5‐S6 loop. Conductance‐voltage (G‐V) curves for Nav1.4 (hSkM1, top left), Nav1.5 (hH1, top right), and chimeras in which the DIS5‐S6 loop of Nav1.5 replaced the Nav1.4 loop (hSkM1P1, bottom left) or vice versa (hH1P1, bottom right) as expressed in fully sialylating Pro5 (circles) and nonsialylating Lec2 (squares) cells. Figure adapted, with permission, from ES Bennett. J Physiol 538: 675‐690, 2002 18.



Figure 7.

The fully sialylated β1‐subunit modulates gating of three Nav α subunits. Conductance‐voltage (G‐V) curves for four Nav α‐subunits ± the β1‐subunit under conditions of full and reduced sialylation. Filled symbols: α‐subunit alone. Open symbols: α + β1. Circles: in Pro5 cells (+SA). Squares: in Lec2 cells (−SA). Solid lines: α‐subunit alone. Dashed lines: α + β1. A, Nav1.4. B, Nav1.5. C, Nav1.7. D, Nav1.2. Figure adapted, with permission, from D Johnson et al. J Biol Chem 279: 44303‐44310, 2004 106.



Figure 8.

The β1‐subunit had no effect on Nav α‐subunit gating following removal of β1 N‐glycosylation sites. Conductance‐voltage (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 coexpressed with β1 (filled squares with dashed lines; n = 9‐12 for each) or with β1‐ΔN, a mutant β1 in which all four putative N‐linked glycosylation Asparagine residues were mutated to Serine residues (filled triangles with dotted lines; n = 4‐6 for each). Removal of β1 N‐linked sugars prevented fully the β1 sialic acid‐induced hyperpolarizing shifts in channel gating. Filled triangles with dotted line, β1‐ΔN. All other symbols and lines are as described in Figure 7. Figure and legend adapted, with permission, from D Johnson et al. J Biol Chem 279: 44303‐44310, 2004 106.



Figure 9.

The impact of channel sialic acids on Nav gating is apparently a saturating phenomenon. Voltage‐dependent steady state and kinetic gating for hSkM1P1 ± β1 ± SA. Circles, hSkM1P1 alone; squares, hSkM1P1 ± β1. Filled symbols, in Pro5 cells; open symbols, in Lec2 cells. n = 8‐11 for each condition. 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) Conductance‐voltage (G‐V) relationship. (B) Steady‐state channel availability curve (SSI). Peak currents were measured during a short, maximally depolarizing pulse that was preceded by a 500 ms prepulse to increasingly depolarized potentials. (C) Fast inactivation time constants. Fractional current that recovered from fast inactivation during recovery prepulses of variable duration was used to determine time constants. (D) Time constants for recovery from fast inactivation. Figure and legend adapted, with permission, from D Johnson et al. J Biol Chem 279: 44303‐44310, 2004 106.



Figure 10.

Model predicting the putative saturating effects of α and β1 sialic acids on Nav gating. (A) Examples of possible interactions of β1 sialic acids with Nav1.4 (left) and the glycosylation‐deficient chimera, hSkM1P1 (right). The data suggest that β1 sialic acids cannot contribute further to the gating of Nav1.4 but do contribute to the gating of the other α‐subunits through an apparent electrostatic mechanism. Thus, the left panel shows ineffectual β1 sialic acids as distant from Nav1.4, whereas the right panel illustrates that fewer α‐subunit functional DIS5‐S6 sialic acids may allow β1 sialic acids to interact more intimately with the α‐subunit and contribute to channel gating. (B) A theoretical concentration response curve comparing relative contributions to Va by functional sialic acids associated with various αβ1 combinations. A typical sigmoidal curve is shown, with the various αβ1‐subunit combinations aligned along the curve; the location of each is not precise but is consistent, relatively, with the data shown in Figures 7‐9. For example, Nav1.4 must contain essentially saturating levels of functional sialic acids in this experimental system. Thus, Nav1.4 alone and Nav1.4 ± β1 are pictured along the maximum of the curve. In a second example, Nav1.5 alone cannot contain many DIS5‐S6 functional sialic acids, and therefore Nav1.5 expressed alone must lie somewhere along the minimum portions of the curve. When β1 is coexpressed with Nav1.5, the levels of functional sialic acids increase, causing a hyperpolarization in Va, and hence, Nav1.5 + β1 is shown somewhere along the rising slope of the curve. Although the data presented in Johnson et al. J Biol Chem, 2004, showed uniform effects of β1 sialic acids on measured voltage‐dependent Nav gating parameters, there is no reason, a priori, for this to occur. The impact of β1 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 β1 sialic acids with a specific α‐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 β1 sialic acids on Va and Vi. “P1” represents hSkM1P1. Figure and legend adapted, with permission, from D Johnson et al. J Biol Chem 279: 44303‐44310, 2004 106.



