The Journal of Membrane Biology

Leveille-Webster, C.; Arias, I.

doi: 10.1007/BF00234655pmid: 7731035

The initial discovery of p-glycoprotein in the plasma membrane of MDR cancer cell lines was followed quickly by the cloning of its gene. Sequence analysis of cloned cDNAs revealed that p-glycoprotein was a member of the ABC family of membrane transporters. Subsequent biochemical characterization demonstrated the binding of chemotherapeutic drugs and ATP to p-glycoprotein. P-glycoprotein-mediated drag transport and drug-stimulated ATPase activity were documented in plasma membrane vesicles and in proteoliposomes containing the partially purified protein. P-glycoprotein was shown to be phosphorylated and the effect of this modification on the protein's biological function was explored. P-glycoproteins were found in many normal tissues and their overexpression was documented in numerous cancers. An important role for p-glycoprotein in intrinsic and acquired drug resistance in clinical oncology was established. Despite all that has been learned about p-glycoprotein over the last few years, additional studies will be necessary to address the many questions that have been left unanswered. Determination of p-glycoprotein structure and membrane topology should help elucidate the nature of chemotherapeutic drug binding sites and the mechanism whereby drug movement is coupled to ATP hydrolysis. Complete purification and functional reconstitution of p-glycoprotein into defined lipid vesicles will permit further characterization of drug transport and ATPase activity and give us the means by which p-glycoprotein's apparent dual function as a transporter and a channel can be clarified. Structural and functional studies on p-glycoprotein will also provide information needed to develop specified inhibitors that can be used clinically to overcome MDR in cancer patients. Further study of the mechanisms whereby p-glycoprotein expression is induced and regulated during malignant transformation is indicated. The development of biliary phospholipid deficiency in mdr2 knockout mice and xenobiotic hypersensitivity in mdr3 knockout mice have given us the first clues into the normal physiologic roles for the p-glycoproteins. The search for endogenous substrates for the p-glycoproteins will continue to be an area of active investigation.

Hager, K.; Hazama, A.; Kwon, H.; Loo, D.; Handler, J.; Wright, E.

doi: 10.1007/BF00234656pmid: 7537337

The two-microelectrode voltage clamp technique was used to examine the kinetics and substrate specificity of the cloned renal Na+/myo-inositol cotransporter (SMIT) expressed in Xenopus oocytes. The steady-state myo-inositol-induced current was measured as a function of the applied membrane potential (V m ), the external myo-inositol concentration and the external Na+ concentration, yielding the kinetic parameters: K 0.5 MI , K 0.5 Na , and the Hill coefficient n. At 100 mM NaCl, K 0.5 MI was about 50 μm and was independent of V m . At 0.5 mm myo-inositol, K 0.5 Na ranged from 76 mm at V m =−50 mV to 40 mm at V m =−150 mV. n was voltage independent with a value of 1.9±0.2, suggesting that two Na+ ions are transported per molecule of myo-inositol. Phlorizin was an inhibitor with a voltage-dependent apparent K I of 64 μm at V m =−50 mV and 130 μm at V m = −150 mV. To examine sugar specificity, sugar-induced steady-state currents (at V m =−150 mV) were recorded for a series of sugars, each at an external concentration of 50 mm. The substrate selectivity series was myo-inositol, scyllo-inositol > l-fucose > l-xylose > l-glucose, d-glucose, α-methyl-d-glucopyranoside > d-galactose, d-fucose, 3-O-methyl-d-glucose, 2-deoxy-d-glucose > d-xylose. For comparison, oocytes were injected with cRNA for the rabbit intestinal Na+/glucose cotransporter (SGLT1) and sugar-induced steady-state currents (at V m =−150 mV) were measured. For oocytes expressing SGLT1, the sugar selectivity was: d-glucose, α-methyl-d-glucopyranoside, d-galactose, d-fucose, 3-O-methyl-d-glucose > d-xylose, l-xylose, 2-deoxy-d-glucose > myo-inositol, l-glucose, l-fucose. The ability of SMIT to transport glucose and SGLT1 to transport myo-inositol was independently confirmed by monitoring the Na+-dependent uptake of 3H-d-glucose and 3H-myo-inositol, respectively. In common with SGLT1, SMIT gave a relaxation current in the presence of 100 mm Na+ that was abolished by phlorizin (0.5 mm). This transient current decayed with a voltage-sensitive time constant between 10 and 14 msec. The presteady-state current is apparently due to the reorientation of the cotransporter protein in the membrane in response to a change in V m . The kinetics of SMIT is accounted for by an ordered six-state nonrapid equilibrium model.

