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Cholesterol Modulation of BK (MaxiK; Slo1) Channels
Published in Qiu-Xing Jiang, New Techniques for Studying Biomembranes, 2020
Alex M. Dopico, Anna N. Bukiya, Kelsey North
From the very first reports on CLR modulation of BK channel function in rat aortic myocyte membranes coincidental changes in BK steady-activity and bilayer physical properties in response to modification of membrane CLR levels were documented (Bolotina et al., 1989; Bregestovski and Bolotina, 1989). A pioneer work showed that treatment of aortic smooth muscle cells with mevinolin, a CLR-depleting agent, led to a nine-fold increase in the channel open probability (Po) and a nearly two-fold increase in the rotational diffusion coefficient of diphenylhexatriene (DPH) (Bolotina et al., 1989), the latter being an indicator of lipid packing and order in the apolar regions of the membrane (Van Blitterswijk et al., 1981). Conversely, addition of CLR to aortic smooth muscle cell membranes resulted in a two-fold decrease in BK Po and a nearly two-fold decrease in DPH rotational diffusion. These data led authors conclude that modifications in the kinetic properties of BK channels by membrane CLR were “presumably due to changes in plasma membrane fluidity” (Bolotina et al., 1989) which, in more recent and accurate terminology, implies an increase in membrane lipid order parameters. Arguments against a major involvement of lipid order/packing in CLR modification of BK function are also indirect: first, while CLR inhibition of BK channels reconstituted into planar phospholipid bilayers shows a monotonic dependence on CLR molar fraction (0–50 mol%; Crowley et al., 2003; Bukiya et al., 2008), CLR distribution and formation of packed complexes with phospholipids into superlattices within the lipid bilayer (Chong et al., 2009) and membrane-associated properties, such as membrane packing, vary with CLR molar fraction in a nonmonotonic manner (Sugár and Chong, 2012). In addition, generalized polarization of Laurdan in large unilamellar vesicles where CLR was probed at small increments within a biologically relevant range (20–50 mol%) shows that fluorescence anisotropy does not change monotonically with increased CLR levels either (Venegas et al., 2007). Second, structure-activity relationship (SAR) data show that CLR analogs (CLR and epiCLR) having similar effect on bulk bilayer order, as measured by fluorescence polarization anisotropy (Gimpl et al., 1997; Xu and London, 2000), drastically differ in their inhibitory efficacy of BK channel activity (Bukiya et al., 2011a). The same study also reveals that coprostanol (COPR), while having an anti-CLR effect on bilayer lipid order (Xu and London, 2000) is an effective inhibitor of BK channel activity (Bukiya et al., 2011a).
Novel temporin L antimicrobial peptides: promoting self-assembling by lipidic tags to tackle superbugs
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Rosa Bellavita, Annarita Falanga, Elisabetta Buommino, Francesco Merlino, Bruno Casciaro, Floriana Cappiello, Maria Luisa Mangoni, Ettore Novellino, Maria Rosaria Catania, Rossella Paolillo, Paolo Grieco, Stefania Galdiero
Membrane fluidity was determined using LUVs containing the fluorescent probe Laurdan43. Laurdan was encapsulated into lipid films (0.1 mM) at a concentration of 0.001 mM. After lipid films with Laurdan were lyophilised, hydrated with PBS 1X buffer, pH 7.4, and vortexed for 1 h, they were freeze-thawed 6 times and extruded 10 times through polycarbonate membranes with 0.1 µm diameter pores, obtaining LUVs. The variation of fluidity membrane in presence of peptide was evaluated at 5 and 30 µM, under and above the CAC. The peptide, dissolved in water (2 mM peptide stock solution), was added to LUVs at specific P/L molar ratio and after 10 min, the fluorescence spectra were recorded using a 1 cm path length quartz cell, thermostated at 25 °C. Spectra were corrected for the baseline signal. Laurdan emission spectra were recorded from 400 to 550 nm with λex 365 nm in the absence or presence of peptide. Laurdan emission can shift from 440 nm, in the ordered phase, to 490 nm in the disordered phase. The Generalised Polarisation (GP) is a parameter commonly used to quantify the change in the lipid fluidity. It was calculated as GP = (I440 − I490)/(I440 + I490), where I440 and I490 are the fluorescence intensities at the maximum emission wavelength in the ordered (λem 440 nm) and disordered (λem 490 nm) phases43.