Figure 11.

The sialic acid‐dependent effects of β1 and β2 on Nav1.5 gating are additive. Filled symbols, Pro5 cells (+SA); open symbols, Lec2 cells (−SA); gold circles/lines, Nav1.5; red squares/lines, Nav1.5 + β2; cyan diamonds/lines, Nav1.5 + β1; purple triangles/lines, Nav1.5 + β1 + β2. n = 9‐12 for each condition tested. (A) Conductance‐voltage (G‐V) relationships. (B) Channel availability (SSI) curves. (C) Fast inactivation time constant. (D) Time constants of the recovery from fast inactivation. Figure and legend adapted, with permission, from D Johnson and ES Bennett. J Biol Chem 281: 25875‐25881, 2006 105.



Figure 12.

Sialic acids modulate Kv1.1 gating through apparent electrostatic mechanisms. Bar graphs illustrating the impact of changes in [Cao2+] on activation gating of Kv1.1 under three conditions of glycosylation (full, in Pro5; reduced sialylation, in Lec2; terminal galactose and sialic acid residues missing, in Lec8).

Figure adapted, with permission, from WB Thornhill et al. J Biol Chem 271: 19093‐19098, 1996 194. Reprinted with permission.


Figure 13.

Kv1.2 gating can be modulated by the addition or removal of N‐glycans. Conductance‐voltage (G‐V) curves for wild‐type Kv1.2 (contains one N‐glycosylation site) and two mutant Kv1.2 constructs in which N‐glycosylation sites were added (Kv1.2Tri‐N) or removed (Kv1.2N207Q) through mutagenesis.

Figure adapted, with permission, from I Watanabe et al. Brain Res 1144: 1‐18, 2007 211. Reprinted with permission.


Figure 14.

Cardiac transient outward K+ current, Ito, is sensitive to desialylation. (A) Whole cell IK traces from adult ventricular myocytes ± neuraminidase treatment. (B) Peak Ito density ± neuraminidase. (C) Conductance‐voltage (G‐V) relationships for Ito ± neuraminidase. (D) Steady‐state channel availability (SSI) curves for Ito ± neuraminidase. Figure adapted, with permission, from CA Ufret‐Vincenty et al. Am J Physiol Cell Physiol 281: C464‐C474, 2001b 200.



Figure 15.

Adult dorsal root ganglia (DRG) neuronal Nav is less sialylated and less sensitive to neuraminidase treatment than the neonatal Nav. Comparison of INa in neonatal and adult DRG neurons. (A) Current‐voltage (I‐V) relationships. (B) Conductance‐voltage (G‐V) relationships. (C) Steady‐state channel availability (SSI) curves.

Adapted, with permission, from L Tyrrell et al. J Neuroscience 21: 24 9629‐9637, 2001 198. Reprinted with permission.


Figure 16.

Neonatal ventricular Nav gate at more depolarized potentials. (A) Conductance‐voltage (G‐V) relationships. Inset: whole cell Na+ current (INa) from an isolated adult ventricular myocyte. (B) Steady‐state inactivation (SSI). Inset: INa measured during a series of +40 mV test pulses, each following a prepulse to a larger depolarization. (C) Time constant of fast inactivation (τh). Inset: INa during a −50 mV test pulse. Solid traces: neonatal atrial (NA), adult atrial (AA), and adult ventricular (NV) myocytes. Dotted trace: neonatal ventricular (NV) myocyte. (D) Voltage dependence of the recovery time constants. Inset: plotted recovery data and fits for all four conditions at a −130 mV recovery potential. Solid and dotted curves as described in (C). n = 6‐17. Figure and legend adapted, with permission, from PJ Stocker and ES Bennett. J Gen Physiol 127: 253‐265, 2006 180.



