Eric S. Bennett

Voltage-gated Na + channels confer invasive properties on human prostate cancer cells

Prostate cancer is the second leading cause of cancer deaths in American males, resulting in an estimated 37,000 deaths annually, typically the result of metastatic disease. A consequence of the unsuccessful androgen ablation therapy used initially to treat metastatic disease is the emergence of androgen-insensitive prostate cancer, for which there is currently no prescribed therapy. Here, three related human prostate cancer cell lines that serve as a model for this dominant form of prostate cancer metastasis were studied to determine the correlation between voltage-gated sodium channel expression/function and prostate cancer metastatic (invasive) potential: the non-metastatic, androgen-dependent LNCaP LC cell line and two increasingly tumorogenic, androgen-independent daughter cell lines, C4 and C4-2. Fluorometric in vitro invasion assays indicated that C4 and C4-2 cells are more invasive than LC cells. Immunoblot analysis showed that voltage-gated sodium channel expression increases with the invasive potential of the cell line, and this increased invasive potential can be blocked by treatment with the specific voltage-gated sodium channel inhibitor, tetrodotoxin (TTX). These data indicate that increased voltage-gated sodium channel expression and function are necessary for the increased invasive potential of these human prostate cancer cells. When the human adult skeletal muscle sodium channel Nav1.4 was expressed transiently in each cell line, there was a highly significant increase in the numbers of invading LC, C4, and C4-2 cells. This increased invasive potential was reduced to control levels by treatment with TTX. These data are the first to indicate that the expression of voltage-gated sodium channels alone is sufficient to increase the invasive potential of non-metastatic (LC cells) as well as more aggressive cells (i.e., C4 and C4-2 cells). Together, the data suggest that increased voltage-gated sodium channel expression alone is necessary and sufficient to increase the invasive potential of a set of human prostate cancer cell lines that serve as a model for prostate cancer metastasis.

Regulated and aberrant glycosylation modulate cardiac electrical signaling

Millions afflicted with Chagas disease and other disorders of aberrant glycosylation suffer symptoms consistent with altered electrical signaling such as arrhythmias, decreased neuronal conduction velocity, and hyporeflexia. Cardiac, neuronal, and muscle electrical signaling is controlled and modulated by changes in voltage-gated ion channel activity that occur through physiological and pathological processes such as development, epilepsy, and cardiomyopathy. Glycans attached to ion channels alter channel activity through isoform-specific mechanisms. Here we show that regulated and aberrant glycosylation modulate cardiac ion channel activity and electrical signaling through a cell-specific mechanism. Data show that nearly half of 239 glycosylation-associated genes (glycogenes) were significantly differentially expressed among neonatal and adult atrial and ventricular myocytes. The N-glycan structures produced among cardiomyocyte types were markedly variable. Thus, the cardiac glycome, defined as the complete set of glycan structures produced in the heart, is remodeled. One glycogene, ST8sia2, a polysialyltransferase, is expressed only in the neonatal atrium. Cardiomyocyte electrical signaling was compared in control and ST8sia2(−/−) neonatal atrial and ventricular myocytes. Action potential waveforms and gating of less sialylated voltage-gated Na+ channels were altered consistently in ST8sia2(−/−) atrial myocytes. ST8sia2 expression had no effect on ventricular myocyte excitability. Thus, the regulated (between atrium and ventricle) and aberrant (knockout in the neonatal atrium) expression of a single glycogene was sufficient to modulate cardiomyocyte excitability. A mechanism is described by which cardiac function is controlled and modulated through physiological and pathological processes that involve regulated and aberrant glycosylation.

