Electrogenic H+/OH- movement across phospholipid vesicles measured by spin-labeled hydrophobic ions.

Transmembrane pH gradients created across phospholipid vesicles give rise to time-dependent potentials as determined from the EPR spectra of phosphonium ion spin labels in the system. From the time-dependent spectra, the transmembrane H+/OH- current is obtained and hence the current-voltage curve for the vesicle membrane is obtained. The current-voltage curve is linear with a membrane resistance of 3 +/- 2 X 10(9) omega cm2 corresponding to a membrane permeability of 5 +/- 2 X 10(-7) cm/s. This unusually high permeability is further increased by small amounts of lipid oxidation, CHCl3 or the general anesthetic halothane.     

Transmembrane currents in frog olfactory cilia

Summary We have measured transmembrane currents in intact single cilia from frog olfactory receptor neurons. A single cilium on a neuron was sucked into a patch pipette, and a high-resistance seal was formed near the base of the cilium. Action potentials could be induced by applying suction or a voltage ramp to the ciliary membrane. A transient current was seen in some cells on stimulation with odorants. After excision from the cell, most of the cilia showed increased conductance in a bath containing cAMP, indicating that the cytoplasmic face of the ciliary membrane was accessible to the bath. The estimated resistance of a single cilium was surprisingly low.     

Binding of spin-labeled fatty acids and lysophospholipids to hydrophobic region of calmodulin.

In the presence of bovine brain calmodulin activated by calcium, the sharp triplet electron spin resonance (ESR) lines of free doxyl stearic acids decreased, and the broad resonance lines increased concomitantly, suggesting that the doxyl stearic acids bound to calmodulin calcium-dependently. The bound molecules were displaced by a calmodulin inhibitor, W-7, whereas their nitroxide radicals were hardly reduced by ascorbic acid, suggesting that the spin-labeled fatty acids bind to hydrophobic regions of calmodulin, and consequently inhibit calmodulin-dependent phosphodiesterase activity. These binding characteristics to calmodulin were different from those to bovine serum albumin. Moreover, the ESR spectra of two spin-labeled derivatives of lysophospholipid having a spin-labeled acyl group or a spin-labeled polar head group showed that it is the acyl chain of lysophospholipid that interacts with the hydrophobic region of calmodulin. The interactions of fatty acids and lysophospholipids with calmodulin seem to be quite different from those of acidic phospholipids, described previously [Suzuki, T., Katoh, H., & Uchida, M.K. (1986) Biochim. Biophys. Acta, 873, 379-386]. Thus, from the results of ESR study, we can obtain information on the function of fatty acids and lysophospholipids on calmodulin. Instead of enzyme assay, ESR spectroscopy is a useful means to examine lipid-protein interaction.     

Transmembrane insertion of the Colicin Ia hydrophobic hairpin

Colicin Ia is a bactericidal protein that forms voltage-dependent, ion-conducting channels, both in the inner membrane of target bacteria and in planar bilayer membranes. Its amino acid sequence is rich in charged residues, except for a hydrophobic segment of 40 residues near the carboxyl terminus. In the crystal structure of colicin Ia and related colicins, this segment forms an α-helical hairpin. The hydrophobic segment is thought to be involved in the initial association of the colicin with the membrane and in the formation of the channel, but various orientations of the hairpin with respect to the membrane have been proposed. To address this issue, we attached biotin to a residue at the tip of the hydrophobic hairpin, and then probed its location with the biotin-binding protein streptavidin, added to one side or the other of a planar bilayer. Streptavidin added to the same side as the colicin prevented channel opening. Prior addition of streptavidin to the opposite side protected channels from this effect, and also increased the rate of channel opening; it produced these effects even before the first opening of the channels. These results suggest a model of membrane association in which the colicin first binds with the hydrophobic hairpin parallel to the membrane; next the hairpin inserts in a transmembrane orientation; and finally the channel opens. We also used streptavidin binding to obtain a stable population of colicin molecules in the membrane, suitable for the quantitative study of voltage-dependent gating. The effective gating charge thus determined is pH-independent and relatively small, compared with previous results for wild-type colicin Ia. Received: 12 November 1996/Revised: 23 January 1997     

Transmembrane ion currents in pulmonary artery smooth muscle

Transmembrane ionic currents were investigated in the rabbit pulmonary artery smooth muscle under voltage clamp conditions with the use of the double sucrose gap method. With depolarizing pulses, there developed a fast inactivated outward current that was followed by a steady-state outward current. Tetraethylammonium (TEA) partly suppressed the outward current, and the fast inward current that preceded the fast outward one could be seen in these conditions. Appearance of the fast inward current in TEA-containing solution suggests the overlapping of the fast inward and outward currents. It appears that the resultant transmembrane current has an outward direction since in normal conditions the permeability of the fast potassium channels exceeds that of calcium channels. Conditioning hyperpolarization increased and depolarization decreased the fast outward current indicating that at the resting membrane potential a part of the potassium channels is inactivated and this inactivation is removed by hyperpolarization.     

