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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.

ABSTRACT: 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.

SUBMITTER: Zimmermann D

PROVIDER: S-EPMC2755742 | biostudies-other | 2008 Jan

REPOSITORIES: biostudies-other

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Project description:We determine membrane capacitance, C as a function of dc voltage for the human embryonic kidney (HEK) cell. C was calculated from the admittance, Y, obtained during a voltage ramp when the HEK cell was held in whole-cell patch-clamp configuration. Y was determined at frequencies of 390.625 and from the measured current, i obtained with a dual-sinusoidal stimulus. We find that the fractional increase in the capacitance, C is small ( < 1%) and grows with the square of the voltage, Psi. C can be described by: C=C(0)(1+alpha(Psi+psi(s))2)[where C(0): Capacitance at 0 volts, psi(s): Difference in surface potential between cytoplasmic and extracellular leaflets and alpha: Proportionality constant]. We find that alpha and psi(s) are 0.120 (+/- 0.01) V(-2) and -0.073 (+/-0.017 V in solutions that contain ion channel blockers and 0.108 (+/- 0.29) V(-2) and -0.023 (+/- 0.009) V when 10 mM sodium salicylate was added to the extracellular solution. This suggests that salicylate does not affect the rate at which C grows with Psi, but reduces the charge asymmetry of the membrane. We also observe an additional linear differential capacitance of about (-46 fFV(-1)) in about 60% of the cells, this additional component acts simultaneously with the quadratic component and was not observed when salicylate was added to the solution. We suggest that the voltage dependent capacitance originates from electromechanical coupling either by electrostriction and/or Maxwell stress effects and estimate that a small electromechanical force (approximately equal to 1 pN) acts at physiological potentials. These results are relevant to understand the electromechanical coupling in outer hair cells (OHCs) of the mammalian cochlea, where an asymmetric bell-shaped C versus Psi relationship is observed upon application of a similar field. Prestin, a membrane protein expressed in OHCs is required to observe this function. When we compare the total charge contributions from HEK cell membrane (7 x 10(4) electrons, 10 pF cell) with that determined for prestin transfected cells (up to 5 x 10(6) electrons) we conclude that the charge contributions from the collective motion of membrane proteins and lipids in the field is dwarfed relative to that when prestin is present. We suggest that the capacitance-voltage relationships should be similar to that observed for HEK cells for OHCs that do not express prestin in their membranes.

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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.

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