Isoform-specific effects of sialic acid on voltage-dependent Na+ channel gating: Functional sialic acids are localized to the S5-s6 loop of domain I

The isoform specific role of sialic acid in human voltage‐gated sodium channel gating was investigated through expression and chimeric analysis of two human isoforms, Nav1.4 (hSkM1), and Nav1.5 (hH1) in Chinese hamster ovary (CHO) cell lines. Immunoblot analyses indicate that both hSkM1 and hH1 are glycosylated and that hSkM1 is more glycosylated than hH1. Four sets of voltage‐dependent parameters, the voltage of half‐activation (Va), the voltage of half‐inactivation (Vi), the time constants for fast inactivation (τh), and the time constants for recovery from inactivation (τrec), were measured for hSkM1 and hH1 expressed in two CHO cell lines, Pro5 and Lec2, to determine the effect of changing sialylation on channel gating under conditions of full (Pro5) or reduced (Lec2) sialylation. For all parameters measured, hSkM1 gating showed a consistent 11–15 mV depolarizing shift under conditions of reduced sialylation, while hH1 showed no significant change in any gating parameter. Shifts in channel Va with changing external [Ca2+] indicated that sialylation of hSkM1, but not hH1, directly contributes to a negative surface potential. Functional analysis of two chimeras, hSkM1P1 and hH1P1, indicated that the responsible sialic acids are localized to the hSkM1 S5‐S6 loop of domain I. When hSkM1 IS5‐S6 was replaced by the analogous hH1 loop (hSkM1P1), changing sialylation had no significant effect on any voltage‐dependent parameter. Conversely, when hSkM1 IS5‐S6 was added to hH1 (hH1P1), all four parameters shifted by 6–7 mV in the depolarized direction under conditions of reduced sialylation. In summary, the gating of two human sodium channel isoforms show very different dependencies on sialic acid, with hSkM1 gating uniformly altered by sialic acid levels through an apparent electrostatic mechanism, while hH1 gating is unaffected by changing sialylation. Sialic acid‐dependent gating can be removed or created by replacing or inserting hSkM1 IS5‐S6, respectively, indicating that the functionally relevant sialic acid residues are localized to the first domain of the channel.

Isoform-specific Effects of the β2 Subunit on Voltage-gated Sodium Channel Gating

Voltage-gated sodium channels (Nav) are complex glycoproteins comprised of an α subunit and often one to several β subunits. We have shown that sialic acid residues linked to Nav α and β1 subunits alter channel gating. To determine whether β2-linked sialic acids similarly impact Nav gating, we co-expressed β2 with Nav1.5 or Nav1.2 in Pro5 (complete sialylation) and in Lec2 (essentially no sialylation) cells. β2 sialic acids caused a significant hyperpolarizing shift in Nav1.5 voltage-dependent gating, thus describing for the first time an effect of β2 on Nav1.5 gating. In contrast, β2 caused a sialic acid-independent depolarizing shift in Nav1.2 gating. A deglycosylated mutant, β2-ΔN, had no effect on Nav1.5 gating, indicating further the impact of β2 N-linked sialic acids on Nav1.5 gating. Conversely, β2-ΔN modulated Nav1.2 gating virtually identically to β2, confirming that β2 N-linked sugars have no impact on Nav1.2 gating. Thus, β2 modulates Nav gating through multiple mechanisms possibly determined by the associated α subunit. β1 and β2 were expressed together with Nav1.5 or Nav1.2 in Pro5 and Lec2 cells. Together β1 and β2 produced a significantly larger sialic acid-dependent hyperpolarizing shift in Nav1.5 gating. Under fully sialylating conditions, the Nav1.2·β1·β2 complex behaved like Nav1.2 alone. When sialylation was reduced, only the sialic acid-independent depolarizing effects of β2 on Nav1.2 gating were apparent. Thus, the varied effects of β1 and β2 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.

