The Journal of Membrane Biology

Musa-Aziz, R.; Oliveira-Souza, M.; Mello-Aires, M.

doi: 10.1007/s00232-005-0762-7pmid: 16283585

The effect of ANG II on pHi, [Ca2+]i and cell volume was investigated in T84 cells, a cell line originated from colon epithelium, using the probes BCECF-AM, Fluo 4-AM and acridine orange, respectively. The recovery rate of pHi via the Na+/H+ exchanger was examined in the first 2 min following the acidification of pHi with a NH4Cl pulse. In the control situation, the pHi recovery rate was 0.118 ± 0.001 (n = 52) pH units/min and ANG II (10−12 M or 10−9 M) increased this value (by 106% or 32%, respectively) but ANG II (10−7 M) decreased it to 47%. The control [Ca2+]i was 99 ± 4 (n = 45) nM and ANG II increased this value in a dose-dependent manner. The ANG II effects on cell volume were minor and late and should not interfere in the measurements of pHi recovery and [Ca2+]i. To document the signaling pathways in the hormonal effects we used: Staurosporine (a PKC inhibitor), W13 (a calcium-dependent calmodulin antagonist), H89 (a PKA inhibitor) or Econazole (an inhibitor of cytochrome P450 epoxygenase). Our results indicate that the biphasic effect of ANG II on Na+/H+ exchanger is a cAMP-independent mechanism and is the result of: 1) stimulation of the exchanger by PKC signaling pathway activation (at 10−12 – 10−7 M ANG II) and by increases of [Ca2+]i in the lower range (at 10−12 M ANG II) and 2) inhibition of the exchanger at high [Ca2+]i levels (at 10−9 – 10−7 M ANG II) through cytochrome P450 epoxygenase-dependent metabolites of the arachidonic acid signaling pathway.

Álvarez, R.M.S.; Cutin, E.H.; Farías, R.N.

doi: 10.1007/s00232-005-0763-6pmid: 16283586

The conformational changes of 3,5,3′-triiodo L-thyronine induced by interaction with phospholipids were analyzed by Raman spectroscopy. The spectra were interpreted in terms of two conformers of this hormone in equilibrium in the lipid medium, depending on the orientation of the 3′-iodine with respect to the ring α. Theoretical geometry optimizations on both conformers in vacuo and in different solvents, together with the respective calculated energies support the experimental results. The presence of only one iodine atom in the phenolic ring allows assumption of a higher flexibility of 3,5,3′-triiodo L-thyronine and a better accommodation into the lipid medium compared to 3,5,3′,5′-tetraiodo L-thyronine. The possible physiological implications of structural differences that appear in membrane models between 3,5,3′-triiodo L-thyronine and 3,5,3′,5′-tetraiodo L-thyronine are discussed.

Teisseyre, A.; Michalak, K.

doi: 10.1007/s00232-005-0764-5pmid: 16283587

In the present study, the whole-cell patch-clamp technique was applied to follow the inhibitory effect of genistein — a tyrosine kinase inhibitor and a natural anticancer agent—on the activity of voltage-gated potassium channels Kv1.3 expressed in human T lymphocytes (TL). Obtained data provide evidence that genistein application in the concentration range of 1–80 μM reversibly decreased the whole-cell potassium currents in TL in a concentration-dependent manner to about 0.23 of the control value. The half-blocking concentration range of genistein was from 10 to 40 μM. The current inhibition was correlated in time with a significant decrease of the current activation rate. The steady-state activation of the currents was unchanged upon application of genistein, as was the inactivation rate. The inhibitory effect of genistein on the current amplitude and activation kinetics was voltage-independent. The current inhibition was not changed significantly in the presence of 1 mM of sodium orthovanadate, a tyrosine phosphatase inhibitor. Application of daidzein, an inactive genistein analogue, did not affect significantly either the current amplitudes or the activation kinetics. Possible mechanisms of the observed phenomena and their significance for genistein-induced inhibition of cancer cell proliferation are discussed.

Ramanan, S.V.; Valiunas, V.; Brink, P.R.

doi: 10.1007/s00232-005-0765-4pmid: 16283588

Non-stationary fluctuation analysis was applied to macroscopic records of junctional currents arising from homotypic Cx37 and Cx43 gap junction channels expressed in RIN cells. The data were analyzed by a modification of existing analytical methods that takes endemic uncoupling into account. The results are consistent with both channels having open probabilities ranging from 0.7 to near unity for low transjunctional voltages. The analysis also yielded estimates of single-channel conductances for the two channel types similar to those seen in single-channel recordings. The results presented here show that fluctuation analysis can be used to extract single-channel gap junctional conductances from macroscopic double whole-cell recordings. These results also constitute empirically determined estimates of the open probability that are not model-dependent.

Stimac, R.; Kerek, F.; Apell, H.-J.

doi: 10.1007/s00232-005-0767-2pmid: 16283589

The previously reported class of potent inorganic inhibitors of Na,K-ATPase, named MCS factors, was shown to inhibit not only Na,K-ATPase but several P-type ATPases with high potency in the sub-micromolar range. These MCS factors were found to bind to the intracellular side of the Na, K-ATPase. The inhibition is not competitive with ouabain binding, thus excluding its role as cardiac-steroid-like inhibitor of the Na,K-ATPase. The mechanism of inhibition of Na,K-ATPase was investigated with the fluorescent styryl dye RH421, a dye known to report changes of local electric fields in the membrane dielectric. MCS factors interact with the Na,K-ATPase in the E1 conformation of the ion pump and induce a conformational rearrangement that causes a change of the equilibrium dissociation constant for one of the first two intracellular cation binding sites. The MCS-inhibited state was found to have bound one cation (H+, Na+ or K+) in one of the two unspecific binding sites, and at high Na+ concentrations another Na+ ion was bound to the highly Na+-selective ion-binding site.

Pottosin, I.I.; Martínez-Estévez, M.; Dobrovinskaya, O.R.; Muñiz, J.

doi: 10.1007/s00232-005-0766-3pmid: 16283590

Voltage-dependent activation of slow vacuolar (SV) channels has been studied on isolated patches from red beet (Beta vulgaris L.) vacuoles. Isoosmotic variation of vacuolar K+ from 10 to 400 mM in Ca2+-free solutions at the vacuolar side shifted the SV channel activation threshold to more positive voltages. The effect of K+ could be mimicked by additions of choline or N-methyl D-glucamine and could be explained by unspecific screening of the negative surface charge. Fitting the dependence of voltage shift on K+ concentration to the Gouy-Chapman model yields a surface charge density of 0.36 ± 0.05 e−/nm2. Negative surface potential also tended to increase the local concentration of permeable ions (K+), resulting in anomalously high single-channel conductance, ∼200 pS in 10 mM KCl. An increase of ionic strength due to addition of impermeable cations greatly reduced the unitary conductance. Large positive shift of the SV channel voltage dependence, caused by physiological (0.5 mM) free vacuolar Ca2+, was partly ameliorated by increasing luminal K+. We interpreted these results as follows: K+ induced a reduction of surface potential, hence i) causing a positive shift of the voltage dependence and ii) a dilution of Ca2+ in the membrane vicinity, thus reducing the inhibitory effect of vacuolar Ca2+ and causing a negative shift of the SV channel voltage dependence, with a sum of the two shifts being negative.