Negative hydrophobic ions as transport-mediators for positive ions. Evidence for a carrier mechanism

The permeability of hydrophobic cations, such as tetraphenylarsonium across biological membranes and artificial lipid membranes is strongly increased in the presence of trace amounts of hydrophobic anions like tetraphenylborate (Liberman, Y.A. and Topaly, V.P. (1969) Biofizika 14, 452–461). Voltage-jump relaxation experiments performed on thin lipid membranes support the idea that the anions, A^?, act as carriers for the cations, B⁺, by the formation of neutral ion pairs, A^?B⁺. Their permeability is not affected by the electric dipole potential, which hinders the movement of free cations, B⁺.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.     

Electrostatic interactions among hydrophobic ions in lipid bilayer membranes.

We have shown that the absorption of tetraphenylborate into black lipid membranes formed from either bacterial phosphatidylethanolamine or glycerolmonooleate produces concentration-dependent changes in the electrostatic potential between the membrane interior and the bulk aqueous phases. These potential changes were studied by a variety of techniques: voltage clamp, charge pulse, and "probe" measurements on black lipid membranes; electrophroetic mobility measurements on phospholipid vesicles; and surface potential measurements on phospholipid monolayers. The magnitude of the potential changes indicates that tetraphenylborate absorbs into a region of the membrane with a low dielectric constant, where it produces substantial boundary potentials, as first suggested by Markin et al. (1971). Many features of our data can be explained by a simple three-capacitor model, which we develop in a self-consistent manner. Some discrepancies between our data and the simple model suggest that discrete charge phenomena may be important within these thin membranes.     

Transmembrane helices of membrane proteins may flex to satisfy hydrophobic mismatch

A novel mechanism for membrane modulation of transmembrane protein structure, and consequently function, is suggested in which mismatch between the hydrophobic surface of the protein and the hydrophobic interior of the lipid bilayer induces a flexing or bending of a transmembrane segment of the protein. Studies on model hydrophobic transmembrane peptides predict that helices tilt to submerge the hydrophobic surface within the lipid bilayer to satisfy the hydrophobic effect if the helix length exceeds the bilayer width. The hydrophobic surface of transmembrane helix 1 (TM1) of lactose permease, LacY, is accessible to the bilayer, and too long to be accommodated in the hydrophobic portion of a typical lipid bilayer if oriented perpendicular to the membrane surface. Hence, nuclear magnetic resonance (NMR) data and molecular dynamics simulations show that TM1 from LacY may flex as well as tilt to satisfy the hydrophobic mismatch with the bilayer. In an analogous study of the hydrophobic mismatch of TM7 of bovine rhodopsin, similar flexing of the transmembrane segment near the conserved NPxxY sequence is observed. As a control, NMR data on TM5 of lacY, which is much shorter than TM1, show that TM5 is likely to tilt, but not flex, consistent with the close match between the extent of hydrophobic surface of the peptide and the hydrophobic thickness of the bilayer. These data suggest mechanisms by which the lipid bilayer in which the protein is embedded modulates conformation, and thus function, of integral membrane proteins through interactions with the hydrophobic transmembrane helices.     

Transmembrane electrical characteristics of cultured human skeletal muscle cells

Skeletal muscle explants from normal subjects were established from biopsy material on collagen. Cellular outgrowth appeared within 3-4 days, and fusion of myoblasts was observed in 5-10 days. Multinucleated myotubes were impaled under high optical magnification, at 37 degrees C, with conventional glass microelectrodes. The mean resting potential was -44.4 mV +/- 2.4 (n = 399); -33 +/- 2.3 mV at 9 days (n = 10) vs -48 +/- 2.5 mV (n = 15) at 27 days. The average input resistance (Rin) was 9.7 M omega (n = 83). Action potentials could be elicited by electrical stimulation and had a mean amplitude of 55.9 +/- 2.1 mV with a mean maximum rate of rise (Vmax) of 72.1 +/- 7.5 V/s. The mean overshoot was 13.9 +/- 2.3 mV, and the action potential duration determined at 50% of repolarization (APD50) was 8.0 msec (n = 7). The resting membrane potential showed a depolarization of 23 mV/decade for extracellular potassium ion concentration ([K]o) between 4.5-100 mM. Thus, we have established the normal resting potential and maximum rate of rise of the action potential for human myotubes in culture. We have shown that the values for these are less than those previously reported in cultured avian and rodent cells. In addition, we have shown that the response in our system of the resting potential to change in extracellular potassium concentration is blunted compared to studies using isolated muscle, suggesting an increase in ratio of sodium to potassium permeability. Cultured human muscle cells depolarized in the presence of ouabain.     

