Stephen B. Hladky

Ion transfer across lipid membranes in the presence of gramicidin A: I. Studies of the unit conductance channel

The conductance induced by gramicidin A in lipid bilayer membranes has been shown to be made up of discrete, well-defined units. In 0.1 M NaCl, and for 100 mV applied, the integral conductance of the unit channel at 20 °C is 5.8·10−12 Ω−1. The channels are formed by transitions involving inactive gramicidin molecules already in the membrane. The precise nature of a transition is not certain, but circumstantial evidence suggests that the final conducting structure consists of at least two polypeptide molecules. From the temperature coefficient of the average duration of the channels it has been shown that the activation energy for channel closure must be ≳ 19 kcal mole−1. The frequency of occurrence and the average duration of the channels both become larger the thinner the membrane. The equilibrium between non-conducting and conducting species therefore shifts towards the conducting species as the membrane thickness decreases. With one exception, the same unit channel conductance was observed for a range of membranes having hydrocarbon thicknesses from 26 to 64 A and, from this and other evidence, it has been concluded that the conducting channel is a pore, rather than a carrier. The length of the pore has been estimated to be less than 35 A. The pore passes univalent cations but completely excludes polyvalent cations and anions. The selectivity between the univalent cations is not great, and the sequence of the various ion conductances is similar to that for the corresponding electrolytes in aqueous solution. The activation energies for the conduction of the ions are also similar to those in aqueous solution. The current-voltage relationships for the single channel tend to be curved towards the current axis at high electrolyte concentrations, linear at intermediate concentrations and curved towards the voltage axis at low concentrations. For each of the electrolytes studied the conductance of the single channel tends towards a limiting value at high concentrations. It is noted that one of the dimeric helical structures (that which contains the πL,D6 helix) proposed by Urry et al.22 could be consistent with some of the properties of the single channel.

Mechanisms of fluid movement into, through and out of the brain: evaluation of the evidence

Interstitial fluid (ISF) surrounds the parenchymal cells of the brain and spinal cord while cerebrospinal fluid (CSF) fills the larger spaces within and around the CNS. Regulation of the composition and volume of these fluids is important for effective functioning of brain cells and is achieved by barriers that prevent free exchange between CNS and blood and by mechanisms that secrete fluid of controlled composition into the brain and distribute and reabsorb it. Structures associated with this regular fluid turnover include the choroid plexuses, brain capillaries comprising the blood-brain barrier, arachnoid villi and perineural spaces penetrating the cribriform plate. ISF flow, estimated from rates of removal of markers from the brain, has been thought to reflect rates of fluid secretion across the blood-brain barrier, although this has been questioned because measurements were made under barbiturate anaesthesia possibly affecting secretion and flow and because CSF influx to the parenchyma via perivascular routes may deliver fluid independently of blood-brain barrier secretion. Fluid secretion at the blood-brain barrier is provided by specific transporters that generate solute fluxes so creating osmotic gradients that force water to follow. Any flow due to hydrostatic pressures driving water across the barrier soon ceases unless accompanied by solute transport because water movements modify solute concentrations. CSF is thought to be derived primarily from secretion by the choroid plexuses. Flow rates measured using phase contrast magnetic resonance imaging reveal CSF movements to be more rapid and variable than previously supposed, even implying that under some circumstances net flow through the cerebral aqueduct may be reversed with net flow into the third and lateral ventricles. Such reversed flow requires there to be alternative sites for both generation and removal of CSF. Fluorescent tracer analysis has shown that fluid flow can occur from CSF into parenchyma along periarterial spaces. Whether this represents net fluid flow and whether there is subsequent flow through the interstitium and net flow out of the cortex via perivenous routes, described as glymphatic circulation, remains to be established. Modern techniques have revealed complex fluid movements within the brain. This review provides a critical evaluation of the data.

Ion movements in gramicidin pores. An example of single-file transport

Experimental results on ion movement through gramicidin membrane channels are presented and discussed in terms of ion transport in the simplest single-file pore (for review see Urban, B.W. and Hladky, S.B. (1979) Biochim. Biophys. Acta 554, 410-429). Single-channel conductance and bi-ionic potential data for Na+, K+, Cs+, NH4+ and Tl+ are used to assign values to the rate constants of the model. Not all of the rate constants can be determined uniquely and simplifications are introduced to reduce the number of free parameters. The simplified model gives good quantitative fits to the experimental results for Na+, K+, Cs+ and NH4+. For Tl+, although the model accounts qualitatively for the salient features of the results, the quantitative agreement is less satisfactory. Predictions calculated from the model and the fitted rate constants are compared with independent data from blocking and tracer-flux measurements. In agreement with experiment, the model shows that only Tl+ blocks the Na+ conductance significantly. Furthermore, the exponent, n, in the tracer flux ratio rises, as observed, well above unity. The values for the rate constants suggest internal consistency of the model in that entry is always slower to singly occupied pores than to empty pores while exit is always faster from doubly as compared to singly occupied pores. The agreement between model prediction and experimental results suggests that the main features of ion transport in the gramicidin channel arise from cation-cation interaction in a single-file pore.

