Methyl-β-cyclodextrin reversibly alters the gating of lipid rafts-associated Kv1.3 channels in Jurkat T lymphocytes

Abstract

The voltage-dependent Kv1.3 potassium channels mediate a variety of physiological functions in human T lymphocytes. These channels, along with their multiple regulatory components, are localized in cholesterol-enriched microdomains of plasma membrane (lipid rafts). In this study, patch-clamp technique was applied to explore the impact of the lipid-raft integrity on the Kv1.3 channel functional characteristics. T lymphoma Jurkat cells were treated for 1 h with cholesterol-binding oligosaccharide methyl-beta-cyclodextrin (MbetaCD) in 1 or 2 mM concentration, resulting in depletion of cholesterol by 63 +/- 5 or 75 +/- 4%, respectively. Treatment with 2 mM MbetaCD did not affect the cells viability but retarded the cell proliferation. The latter treatment caused a depolarizing shift of the Kv1.3 channel activation and inactivation by 11 and 6 mV, respectively, and more than twofold decrease in the steady-state activity at depolarizing potentials. Altogether, these changes underlie the depolarization of membrane potential, recorded in a current-clamp mode. The effects of MbetaCD were concentration- and time-dependent and reversible. Both development and recovery of the MbetaCD effects were completed within 1-2 h. Therefore, cholesterol depletion causes significant alterations in the Kv1.3 channel function, whereas T cells possess a potential to reverse these changes.

TRESK-like potassium channels in leukemic T cells

“…1a), with characteristics identical to those described previously [35]. Later on, two possible scenarios could be realized: (a) at all times only Kv1.3 current was detected, or (b) in addition to Kv1.3 current, a run up of an instantaneous current was observed (Fig.…”

“…Fabrication of patch pipettes for whole cell measurements was as described previously [35], whereas for excised patches higher resistance patch electrodes were used. When filled with a standard solution containing (in mM): 134 KCl, 2 MgCl 2 , 1 CaCl 2 , 10 EGTA, 10 HEPES-KOH (pH 7.4; 7.5-nM-free Ca 2+ ) and inserted in bath solution contained (in mM): 150 NaCl, 5 KCl, 1 MgCl 2 , 2.5 CaCl 2 , 10 HEPES-NaOH (pH 7.4), the resistance of patch electrodes was 3-5 and 6-8 MΩ for whole cell and outside-out patches, respectively.…”

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).…”

The Epithelial Sodium Channel (ENaC) Traffics to Apical Membrane in Lipid Rafts in Mouse Cortical Collecting Duct Cells

“…In some cases rafts and/or cholesterol have been found to have functional effects on channel kinetics independent of trafficking, suggesting a role for protein-lipid interactions in modulating channel activities (12). We have previously shown that the epithelial Na ϩ channel (ENaC) 3 is present in lipid rafts in A6 renal epithelial cells, which endogenously express this channel, and suggested that raft distribution was related to hormonal regulation (13).…”