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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Background: Glycosylation results from the coordinated activities of glycogene products that can modulate ion channel function. Results: Gene ablation of the sialyltransferase ST3Gal4 impacts sialylation and gating of specific cardiac voltage-gated K

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Dilated cardiomyopathy (DCM), the third most common cause of heart failure, is often associated with arrhythmias and sudden cardiac death if not controlled. The majority of DCM is of unknown etiology. Protein sialylation is altered in human DCM, with responsible mechanisms not yet described. Here we sought to investigate the impact of clinically relevant changes in sialylation on cardiac function using a novel model for altered glycoprotein sialylation that leads to DCM and to chronic stress-induced heart failure (HF), deletion of the sialyltransferase, ST3Gal4. We previously reported that 12- to 20-week-old ST3Gal4−/− mice showed aberrant cardiac voltage-gated ion channel sialylation and gating that contribute to a pro-arrhythmogenic phenotype. Here, echocardiography supported by histology revealed modest dilated and thinner-walled left ventricles without increased fibrosis in ST3Gal4−/− mice starting at 1 year of age. Cardiac calcineurin expression in younger (16–20 weeks old) ST3Gal4−/− hearts was significantly reduced compared to WT. Transverse aortic constriction (TAC) was used as a chronic stressor on the younger mice to determine whether the ability to compensate against a pathologic insult is compromised in the ST3Gal4−/− heart, as suggested by previous reports describing the functional implications of reduced cardiac calcineurin levels. TAC’d ST3Gal4−/− mice presented with significantly reduced systolic function and ventricular dilation that deteriorated into congestive HF within 6 weeks post-surgery, while constricted WT hearts remained well-adapted throughout (ejection fraction, ST3Gal4−/− = 34 ± 5.2 %; WT = 53.8 ± 7.4 %; p < 0.05). Thus, a novel, sialo-dependent model for DCM/HF is described in which clinically relevant reduced sialylation results in increased arrhythmogenicity and reduced cardiac calcineurin levels that precede cardiomyopathy and TAC-induced HF, suggesting a causal link among aberrant sialylation, chronic arrhythmia, reduced calcineurin levels, DCM in the absence of a pathologic stimulus, and stress-induced HF.

Activity of human ether-a-go-go-related gene (hERG) 1 voltage-gated K(+) channels is responsible for portions of phase 2 and phase 3 repolarization of the human ventricular action potential. Here, we questioned whether and how physiologically and pathophysiologically relevant changes in surface N-glycosylation modified hERG channel function. Voltage-dependent hERG channel gating and activity were evaluated as expressed in a set of Chinese hamster ovary (CHO) cell lines under conditions of full glycosylation, no sialylation, no complex N-glycans, and following enzymatic deglycosylation of surface N-glycans. For each condition of reduced glycosylation, hERG channel steady-state activation and inactivation relationships were shifted linearly by significant depolarizing ∼9 and ∼18 mV, respectively. The hERG window current increased significantly by 50-150%, and the peak shifted by a depolarizing ∼10 mV. There was no significant change in maximum hERG current density. Deglycosylated channels were significantly more active (20-80%) than glycosylated controls during phases 2 and 3 of action potential clamp protocols. Simulations of hERG current and ventricular action potentials corroborated experimental data and predicted reduced sialylation leads to a 50-70-ms decrease in action potential duration. The data describe a novel mechanism by which hERG channel gating is modulated through physiologically and pathophysiologically relevant changes in N-glycosylation; reduced channel sialylation increases hERG channel activity during the action potential, thereby increasing the rate of action potential repolarization.

Background Voltage-gated Na+ channels (Nav) are responsible for the initiation and conduction of neuronal and muscle action potentials. Nav gating can be altered by sialic acids attached to channel N-glycans, typically through isoform-specific electrostatic mechanisms. Methods Using two sets of Chinese Hamster Ovary cell lines with varying abilities to glycosylate glycoproteins, we show for the first time that sialic acids attached to O-glycans and N-glycans within the Nav1.4 D1S5-S6 linker modulate Nav gating. Results All measured steady-state and kinetic parameters were shifted to more depolarized potentials under conditions of essentially no sialylation. When sialylation of only N-glycans or of only O-glycans was prevented, the observed voltage-dependent parameter values were intermediate between those observed under full versus no sialylation. Immunoblot gel shift analyses support the biophysical data. Conclusions The data indicate that sialic acids attached to both N- and O-glycans residing within the Nav1.4 D1S5-S6 linker modulate channel gating through electrostatic mechanisms, with the relative contribution of sialic acids attached to N- versus O-glycans on channel gating being similar. General Significance Protein N- and O-glycosylation can modulate ion channel gating simultaneously. These data also suggest that Nav gating might be altered simply through exposure to different levels of sugar residues that serve as substrates for N- and O-glycosylation. Thus, in disease states that affect sugar substrate levels, the glycosylation process would be impacted, likely resulting in changes in Nav sialylation levels that would lead to modulated channel gating, AP waveforms and conduction.