Differential Sialylation Modulates Voltage-gated Na+ Channel Gating throughout the Developing Myocardium

Voltage-gated sodium channel function from neonatal and adult rat cardiomyocytes was measured and compared. Channels from neonatal ventricles required an ∼10 mV greater depolarization for voltage-dependent gating events than did channels from neonatal atria and adult atria and ventricles. We questioned whether such gating shifts were due to developmental and/or chamber-dependent changes in channel-associated functional sialic acids. Thus, all gating characteristics for channels from neonatal atria and adult atria and ventricles shifted significantly to more depolarized potentials after removal of surface sialic acids. Desialylation of channels from neonatal ventricles did not affect channel gating. After removal of the complete surface N-glycosylation structures, gating of channels from neonatal atria and adult atria and ventricles shifted to depolarized potentials nearly identical to those measured for channels from neonatal ventricles. Gating of channels from neonatal ventricles were unaffected by such deglycosylation. Immunoblot gel shift analyses indicated that voltage-gated sodium channel α subunits from neonatal atria and adult atria and ventricles are more heavily sialylated than α subunits from neonatal ventricles. The data are consistent with approximately 15 more sialic acid residues attached to each α subunit from neonatal atria and adult atria and ventricles. The data indicate that differential sialylation of myocyte voltage-gated sodium channel α subunits is responsible for much of the developmental and chamber-specific remodeling of channel gating observed here. Further, cardiac excitability is likely impacted by these sialic acid–dependent gating effects, such as modulation of the rate of recovery from inactivation. A novel mechanism is described by which cardiac voltage-gated sodium channel gating and subsequently cardiac rhythms are modulated by changes in channel-associated sialic acids.

Modulation of voltage-gated ion channels by sialylation.

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.

Ethanol administration exacerbates the abnormalities in hepatic lipid oxidation in genetically obese mice

Alcohol consumption synergistically increases the risk and severity of liver damage in obese patients. To gain insight into cellular or molecular mechanisms underlying the development of fatty liver caused by ethanol-obesity synergism, we have carried out animal experiments that examine the effects of ethanol administration in genetically obese mice. Lean wild-type (WT) and obese (ob/ob) mice were subjected to ethanol feeding for 4 wk using a modified Lieber-DeCarli diet. After ethanol feeding, the ob/ob mice displayed much more pronounced changes in terms of liver steatosis and elevated plasma levels of alanine aminotransferase and aspartate aminotransferase, indicators of liver injury, compared with control mice. Mechanistic studies showed that ethanol feeding augmented the impairment of hepatic sirtuin 1 (SIRT1)-AMP-activated kinase (AMPK) signaling in the ob/ob mice. Moreover, the impairment of SIRT1-AMPK signaling was closely associated with altered hepatic functional activity of peroxisome proliferator-activated receptor γ coactivator-α and lipin-1, two vital downstream lipid regulators, which ultimately contributed to aggravated fatty liver observed in ethanol-fed ob/ob mice. Taken together, our novel findings suggest that ethanol administration to obese mice exacerbates fatty liver via impairment of the hepatic lipid metabolism pathways mediated largely by a central signaling system, the SIRT1-AMPK axis.

Gating of the shaker potassium channel is modulated differentially by N-glycosylation and sialic acids

N-linked glycans, including sialic acids, are integral components of ion channel complexes. To determine if N-linked sugars can modulate a rapidly inactivating K+ channel, the glycosylated Drosophila melanogaster Shaker K+ channel (ShB) and the N-glycosylation-deficient mutant (ShNQ), were studied under conditions of full and reduced sialylation. Through an apparent electrostatic mechanism, full sialylation induced uniform and significant hyperpolarizing shifts in all measured voltage-dependent ShB gating parameters compared to those measured under conditions of reduced sialylation. Steady-state gating of ShNQ was unaffected by changes in sialylation and was nearly identical to that observed for ShB under conditions of reduced sialylation, indicating that N-linked sialic acids were wholly responsible for the observed effects of sialic acid on ShB gating. Interestingly, the rates of transition among channel states and the voltage-independent rates of activation and inactivation were significantly slower for ShNQ compared to ShB. Both effects were independent of sialylation, indicating that N-linked sugars other than sialic acids alter ShB gating kinetics but have little to no effect on the steady-state distribution of channels among states. The effect of sialic acids on channel gating, particularly inactivation gating, and the impact of other N-linked sugars on channel gating kinetics are unique to the ShB isoform. Thus, ShB gating is modulated by two complementary but distinct sugar-dependent mechanisms, (1) an N-linked sialic acid-dependent surface charge effect and (2) a sialic acid-independent effect that is consistent with N-linked sugars affecting the stability of ShB among its functional states.