Figure 17.

Removal of Nav1.5 glycosylation and sialylation can account fully for gating difference in Nav1.5 among neonatal and adult atrial and ventricular Nav. Conductance‐voltage (G‐V) (A), and steady‐state inactivation (SSI) (B) curves, fast inactivation (C), and recovery (D) kinetics for all channel types following treatment with PNGase‐F (open symbols, triangles, NA; inverted triangles, AA; circles, NV; squares, AV), and for Nav from neonatal ventricles under control/untreated (solid circles) and neuraminidase‐treated (dotted, open circles) conditions. Figure and legend adapted, with permission, from PJ Stocker and ES Bennett. J Gen Physiol 127: 253‐265, 2006 180.



Figure 18.

Gel‐shift analyses suggest that Nav1.5 sialylation levels are regulated between cardiac chambers and during ventricular development. (A‐C) Immunoblots of Nav α subunits from neonatal and adult atria and ventricles ± glycosidase treatments. (A) Untreated lysates. (B) Lysates ± PNGase‐F treatment. (C) Lysates ± neuraminidase treatment. (D) Bar graph of average measured Nav α‐subunit Mr ± PNGase‐F ± neuraminidase. Significance was determined for the untreated data using a two‐tailed Student's t test comparing the Mr for the α‐subunit from neonatal ventricles to the Mr for the α‐subunits from neonatal atria and adult atria and ventricles. *, significant (P < 0.01). Figure and legend adapted, with permission, from PJ Stocker and ES Bennett. J Gen Physiol 127: 253‐265, 2006 180.



Figure 19.

Suggested mechanisms by which differential sialylation modulates voltage‐gated ion channel (VGIC) gating. Simplified model depicts subunit‐ and cell‐specific differential sialylation relevant to Now gating. Each mechanism potentially can produce a modest spectrum of channel gating motifs [shown for illustrative purposes only as different conductance‐voltage (G‐V) relationships]. Top left: subunit‐specific differential sialylation. Each α‐ (and β‐) subunit will have an inherent “glycosylation signature” that predetermines the level/location of glycosylation that can be attached to channel. Top right: cell‐specific differential sialylation. The relative activity of one to several of the 20 sialyltransferases (STs) will determine the amount and location of sialic acids added to one α‐ (and/or β‐) subunit. When combined together (bottom), perhaps hundreds of gating motifs could be realized. For example, by assuming that each of the Nav α‐subunits might be sialylated differently by the activity of each of the 20 known STs, 200 different gating motifs might be possible. This model is likely oversimplified, as it does not account for the even greater number of gating motifs that might be realized through regulated expression of the Nav β‐subunits, the activity of multiple STs in various combinations, or for the varied expression/activity of other glycogenes. Thus, with the regulated expression of specific α‐ and β‐subunits combined with the regulated expression and activity of specific STs, Nav gating might be modulated across a large and finely tuned spectrum of voltages. Figure and legend adapted, with permission, from PJ Stocker and ES Bennett. J Gen Physiol 127: 253‐265, 2006 180.



Figure 20.

Mutant polysialyltransferase modulates activity of Drosophila Nav. (A) Tetrodotoxin (TTX) sensitivity of the Drosophila excitatory junction potentials (EJP) is reduced in mutant DSiaT (S23), suggesting reduced Para (Drosophila Nav) activity in the mutant. (B) The TTX sensitivity of the EJP is rescued by the DSiaT rescue mutant.

Figure adapted, with permission, from E Repnikova et al. J Neuroscience 30: 186466‐6476, 2010 162. Reprinted with permission.


Figure 21.

Hippocampal Nav gating is modulated by sialylation. Nucleated patch (A and B) and whole cell recording (C and D) measurements of isolated hippocampal neuronal Nav conductance‐voltage (G‐V) (A and C) and SSI (B and D) ± neuraminidase treatment. In (C) and (D), G‐V and SSI curves measured following treatment with neuraminidase + inhibitor are essentially the same as the untreated curves.