Kuno, M.; Shibata, T.; Kawawaki, J.; Kyogoku, I.

doi: 10.1007/BF00234657pmid: 7731031

Electrophysiological properties of mouse bone marrow-derived mast cells (BMMC) were studied under the whole-cell clamp configuration. About one third of the cells were quiescent, but others expressed either inward or outward currents. Inwardly rectifying (IR) currents were predominant in 14% of the cells, and outwardly rectifying (OR) currents in 24%. The rest (22%) of the cells exhibited both inward and outward currents. The IR currents were eliminated by 1 mm Ba2+, and were partially inhibited by 100 μm quinidine. The reversal potential was dependent on extracellular K+, thereby indicating that K+ mediated the IR currents. The negative conductance region was seen at potentials positive to E K. The OR currents did not apparently depend on the extracellular K+ concentration, but were reduced by lowering the extracellular Cl− concentration. The OR currents were partially blocked by 1 mm Ba2+, and were further blocked by a Cl− channel blocker, 4,4′-diisothiocyano-2, 2′-stilbenedisulfonate (DIDS). In addition, the reversal potential of the OR currents was positively shifted by decreasing the ratio of external and internal Cl− concentrations, suggesting that Cl− was a major ion carrier. In cells exhibiting IR currents, the membrane potential varied among cells and tended to depolarize by elevating the external K+ concentration. In cells with OR currents, the resting potential was hyperpolarized in association with an increase in conductance. These results suggest that BMMC have a heterogeneous electrophysiological profile that may underlie a variety of ion channels expressed in different phenotypes of mast cells. Activities of both the inwardly rectifying K+ channel and the outwardly rectifying Cl− channel seem to contribute to the regulation of the membrane potential.

Belachgar, F.; Hulin, P.; Planelles, G.; Anagnostopoulos, T.

doi: 10.1007/BF00234658pmid: 7731032

Intracellular pH (pH i ), membrane potential (V m ) and membrane conductance (G m ) in fused proximal tubular cells of the frog kidney, were determined at three extracellular pH (pH o ) values, 7.5, 8.5 and 6.5. Imposed changes of pH o by ±1 pH unit induced parallel but smaller shifts of pH i . The alkaline milieu hyperpolarized the cells and increased G m , whereas the acid milieu depolarized and lowered G m . We subsequently introduced a weak acid and its conjugate base (acetic acid/acetate), or a weak base and its conjugate acid (NH3/NH 4 + ), at pH o 7.5, 8.5 and 6.5 to shift pH i -without altering pH o , or to shift pH i against imposed changes of pH o . From these experiments, we observed that under some circumstances V m varied with pH o but without G m or pH i changes, whereas under other circumstances changes of G m occurred during alterations of pH i while pH o and V m remained unaltered. At pH i ≈ 6.5 associated with V m ≈ −10 mV, G m dramatically increased to quasi-infinite values. This increase was not an artifact since G m returned to its control value following recovery to the control solution or in the presence of hyperosmotic solution. In conclusion, we demonstrate a differential regulation whereby V m and G m are controlled by pH o and pH i : pH o modulates mainly V m , and pH i modulates chiefly G m . Furthermore, at pH i ≈ 6.5 and V m ≈ −10 mV, our data reveal a large G m that tends towards infinite values in a reversible fashion.