Sialic Acids Attached to O-Glycans Modulate Voltage-gated Potassium Channel Gating

Neuronal, cardiac, and skeletal muscle action potentials are produced and conducted through the highly regulated activity of several types of voltage-gated ion channels. Voltage-gated potassium (Kv) channels are responsible for action potential repolarization. Glycans can be attached to glycoproteins through N- and O-linkages. Previous reports described the impact of N-glycans on voltage-gated ion channel function. Here, we show that sialic acids attached through O-linkages modulate gating of Kv2.1, Kv4.2, and Kv4.3. The conductance-voltage (G-V) relationships for each isoform were shifted uniquely by a depolarizing 8–16 mV under conditions of reduced sialylation. The data indicate that sialic acids modulate Kv channel activation through apparent electrostatic mechanisms that promote channel activity. Voltage-dependent steady-state inactivation was unaffected by changes in sialylation. N-Linked sialic acids cannot be responsible for the G-V shifts because Kv4.2 and Kv4.3 cannot be N-glycosylated, and immunoblot analysis confirmed Kv2.1 is not N-glycosylated. Glycosidase gel shift analysis suggested that Kv2.1, Kv4.2, and Kv4.3 were O-glycosylated and sialylated. To confirm this, azide-modified sugar residues involved specifically in O-glycan and sialic acid biosynthesis were shown to incorporate into all three Kv channel isoforms using Cu(I)-catalyzed cycloaddition chemistry. Together, the data indicate that sialic acids attached to O-glycans uniquely modulate gating of three Kv channel isoforms that are not N-glycosylated. These data provide the first evidence that external O-glycans, with core structures distinct from N-glycans in type and number of sugar residues, can modulate Kv channel function and thereby contribute to changes in electrical signaling that result from regulated ion channel expression and/or O-glycosylation.

N-glycans modulate Kv1.5 gating but have no effect on Kv1.4 gating

Nerve and muscle action potential repolarization are produced and modulated by the regulated expression and activity of several types of voltage-gated K(+) (K(v)) channels. Here, we show that sialylated N-glycans uniquely impact gating of a mammalian Shaker family K(v) channel isoform, K(v)1.5, but have no effect on gating of a second Shaker isoform, K(v)1.4. Each isoform contains one potential N-glycosylation site located along the S1-S2 linker; immunoblot analyses verified that K(v)1.4 and K(v)1.5 were N-glycosylated. The conductance-voltage (G-V) relationships and channel activation rates for two glycosylation-site deficient K(v)1.5 mutants, K(v)1.5(N290Q) and K(v)1.5(S292A), and for wild-type K(v)1.5 expressed under conditions of reduced sialylation, were each shifted linearly by a depolarizing approximately 18 mV compared to wild-type K(v)1.5 activation. External divalent cation screening experiments suggested that K(v)1.5 sialic acids contribute to an external surface potential that modulates K(v)1.5 activation. Channel availability was unaffected by changes in K(v)1.5 glycosylation or sialylation. The data indicate that sialic acid residues attached to N-glycans act through electrostatic mechanisms to modulate K(v)1.5 activation. The sialic acids fully account for effects of N-glycans on K(v)1.5 gating. Conversely, K(v)1.4 gating was unaffected by changes in channel sialylation or following mutagenesis to remove the N-glycosylation site. Each phenomenon is unique for K(v)1 channel isoforms, indicating that sialylated N-glycans modulate gating of homologous K(v)1 channels through isoform-specific mechanisms. Such modulation is relevant to changes in action potential repolarization that occur as ion channel expression and glycosylation are regulated.