Figure adapted, with permission, from D Isaev et al. J Neuroscience 27: 4311587‐11594, 2007 96. Reprinted with permission.


Figure 22.

Hippocampal excitability is modulated by apparent changes in hippocampal protein sialylation. (A) Field recordings ± neuraminidase ± neuraminidase blocker following multiple injections of higher [Ko+] used to induce epileptiform activity in vivo. (B) Summary data of seizure occurrence as a function of injection number. (C) Duration of ictal‐like activity. (D) Maximum population spike frequency.

Figure adapted, with permission, from D Isaev et al. J Neuroscience 27: 4311587‐11594, 2007 96. Reprinted with permission.


Figure 23.

Atrial action potential (AP) waveform is modulated by expression of the polysialyltransferase, ST8Sia2. APs were measured from isolated control and ST8sia2(−/−) neonatal atrial and ventricular myocytes. Top left: typical AP waveforms measured from control (in black) and ST8sia2(−/−) (in red) atrial myocytes. (A‐C) The mean ± SEM AP waveform parameters (relevant portions of AP marked with arrows). Black, control atrium (n = 7); red, ST8sia2 (−/−) atrium (n = 9); dark blue, control ventricle (n = 7); blue, ST8sia2(−/−) ventricle (n = 5). (A) Time to AP peak. (B) AP duration at 50% repolarization (APD50). (C) AP duration at 90% repolarization (APD90). Significance tested using a two‐tailed t test and comparing ST8sia2(−/−) to control parameters. *, significant (P<0.005); #, not significant (P>0.1). Figure and legend adapted, with permission, from ML Montpetit et al. Proc Natl Acad Sci, U S A 106: 16517‐16522, 2009 142.



Figure 24.

Atrial Nav gating is modulated by ST8Sia2 expression. ST8sia2(−/−) and control myocyte Nav gating. Left panels (A, C, E, and G): atrium, ST8sia2(−/−) (red; n = 10); control (black; n = 4). Right panels (B, D, F, and H): ventricle, ST8sia2(−/−) (blue; n = 4); control (dark blue; n = 6). (A and B) Steady‐state activation. Insets: half‐activation voltage. (C and D) Steady‐state channel availability (SSI). Dashed line: ST8sia2(−/−) data shifted along voltage axis by −7.3 mV (to mimic measured shift in half‐activation voltage). Insets: half‐inactivation voltage. (E and F) Fast inactivation time constants. Dashed line: The ST8sia2(−/−) data shifted along voltage axis by −7.3 mV. Insets: typical whole cell Na+ current traces elicited by stepping to a −40 mV test potential. (G and H) Time constants of recovery from fast inactivation at a −130 mV recovery potential. Significance tested using a two‐tailed t test and comparing ST8sia2(−/−) to control parameters. *, significant (P ≤ 0.005); #, not significant (P > 0.1). Figure and legend adapted, with permission, from ML Montpetit et al. Proc Natl Acad Sci, U S A 106: 16517‐16522, 2009 142.



Figure 25.

MLP−/− ventricular myocytes are susceptible to early afterdepolarizations. MLP−/− mice show extended QT intervals (A), increased action potential durations (B), and increased susceptibility to early after depolarizations (C and D).

Figure adapted, with permission, from CA Ufret‐Vincenty et al. J Biol Chem 276: 28197‐28203, 2001 199. Reprinted with permission.


Figure 26.

Gating of MLP−/− ventricular Nav is less sensitive to desialylation than the wild‐type channel. INa for desialylated control myocytes mimicked untreated and neuraminidase‐treated MLP−/− myocyte INa. (A) Current‐voltage (I‐V) relationships. (B) Conductance‐voltage (G‐V) relationships. (D) SSI curves. (E) Fast inactivation times constants.

Figure adapted, with permission, from CA Ufret‐Vincenty et al. J Biol Chem 276: 28197‐28203, 2001 199. Reprinted with permission.
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Article Title: Modulation of Voltage‐Gated Ion Channels by Sialylation
 

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Andrew R. Ednie, Eric S. Bennett. Modulation of Voltage‐Gated Ion Channels by Sialylation. Compr Physiol 2012, 2: 1269-1301. doi: 10.1002/cphy.c110044