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J Membr Biol
2008 Jan 01;2212:107-21. doi: 10.1007/s00232-007-9090-4.
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A combined patch-clamp and electrorotation study of the voltage- and frequency-dependent membrane capacitance caused by structurally dissimilar lipophilic anions.
Zimmermann D
,
Kiesel M
,
Terpitz U
,
Zhou A
,
Reuss R
,
Kraus J
,
Schenk WA
,
Bamberg E
,
Sukhorukov VL
.
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Interactions of structurally dissimilar anionic compounds with the plasma membrane of HEK293 cells were analyzed by patch clamp and electrorotation. The combined approach provides complementary information on the lipophilicity, preferential affinity of the anions to the inner/outer membrane leaflet, adsorption depth and transmembrane mobility. The anionic species studied here included the well-known lipophilic anions dipicrylamine (DPA(-)), tetraphenylborate (TPB(-)) and [W(2)(CO)(10)(S(2)CH)](-), the putative lipophilic anion B(CF(3))(4)(-) and three new heterocyclic W(CO)(5) derivatives. All tested anions partitioned strongly into the cell membrane, as indicated by the capacitance increase in patch-clamped cells. The capacitance increment exhibited a bell-shaped dependence on membrane voltage. The midpoint potentials of the maximum capacitance increment were negative, indicating the exclusion of lipophilic anions from the outer membrane leaflet. The adsorption depth of the large organic anions DPA(-), TPB(-) and B(CF(3))(4)(-) increased and that of W(CO)(5) derivatives decreased with increasing concentration of mobile charges. In agreement with the patch-clamp data, electrorotation of cells treated with DPA(-) and W(CO)(5) derivatives revealed a large dispersion of membrane capacitance in the kilohertz to megahertz range due to the translocation of mobile charges. In contrast, in the presence of TPB(-) and B(CF(3))(4)(-) no mobile charges could be detected by electrorotation, despite their strong membrane adsorption. Our data suggest that the presence of oxygen atoms in the outer molecular shell is an important factor for the fast translocation ability of lipophilic anions.
Fig. 1. Structures of the lipophilic anions studied here (notation, salt formula, chemical name): WO−, Et4N+[W(CO)5(SCNOC6H4)]−, tetraethylammonium-benzoxazolidin-2-thion-1-yl-pentacarbonyltungstate; WN−, Et4N+[W(CO)5(SCN2HC6H4)]−, tetraethylammonium-benzimidazolidin-2-thion-1-yl-pentacarbonyltungstate; WS−, Et4N+[W(CO)5(SCNSC6H4)]−, tetraethylammonium-benzothiazolidin-2-thion-1-yl-pentacarbonyltungstate; WW−, Et4N+[W2(CO)10(μ-S2CH)]− tetraethylammonium decacarbonyl-μ-dithioformiato-ditungstate; DPA−, dipicrylamine (2,2',4,4',6,6'-hexanitrodiphenylamine); TPB−, Na+[B(C6H5)4]− sodium tetraphenylborate; B(CF3)4−, Cs+[B(CF3)4]−, cesium tetrakis-(trifluormethyl)-borate
Fig. 2. Patch-clamp measurements on HEK cells in the whole-cell configuration. a A typical voltage protocol applied to a HEK cell and the resulting current recording. Voltage ramps (ramp) are started from different holding potentials. Note that capacitive peak currents resulting after a voltage step (step) are shown only partly. b Current recordings after subjecting a HEK cell to a +2 V/s ramp starting from a holding potential at −75 mV in the presence and absence of 20 μm WO−, respectively. Dotted lines were used to determine the amplitude of corresponding capacitive currents, Ic
Fig. 3. Voltage dependence of the area-specific membrane capacitance increment (ΔCLI) induced in HEK cells by the adsorbed lipophilic anions WO− (a), WW− (b), DPA− (c) and TPB− (d). Membrane capacitance was measured by whole-cell patch clamp. The plotted ΔCLI data (symbols) were calculated by subtracting the capacitance values of control cells from those of cells treated with various anion concentrations: 5 μm (open circles), 20 μm (open squares) and 50 μm (filled circles). Curves are least-square fits of the first derivative of the Boltzmann function (equation 1). The fitted parameters are summarized in Table 1
Fig. 4. Voltage dependence of the area-specific membrane capacitance increment (ΔCLI) induced in Xenopus oocytes by the indicated lipophilic anions. Membrane capacitance was measured by the TEVC technique. The plotted ΔCLI data (symbols) were calculated by subtracting the capacitance values of control cells from those of treated cells. Curves are least-square fits of the first derivative of the Boltzmann function (equation 1). For further details, see Table 2
Fig. 5. Analysis of the electric properties of Jurkat (a, b) and HEK (c, d) cells by electrorotation. a, c The ROT spectra of untreated control cells (open symbols) were fitted by the single-shell model (dotted curves). The radius-normalized fc1 data of control Jurkat and HEK cells measured by the CRF technique and plotted vs. σe are shown in b and d, respectively. The lines are calculated by regression of equation 4 to the data points (bn = 320 cells, dn = 180 cells). The fitted Cm and Gm values are summarized in Table 3. The effects of increasing concentration of WO− on the ROT spectra of Jurkat and HEK cells are shown in a and c (filledsymbols), respectively. All spectra were measured at σe ≈ 60 mS/m. The solution osmolalities were 100 (a, b) and 300 (c, d) mOsm. fc1, fc2 and fLI in a denote the plasma membrane, cytosolic and mobile charge peaks, respectively. Dashed and solid curves in a and c represent best fits of the mobile charge model (equations 2, 3 and 5) to the ROT spectra of cells treated with WO−. The fitted parameters for HEK and Jurkat cells are given in Tables 4 and 5, respectively
Fig. 6. The apparent capacitance increment (ΔCLI) (a), translocation rate constant (ki) (b), midpoint potential (Vmid) (c) of the induced capacitance and slope parameter (α) (d) for the lipophilic anion WW− adsorbed to the plasma membrane of HEK cells as functions of WW− concentration in bath medium. The data were obtained by electrorotation (ΔCLI and ki, opensymbols) and voltage clamp (ΔCLI, Vmid and α,(filled symbols). Note that the two independent techniques yielded comparable ΔCLI values (a)
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