An ω-3, but Not an ω-6 Polyunsaturated Fatty Acid Decreases Membrane Dipole Potential and Stimulates Endo-Lysosomal Escape of Penetratin

Abstract

Although the largely positive intramembrane dipole potential (DP) may substantially influence the function of transmembrane proteins, its investigation is deeply hampered by the lack of measurement techniques suitable for high-throughput examination of living cells. Here, we describe a novel emission ratiometric flow cytometry method based on F66, a 3-hydroxiflavon derivative, and demonstrate that 6-ketocholestanol, cholesterol and 7-dehydrocholesterol, saturated stearic acid (SA) and ω-6 γ-linolenic acid (GLA) increase, while ω-3 α-linolenic acid (ALA) decreases the DP. These changes do not correlate with alterations in cell viability or membrane fluidity. Pretreatment with ALA counteracts, while SA or GLA enhances cholesterol-induced DP elevations. Furthermore, ALA (but not SA or GLA) increases endo-lysosomal escape of penetratin, a cell-penetrating peptide. In summary, we have developed a novel method to measure DP in large quantities of individual living cells and propose ALA as a physiological DP lowering agent facilitating cytoplasmic entry of penetratin.

Cyclodextrins Exert a Ligand-like Current Inhibitory Effect on the KV1.3 Ion Channel Independent of Membrane Cholesterol Extraction

“…On the other hand, iCDs not capable of depleting membrane cholesterol induced no significant changes in membrane fluidity. Similar effects were observed when examining membrane hydration through quantification of Laurdan GP inversely correlating with membrane hydration (Batta et al, 2021;Zakany et al, 2021). Laurdan GP of control cells was significantly reduced by CDs causing cholesterol depletion, while it was not affected by iCDs (Figure 3B).…”

“…Therefore, we examined membrane fluidity, hydration and lipid order using environment-sensitive fluorophores. First, to test membrane fluidity, we treated cells with 1 or 5 mM CDs for 1 h and determined TMA-DPH fluorescence anisotropy negatively correlating with the fluidity of the cell membrane ( Batta et al, 2021 ; Zakany et al, 2021 ). As can be expected from the intimate relationship between membrane cholesterol content and fluidity, TMA-DPH fluorescence anisotropy of control cells was significantly decreased by cholesterol-extracting CDs ( Figure 3A ).…”

“…In order to remove the potential contribution of internalized Laurdan to the measured signal and to ascertain that the calculated GP values reflect the hydration of the plasma membrane where the potassium channels are expressed, membrane lipid order was also investigated with PY3174. Analysis of PY3174 GP, a parameter that positively correlates with the degree of lipid order ( Kwiatek et al, 2013 ; Zakany et al, 2021 ), was restricted to the plasma membrane using segmentation of confocal microscopic images. PY3174 GP of control cells was significantly lowered by conventional CDs, but not the inverted derivatives ( Figure 3C ).…”

“…To measure changes in membrane fluidity, hydration and lipid order in response to CDs, fluorescence anisotropy of 4′-(trimethylammonio)-diphenylhexatriene (TMA-DPH) and generalized polarization (GP) of two indicators, 6-dodecanoyl-N,N-dimethyl-2-naphthylamine (Laurdan) and 4-[2-(6-Dibutylamino-5-fluoro-naphthalen-2-yl)-vinyl]-1-(3-triethylammonio-propyl)-pyridinium dibromide (PY3174), were quantified as described previously and in Supplementary Material ( Kwiatek et al, 2013 ; Kovács et al, 2017 ; Zakany et al, 2021 ).…”

It Takes More than Two to Tango: Complex, Hierarchal, and Membrane-Modulated Interactions in the Regulation of Receptor Tyrosine Kinases

“…However, these techniques are not suitable for the investigation of living cells, which can rather be examined with membrane-incorporating, environment-sensitive fluorophores that change their excitation or emission characteristics in response to alterations of a local biophysical parameter. For example, the fluidity of biological membranes can be tested with TMA-DPH (4′-(trimethylammonio)-diphenylhexatriene) using spectrofluorometry, since its fluorescence anisotropy negatively correlates with the rotational freedom of the fluorophore, i.e., fluidity of the membrane, as shown in living cells with altered levels of glucosylceramide, cholesterol, other sterols, and saturated or polyunsaturated fatty acids [ 149 , 150 , 151 , 152 , 153 ]. Membrane hydration can be estimated using Laurdan (6-dodecanoyl-N,N-dimethyl-2-naphthylamine) since the value of generalized polarization quantifying shifts in its emission spectrum shows an inverse correlation with the degree of water penetration into bilayers [ 154 , 155 ].…”