Outer membrane of Salmonella typhimurium: Transmembrane diffusion of some hydrophobic substances

The outer membrane, which is composed of lipopolysaccharide, phospholipids, and proteins, is a layer of the cell wall of Gram-negative bacteria, and apparently acts as a penetration barrier for various substances. It had been shown by other workers that “deep rough” mutants of Salmonella typhimurium, whose lipopolysaccharides lack most of the saccharide chains, were much more sensitive than the wild type strain to certain antibiotics and dyes, but not to others. We found that the former group of agents are usually hydrophobic and the latter group mostly hydrophilic. All hydrophilic antibiotics had molecular weights lower than 650, and one of them was shown to diffuse through the outer membrane at 0 °C. In contrast, some hydrophobic antibiotics had molecular weights in excess of 1200, and the rate of diffusion of one of them was shown to be extremely dependent both on temperature and on the structure of lipopolysaccharide present. These data and results presented elsewhere suggest, but do not necessarily prove, that most hydrophilic antibiotics diffuse through aqueous pores, whereas hydrophobic antibiotics and dyes mainly penetrate by dissolving into the hydrocarbon interior of the outer membrane. In contrast to the outer membrane of deep rough mutants, that of the wild type strain and less defective rough mutants was unusual among biological membranes in that it was practically impermeable to hydrophobic agents. It is proposed that the difference in hydrophobic permeability between the two types of strain is due to radical differences in the organization of the outer membrane, more specifically to the presence or absence of exposed phospholipid bilayer regions.     

Estimation of transmembrane potentials from phase equilibria of hydrophobic paramagnetic ions.

Positively charged hydrophobic spin labels have been synthesized which respond to transmembrane potentials in sonicated liposomes. Electron paramagnetic resonance spectroscopy is used to show that the distribution of these probes between aqueous and membrane phases changes as a function of transmembrane potential. When liposomes are made more inside-negative, the fraction of membrane associated probe increases while the fraction of probe in the aqueous phase decreases. The results are in quantitative agreement with a simple equilibrium thermodynamic theory which allows estimation of absolute transmembrane potentials in phospholipid vesicles.     

Transmembrane electrical potential and transmembrane pH gradient in the acidophile Thiobacillus ferro-oxidans.

Thiobacillus ferro-oxidans is capable of using the oxidation of Fe2+ by O2 at pH 2.0 as the sole source of energy for growth and CO2 fixation. The bacterium maintains an intracellular pH of 6.5 over a range of external pH from 1.0 to 8.0, as measured by [14C]acetate and [3H]methylamine distribution. The membrane potential was estimated by the distribution of the lipid-soluble cation dibenzyldimethylammonium and the anion SCN-. At pH 2.0 (the pH of growth) during Fe2+ oxidation the transmembrane pH gradient is 4.5 units with an opposing membrane potential of -10mV, giving a proton electrochemical gradient of +256mV. This gradient is actively maintained.     

Transmembrane electrical and pH gradients across human erythrocytes and human peripheral lymphocytes.

Transmembrane electrical and pH gradients have been measured across human erythrocytes and peripheral blood lymphocytes using equilibrium distributions of radioactively labelled lipophilic ions, and of weak acids and weak bases, respectively. The distributions of methylamine, trimethylamine, acetic acid and trimethylacetic acid give calculated transmembrane pH gradients (pHe-pHi) for erythrocytes of between 0.14-0.21 for extracellular pH values of 7.28-7.16. The distributions of trimethylacetic acid. DMO and trimethylamine were determined for lymphocytes, establishing upper and lower limits of the calculated pH gradient over the external pH range of 6.7 to 7.7. Tritiated triphenylmethyl phosphonium ion (TPMP) and 14C-thiocyanate ion (SCN) equilibrium distributions were measured in order to calculate transmembrane electrical potentials, using tetraphenylboron as a catalyst to facilitate TPMP equilibrium. Transmembrane potentials of -7 to -10 mV were calculated from SCN and TPMP, respectively for red cells, and -35 to -52 mV respectively, in the case of lymphocytes. Distributions of TPMP and potassium ions were determined in the presence of valinomycin over a wide range of extracellular potassium concentrations for red cells and the calculated Nernst potentials for TPMP compared to the calculated potential using the Goldman equation for chloride and potassium ions. Distributions of TPMP, SCN and potassium ions were also determined for lymphocyte suspensions as a function of extracellular potassium and the calculated Nernst potentials for TPMP and SCN compared to the calculated potassium diffusion potential.     

The transport of hydrophobic ions across lipid bilayers

The three-capacitor model for hydrophobic ion adsorption in lipid membranes (Andersen, O.S., Feldberg, S., Nakadomari, H., Levy, S. and McLaughlin, S. (1978) Biophys. J. 21, 35-70) is extended to ion transport whereby electrostatic effects from the interfacially adsorbed hydrophobic space charge have been encountered. The phenomena of current saturation with increasing concentrations of hydrophobic ions in the bulk electrolyte and the associated increase of the time constant of current relaxation can be quantitatively understood on the basis of space charge-limited currents as well as the nonexponential current decay.