A potential role for sialic acid in the voltage-dependent gating of rat skeletal muscle sodium channels (rSkM1) was investigated using Chinese hamster ovary (CHO) cells stably transfected with rSkM1. Changes in the voltage dependence of channel gating were observed after enzymatic (neuraminidase) removal of sialic acid from cells expressing rSkM1 and through the expression of rSkM1 in a sialylation-deficient cell line (lec2). The steady-state half-activation voltages (Va) of channels under each condition of reduced sialylation were ∼10 mV more depolarized than control channels. The voltage dependence of the time constants of channel activation and inactivation were also shifted in the same direction and by a similar magnitude. In addition, recombinant deletion of likely glycosylation sites from the rSkM1 sequence resulted in mutant channels that gated at voltages up to 10 mV more positive than wild-type channels. Thus three independent means of reducing channel sialylation show very similar effects on the voltage dependence of channel gating. Finally, steady-state activation voltages for channels subjected to reduced sialylation conditions were much less sensitive to the effects of external calcium than those measured under control conditions, indicating that sialic acid directly contributes to the negative surface potential. These results are consistent with an electrostatic mechanism by which external, negatively charged sialic acid residues on rSkM1 alter the electric field sensed by channel gating elements.

Voltage-gated sodium channels are responsible for the initiation and propagation of nerve, skeletal muscle, and cardiac action potentials. The orchestrated activation and inactivation gating of sodium channels is vital to normal neuronal signalling, skeletal muscle contraction, and normal heart rhythms. Even small syncopations from this normal gating rhythm may alter cellular excitability and whole animal physiology significantly. Because channel gating is dependent directly on the membrane potential, anything that alters this potential will affect gating. For example, external calcium alters the voltage dependence of channel activation such that as external calcium is increased, a greater depolarization is required in order to achieve the same degree of channel opening (Frankenhauser & Hodgkin, 1957; Bennett et al. 1997; Hille, 2001). The surface charge hypothesis predicts that the surface of the membrane near the channel has fixed negative charges that alter the electric field sensed by the gating mechanism of the channel. These negative charges are formally equivalent to internal fixed positive charges that will depolarize the membrane and thereby move the transmembrane potential closer to the threshold for channel opening. Calcium may act to neutralize the effects of these external negative charges, either through direct interaction with these charges or by a screening mechanism, effectively hyperpolarizing the membrane. Therefore, depolarizations sufficient to activate channels in low calcium are no longer adequate in elevated calcium.Patients with misregulated plasma calcium levels (hypoand hypercalcaemia) show symptoms consistent with direct effects of calcium on sodium channel gating 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, Na v1.4 (hSkM1), and Na v1.5 (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 (V a ), the voltage of half-inactivation (V i ), the time constants for fast inactivation (t h ), and the time constants for recovery from inactivation (t rec ), 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 V a with changing external [Ca 2+ ] 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 t...

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 on the gating 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.

In the present study we investigated cardiac hypertrophy and cardiac complications in mice subjected to hyperoxia. Results demonstrate that there is a significant increase in average heart weight to tibia length (22%) in mice subjected to hyperoxia treatment vs. normoxia. Functional assessment was performed in mice subjected to hyperoxic treatment, and results demonstrate impaired cardiac function with decreased cardiac output and heart rate. Staining of transverse cardiac sections clearly demonstrates an increase in the cross-sectional area from hyperoxic hearts compared with control hearts. Quantitative real-time RT-PCR and Western blot analysis indicated differential mRNA and protein expression levels between hyperoxia-treated and control left ventricles for ion channels including Kv4.2 (Ϫ2 Ϯ 0.08), Kv2.1 (2.54 Ϯ 0.48), and Scn5a (1.4 Ϯ 0.07); chaperone KChIP2 (Ϫ1.7 Ϯ 0.06); transcriptional factors such as GATA4 (Ϫ1.5 Ϯ 0.05), Irx5 (5.6 Ϯ 1.74), NFB1 (4.17 Ϯ 0.43); hypertrophy markers including MHC-6 (2.17 Ϯ 0.36) and MHC-7 (4.62 Ϯ 0.76); gap junction protein Gja1 (4.4 Ϯ 0.8); and microRNA processing enzyme Drosha (4.6 Ϯ 0.58). Taken together, the data presented here clearly indicate that hyperoxia induces left ventricular remodeling and hypertrophy and alters the expression of Kv4.2 and MHC6/7 in the heart. ion channel regulation; hyperoxia; heart; hypertrophy; potassium channel; redox PATIENTS in critical or intensive care units (ICU) with acute lung injury or cardiac disease are often administered 100% O 2 for treatment. Recent studies indicate that hyperoxia induces cardiac injury due to dysfunctional lung and compromised pulmonary functioning (37), even though the exact nature of this problem remains unknown. Here, we evaluated changes in expression of the ion channel and key transcriptional factors in the heart that occur with hyperoxia and likely play a role in cardiovascular remodeling.Potassium channels and their auxiliary subunits such as potassium channel interacting protein-2 (KChIP2) are abundantly expressed in the heart (5, 7, 35). It is established that the potassium channels Kv4.2 and Kv1.5 are responsive to oxygen changes (29, 39). In the present study, we investigate whether hyperoxia alters expression of the transcription factors Irx5 and Mef2c, which are implicated to play a direct role in regulating Kv4.2 expression (7,15,22). Cardiac-specific markers used to identify hypertrophy and transcriptional changes (9, 22) were also evaluated by assessing myosin heavy chain-6, and -7 (MHC6, MHC7), zinc finger transcription factor (GATA4), histone-lysine N-methyltransferase (Ezh2), and Six-1 expression levels with hyperoxia.Key inflammatory mediators such as TNF␣ and NFB are central regulators or master switches for many pathological processes (10,20,30). Recent evidence indicates that NFB regulates KChIP2, which in turn regulates Kv4.2 expression (26). Therefore we assessed the levels of Kv4.2, KChIP2, and NFB in the mouse heart subjected to hyperoxia. We hypothesized that hyperoxia induces cardi...

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. action potentials ͉ cardiomyocyte ͉ glycomics ͉ ion channels ͉ sialic acids

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 Na(v1.4) 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.

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.

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.