Localization of Pacemaker Channels in Lipid Rafts Regulates Channel Kinetics

Abstract

Abstract-Lipid rafts are discrete membrane subdomains rich in sphingolipids and cholesterol. In ventricular myocytes a function of caveolae, a type of lipid rafts, is to concentrate in close proximity several proteins of the ␤-adrenergic transduction pathway. We have investigated the subcellular localization of HCN4 channels expressed in HEK cells and studied the effects of such localization on the properties of pacemaker channels in HEK and rabbit sinoatrial (SAN) cells. We used a discontinuous sucrose gradient and Western blot analysis to detect HCN4 proteins in HEK and in SAN cells, and found that HCN4 proteins localize to low-density membrane fractions together with flotillin (HEK) or caveolin-3 (SAN), structural proteins of caveolae. Lipid raft disruption by cell incubation with methyl-␤-cyclodextrin (M␤CD) impaired specific HCN4 localization. It also shifted the midpoint of activation of the HCN4 current in HEK cells and of I f in SAN cells to the positive direction by 11.9 and 10.4 mV, respectively. These latter effects were not due to elevation of basal cyclic nucleotide levels because the cholesterol-depletion treatment did not alter the current response to cyclic nucleotides. In accordance with an increased I f , M␤CD-treated SAN cells showed large increases of diastolic depolarization slope (87%) and rate (58%). We also found that the kinetics of HCN4-and native f-channel deactivation were slower after lipid raft disorganization. In conclusion, our work indicates that pacemaker channels localize to lipid rafts and that disruption of lipid rafts causes channels to redistribute within the membrane and modifies their kinetic properties. Key Words: HCN channels Ⅲ pacemaker current Ⅲ lipid rafts Ⅲ caveolin Ⅲ sinoatrial node T he sinoatrial node (SAN) is the region of the heart from which spontaneous action potentials originate and propagate to determine cardiac rhythm. This particular anatomical district is composed by specialized myocytes (pacemaker cells) whose activity is characterized by a slow diastolic depolarization phase at the end of the action potential. The pacemaker (I f ) current plays a key role in the generation of diastolic depolarization. f-Channels open toward the end of the action potential repolarization process and carry an inward current that depolarizes the membrane and drives the membrane potential up to threshold for initiating a new action potential. Although spontaneous activity is an intrinsic property of the heart, independent of innervation, fine modulation of heart rate is achieved through the release of the neurotransmitters norepinephrine (NE) and acetylcholine (ACh) by sympathetic and parasympathetic branches of the autonomic nervous system. It is well established that in cardiac cells NE and ACh, through specific ␤-adrenergic (␤-AR) and muscarinic (mAChR) receptors, modulate intracellular level of cAMP. Direct binding of cAMP to f-channels shifts their activation curve toward more depolarized potentials, thus increasing the steepness of the diastolic depolarization. 1 cAM...

Caveolin Regulates Kv1.5 Trafficking to Cholesterol-Rich Membrane Microdomains

“…We have shown previously that depletion of cholesterol results in a shift in the steady-state activation and inactivation kinetics of Kv1.5 in the hyperpolarizing direction, opposite that measured with cholesterol enrichment (Martens et al, 2001). It is interesting that most other reports of cholesterol modulation of ion channel activity also find effects on the activation and/or inactivation properties (Barbuti et al, 2004;Martens et al, 2004;Maguy et al, 2006;Pottosin et al, 2007). Together, these data emphasize the importance of membrane cholesterol levels and microdomain localization in regulating the voltage-sensitivity of Kv1.5.…”

“…1 and 5). Previous work, including our own (Martens et al, 2001, has used cholesterol-depleting agents such as ␤-methyl cyclodextrin to disrupt microdomain organization and alter channel function while extrapolating these effects to functional consequence of lipid raft localization (Hnasko and Lisanti, 2003;Barbuti et al, 2004;Davies et al, 2006;Maguy et al, 2006;Pottosin et al, 2007). However, increased awareness to the plurality of their effects, including cytoskeletal disruption, complicates the interpretation of data using these agents (Kwik et al, 2003).…”

Deletion of integrin linked kinase in endothelial cells results in defective RTK signaling caused by caveolin 1 mislocalization

“…Because of the lack of caveolin 1 association with the plasma membrane in Ilk -/-M-ECs, we investigated whether disruption of caveolin 1 assembly and caveolin 1 microdomain formation in WT M-ECs could reproduce the functional defects observed in the Ilk -/-M-ECs. For this purpose, we used beta cyclodextrin (MbetaCD); this compound is a known disruptor of lipid rafts and acts via depletion of cholesterol causing malpositioning of membrane protein complexes (Barbuti et al, 2004;Jang et al, 2001). In agreement with our hypothesis, MbetaCD (2%) prevented VEGF-induced increase of [Ca 2+ ] i in WT M-ESCs (n=11) (Fig.…”

The Funny Current in Cardiac Non-Pacemaker Cells: Functional Role and Pharmacological Modulation

“…In addition to this, membrane sub-localization with interacting proteins, e.g. caveolin-3, may also contribute to differences in channel voltage activation (Barbuti et al, 2004;Barbuti et al, 2007). Despite the observed properties of funny current in the ventricular cardiomyocytes, a contribution to the diastolic phase of ventricular action potential is possible, since membrane resting potential is also more negative in ventricular cells than in sinoatrial node cells.…”