Graduation Year

2009

Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Molecular Pharmacology and Physiology

Major Professor

Eric S. Bennett, Ph.D.

Keywords

Sialylation, Potassium channel, Cardiac conduction, Arrhythmia

Abstract

Neuronal, cardiac, and skeletal muscle electrical signaling is achieved through the highly regulated activity of several types of voltage-gated ion channels to produce an action potential (AP). Voltage-gated potassium (Kv) channels are responsible for repolarization of the AP. Kv channels are uniquely and heavily glycosylated proteins. Previous reports indicate glycosylation modulates gating of some Kv channel isoforms; often, terminal sialic acid residues alter Kv channel gating. Here, we questioned whether alterations in glycosylation impact Kv channel gating, thus altering APs and cardiac excitability. ST3Gal-IV, a sialyltransferase expressed at uniform levels throughout the heart, adds sialic acids to N- and O-glycans through alpha 2-3 linkages. Electrocardiograms (ECGs) suggest that cardiac conduction/rhythm are altered in ST3Gal-IV(-/-) animals, which show an increased incidence of arrhythmic beats.

AP waveform parameters and two components of IK, the transient outward, Ito, and the slowly inactivating, IK,slow, were compared in neonatal control versus ST3Gal-IV(-/-) and glycosidase treated atrial and ventricular myocytes. Action potential durations (APDs) measured from ST3Gal-IV(-/-) and glycosidase treated atrial myocytes were lengthened significantly (~25-150%) compared to control; however, ventricular APDs were unaffected by changes in glycosylation. Consistently, atrial Ito and IK,slow activation were shifted to more depolarized potentials (by ~9-17 mV) in ST3Gal-IV(-/-) and glycosidase treated myocytes, while ventricular K+ currents were unaltered. Those channels responsible for producing Ito and IK,slow were examined under conditions of full and reduced glycosylation.

Sialylation and N-glycosylation uniquely and differently impact gating of two mammalian Shaker family Kv channel isoforms, Kv1.4 and Kv1.5; Kv1.4 gating was unaffected by changes in channel glycosylation, while N-linked sialic acids, acting through electrostatic mechanisms, fully account for glycan effects on Kv1.5 gating. In addition, sialic acids modulate the gating of three Kv channel isoforms that are not N-glycosylated, Kv2.1, Kv4.2, and Kv4.3, through apparent electrostatic mechanisms. Click chemistry was utilized to confirm that these three isoforms are O-glycosylated and sialylated; thus, O-linked sialylation modulates gating of Kv2.1, Kv4.2, and Kv4.3. This study suggests that regulated or aberrant glycosylation alters the gating of channels producing IK in a chamber-specific manner, thus altering the rate of cardiac repolarization and potentially leading to arrhythmias.

Share

COinS