Farías, R.; Chehin, R.; Rintoul, M.; Morero, R.

doi: 10.1007/BF00234659pmid: 7731033

The effect of thyroid hormones on the degree of order or fluidity of dimyristoyl, dipalmitoyl or egg yolk phosphatidyl choline liposomes was evaluated by fluorescence spectroscopy methods. The freedom of molecular motion above the phase transition temperature was decreased, while below the transition, the mobility was actually increased by the incorporation of triiodothyronine to liposomes. While thyroxine decreases the fluidity in the liquid crystalline state, it cannot increase the fluidity in the gel state.

Korchev, Y.; Alder, G.; Bakhramov, A.; Bashford, C.; Joomun, B.; Sviderskaya, E.; Usherwood, P.; Pasternak, C.

doi: 10.1007/BF00234660pmid: 7537338

The conductance of pores induced by Staphylococcus aureus α-toxin in Lettre cells has been compared to that in bilayers composed of synthetic lipids or Lettre cell membrane constituents. Previously described characteristics of toxin-induced conductance changes in lipid bilayers, namely rectification, voltage-dependent closure, and closure at low pH or in the presence of divalent cations (Menestrina, 1986) are displayed also in bilayers prepared from Lettre cell membranes and in patch clamped Lettre cells. It is concluded that endogenous proteins do not affect the properties of α-toxininduced channels significantly and that the relative lack of ion channels in Lettre cells makes them ideal for studies of pore-forming toxins by the patch clamp technique.

Blandino, J.; Viglione, M.; Bradley, W.; Oie, H.; Kim, Y.

doi: 10.1007/BF00234661pmid: 7731034

Sodium channels of human small-cell lung cancer (SCLC) cells were examined with whole-cell and single-channel patch clamp methods. In the tumor cells from SCLC cell line NCI-H146, the majority of the voltage-gated Na+ channels are only weakly tetrodotoxin (TTX)-sensitive (K d =215 mm). With the membrane potential maintained at −60 to −80 mV, these cells produced all-or-nothing action potentials in response to depolarizing current injection (>20 pA). Similar all-ornothing spikes were also observed with anodal break excitation. Removal of external Ca2+ did not affect the action potential production, whereas 5 μm TTX or substitution of Na+ with choline abolished it. Action potentials elicited in the Ca2+-free condition were reversibly blocked by 4 mm MnCl2 due to the Mn2+-induced inhibition of voltage-dependent sodium currents (I Na). Therefore, Na+ channels, not Ca2+ channels, underlie the excitability of SCLC cells. Whole-cell I Na was maximal with step-depolarizing stimulations to 0 mV, and reversed at +45.2 mV, in accord with the predicted Nernst equilibrium potential for a Na+-selective channel. I Na evoked by depolarizing test potentials (−60 to +40 mV) exhibited a transient time course and activation/ inactivation kinetics typical of neuronal excitable membranes; the plot of the Hodgkin-Huxley parameters, m∞ and h∞, also revealed biophysical similarity between SCLC and neuronal Na+ channels. The single channel current amplitude, as measured with the inside-out patch configuration, was 1.0 pA at −20 mV with a slope conductance of 12.1 pS. The autoantibodies implicated in the Lambert-Eaton myasthenic syndrome (LES), which are known to inhibit I Ca and I Na in bovine adrenal chromaffin cells, also significantly inhibited I Na in SCLC cells. These results indicate that (i) action potentials in human SCLC cells result from the regenerative increase in voltage-gated Na+ channel conductance; (ii) fundamental characteristics of SCLC Na+ channels are the same as the classical sodium channels found in a variety of excitable cells; and (iii) in some LES patients, SCLC Na+ channels are an additional target of the pathological IgG present in the patients' sera.