Glycosylation Modulates Cardiac Excitability by Altering Voltage-Gated Potassium Currents

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

Differential stimulation of insulin secretion by GLP-1 and Kisspeptin-10.

β-cells in the pancreatic islet respond to elevated plasma glucose by secreting insulin to maintain glucose homeostasis. In addition to glucose stimulation, insulin secretion is modulated by numerous G-protein coupled receptors (GPCRs). The GPCR ligands Kisspeptin-10 (KP) and glucagon-like peptide-1 (GLP-1) potentiate insulin secretion through Gq and Gs-coupled receptors, respectively. Despite many studies, the signaling mechanisms by which KP and GLP-1 potentiate insulin release are not thoroughly understood. We investigated the downstream signaling pathways of these ligands and their affects on cellular redox potential, intracellular calcium activity ([Ca(2+)]i), and insulin secretion from β-cells within intact murine islets. In contrast to previous studies performed on single β-cells, neither KP nor GLP-1 affect [Ca(2+)]i upon stimulation with glucose. KP significantly increases the cellular redox potential, while no effect is observed with GLP-1, suggesting that KP and GLP-1 potentiate insulin secretion through different mechanisms. Co-treatment with KP and the Gβγ-subunit inhibitor gallein inhibits insulin secretion similar to that observed with gallein alone, while co-treatment with gallein and GLP-1 does not differ from GLP-1 alone. In contrast, co-treatment with the Gβγ activator mSIRK and either KP or GLP-1 stimulates insulin release similar to mSIRK alone. Neither gallein nor mSIRK alter [Ca(2+)]i activity in the presence of KP or GLP-1. These data suggest that KP likely alters insulin secretion through a Gβγ-dependent process that stimulates glucose metabolism without altering Ca(2+) activity, while GLP-1 does so, at least partly, through a Gα-dependent pathway that is independent of both metabolism and Ca(2+)

Percent of insulin content secreted from intact islets after static incubation at 2.8, 10, and 16.7 mM glucose with and without treatment.

<p>Untreated control samples are shown in white. <b>A</b>, Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations in the presence and absence of GLP-1 (20 nM, light gray), the G_βγ inhibitor, gallein (10 µM, checked), or combination treatment with gallein and GLP-1 (striped). <b>B,</b> Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations with GLP-1 (20 nM, light gray), the G_βγ-activating peptide mSIRK (30 µM, checked), or combination treatment with mSIRK and GLP-1 (striped). Data are the mean ± S.E. <i>n</i> = 4–19. *(<i>p</i><0.05) and **(<i>p</i><0.001) indicate significance compared to untreated control, GLP-1 only, or gallein alone.</p

Percent of insulin content secreted from intact islets after incubation at 2.8, 10, or 16.7 mM glucose with and without treatment.

<p>Untreated control samples are shown in white. <b>A</b>, Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations in the presence and absence of KP (10 µM, dark gray), gallein (10 µM, checked), or gallein+KP (striped). <b>B</b>, Percent of insulin content secreted at 2.8, 10, and 16.7 mM glucose concentrations with and without KP (10 µM, dark gray), mSIRK (30 µM, checked), or mSIRK+KP (striped). Data are the mean ± S.E. <i>n</i> = 4–19. *<i>p</i><0.05 and **<i>p</i><0.001 compared to untreated control, KP alone, or mSIRK only.</p

Changes in Fluo4 signal in dispersed β-cells recorded at 10 mM glucose in the presence and absence of KP (1 µM) or with GLP-1 (20 nM) to measure the frequency and amplitude of [Ca²⁺]_i oscillations.

<p><b>A & C</b>, Representative oscillations in [Ca²⁺]_i recorded from dispersed β-cells before (dotted line) and after (solid line) treatment with KP (<b>A</b>) or GLP-1 (<b>C</b>). <b>B & D</b>, The normalized [Ca²⁺]_i oscillation frequency measured pre- and post-treatment with KP (<b>B</b>, dark gray) or GLP-1 (<b>D</b>, light gray). Data are normalized to the data collected from the dispersed β-cells prior to ligand treatment (white). Data are the mean ± S.E. <i>n</i> = 4. <i>p</i><0.05.</p