Differential Targeting of Shaker-like Potassium Channels to Lipid Rafts

Abstract

Ion channel targeting within neuronal and muscle membranes is an important determinant of electrical excitability. Recent evidence suggests that there exists within the membrane specialized microdomains commonly referred to as lipid rafts. These domains are enriched in cholesterol and sphingolipids and concentrate a number of signal transduction proteins such as nitricoxide synthase, ligand-gated receptors, and multiple protein kinases. Here, we demonstrate that the voltagegated K ؉ channel Kv2.1, but not Kv4.2, targets to lipid rafts in both heterologous expression systems and rat brain. The Kv2.1 association with lipid rafts does not appear to involve caveolin. Depletion of cellular cholesterol alters the buoyancy of the Kv2.1 associated rafts and shifts the midpoint of Kv2.1 inactivation by nearly 40 mV without affecting peak current density or channel activation. The differential targeting of Kv channels to lipid rafts represents a novel mechanism both for the subcellular sorting of K ؉ channels to regions of the membrane rich in signaling complexes and for modulating channel properties via alterations in lipid content.The subcellular localization of ion channels is necessary for proper electrical signaling. In cardiac and skeletal myocytes, ion channels show a differential surface distribution (1, 2). Within the brain, voltage-gated K ϩ (Kv) channels often show not only polarized sorting to either axons or dendrites, but also isoform-specific localization within dendrites alone. Thus, there exists specific sorting mechanisms for restricting lateral distribution within a given membrane domain (3). One physiological consequence for such specific localization is that it places various signal transduction molecules near their ion channel substrates (4). Several families of intracellular proteins, PDZs and AKAPs, have been shown to cluster both ion channels and modulatory signaling enzymes. Indeed, great emphasis has been placed on the role of PDZ proteins such as PSD-95 in the targeting and localization of ion channels and neurotransmitter receptors (5). In contrast, the role of membrane lipids in differential targeting and integration of ion channels within the plane of the plasma membrane has not been addressed.Recent advances in the study of cell membrane structure have led to the emerging idea that microdomains exist within the fluid bilayer of the plasma membrane. These dynamic structures, termed lipid rafts, are rich in tightly packed sphingolipids and cholesterol (6). The rafts, which are present in both excitable and non-excitable cells, localize a number of membrane proteins, including multiple signal transduction molecules, while excluding others (7). Different types of rafts are likely to exist based on the presence of specific marker proteins and ultrastructure data (8). Caveolae represent one well studied subpopulation of lipid raft having an invaginated morphology and containing the scaffolding protein, caveolin, which interacts directly with several intracellular proteins (7, 9 -12). Here, we de...

Localization-dependent activity of the Kv2.1 delayed-rectifier K + channel

“…Details with respect to these methods are contained within SI Text and corresponding references (13,14,26,(32)(33)(34)(35)(36). Only low passage number (<45) HEK cells lacking endogenous K + channel activity were used.…”

Apical sorting of a voltage- and Ca 2+ -activated K + channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation

“…4A). Because the stability of the interaction of hSlo-N200A with lipid rafts to detergent extraction might be low, we also have used an alternative detergent-free method for lipid raft isolation based on sodium carbonate extraction at pH 11 (38). This procedure showed more hSlo-N200A floating in low buoyancy fractions (Fig.…”

“…Procedures to obtain permanent transformants; assess protein expression by metabolic labeling; determine polarity by domainspecific biotinylation in cells cultured on Transwell chambers, or by indirect immunofluorescence; and measure relative density of fluorograms all have been described (36,37). Lipid raft association was assessed by isolating low density membrane fractions after either ice-cold detergent extraction or sodium carbonate extraction (100 mM or 500 mM) pH 11 at 4°C for 20 min, as described (38), except that in both cases, floatation of lipid rafts was made on linear sucrose gradients without eliminating cell debris.…”

Kv1.3 potassium channels are localized in the immunological synapse formed between cytotoxic and target cells

“…Several recent studies (17,18) have indicated that voltagegated potassium channels segregate into specialized membrane microdomains modulating their activity. The predominant voltage-gated potassium channel of T lymphocytes is Kv1.3.…”