Modulatory effects of plant phenols on human multidrug-resistance proteins 1, 4 and 5 (ABCC1, 4 and 5).

Plant flavonoids are polyphenolic compounds, commonly found in vegetables, fruits and many food sources that form a significant portion of our diet. These compounds have been shown to interact with several ATP‐binding cassette transporters that are linked with anticancer and antiviral drug resistance and, as such, may be beneficial in modulating drug resistance. This study investigates the interactions of six common polyphenols; quercetin, silymarin, resveratrol, naringenin, daidzein and hesperetin with the multidrug‐resistance‐associated proteins, MRP1, MRP4 and MRP5. At nontoxic concentrations, several of the polyphenols were able to modulate MRP1‐, MRP4‐ and MRP5‐mediated drug resistance, though to varying extents. The polyphenols also reversed resistance to NSC251820, a compound that appears to be a good substrate for MRP4, as predicted by data‐mining studies. Furthermore, most of the polyphenols showed direct inhibition of MRP1‐mediated [3H]dinitrophenyl S‐glutathione and MRP4‐mediated [3H]cGMP transport in inside‐out vesicles prepared from human erythrocytes. Also, both quercetin and silymarin were found to inhibit MRP1‐, MRP4‐ and MRP5‐mediated transport from intact cells with high affinity. They also had significant effects on the ATPase activity of MRP1 and MRP4 without having any effect on [32P]8‐azidoATP[αP] binding to these proteins. This suggests that these flavonoids most likely interact at the transporters substrate‐binding sites. Collectively, these results suggest that dietary flavonoids such as quercetin and silymarin can modulate transport activities of MRP1, ‐4 and ‐5. Such interactions could influence bioavailability of anticancer and antiviral drugs in vivo and thus, should be considered for increasing efficacy in drug therapies.

Membrane conductance and surface potential

Abstract The specific conductances of black lipid membranes of lecithin, glycerylmonooleate and lecithin and cholesterol in aqueous solutions of KCl + nonactin have been measured. The relative conductances of the lecithin and the glycerylmonooleate membranes are accurately accounted for by the difference in their surface potentials. The large effect of cholesterol in depressing the conductance of the lecithin membranes is, however, not accounted for by changes in the surface potential and it is necessary to invoke either changes in the membrane solubility of the nonactin-K+ complex, or changes in the membrane viscosity (as experiences by the ionophore in transit), or both. The surface potentials which, in all the present membranes, arise solely from layers of oriented molecular dipoles, are not affected by the KCl concentration.

Activation of β-catenin signalling by GSK-3 inhibition increases p-glycoprotein expression in brain endothelial cells

This study investigates involvement of β‐catenin signalling in regulation of p‐glycoprotein (p‐gp) expression in endothelial cells derived from brain vasculature. Pharmacological interventions that enhance or that block β‐catenin signalling were applied to primary rat brain endothelial cells and to immortalized human brain endothelial cells, hCMEC/D3, nuclear translocation of β‐catenin being determined by immunocytochemistry and by western blot analysis to confirm effectiveness of the manipulations. Using the specific glycogen synthase kinase‐3 (GSK‐3) inhibitor 6‐bromoindirubin‐3′‐oxime enhanced β‐catenin and increased p‐gp expression including activating the MDR1 promoter. These increases were accompanied by increases in p‐gp‐mediated efflux capability as observed from alterations in intracellular fluorescent calcein accumulation detected by flow cytometry. Similar increases in p‐gp expression were noted with other GSK‐3 inhibitors, i.e. 1‐azakenpaullone or LiCl. Application of Wnt agonist [2‐amino‐4‐(3,4‐(methylenedioxy) benzylamino)‐6‐(3‐methoxyphenyl)pyrimidine] also enhanced β‐catenin and increased transcript and protein levels of p‐gp. By contrast, down‐regulating the pathway using Dickkopf‐1 or quercetin decreased p‐gp expression. Similar changes were observed with multidrug resistance protein 4 and breast cancer resistance protein, both known to be present at the blood–brain barrier. These results suggest that regulation of p‐gp and other multidrug efflux transporters in brain vasculature can be influenced by β‐catenin signalling.

The energy barriers to ion transport by nonactin across thin lipid membranes

Abstract The experimental steady-state current-voltage relations for low concentrations of a neutral carrier and an ion may be fitted theoretically either by assuming a form for the potential dependence of the rate of transfer of complex across the membrane and adjusting the proposed nature of the association-dissociation reactions or by assuming equilibrium for the association and adjusting the potential dependence of the transfer process. Different dependences for the rate of transfer correspond, at least formally, to different shapes for the potential energy barrier which the complex must cross. By comparing measurements of the current-voltage relations for non-actin with Na + , K + , and NH 4 + , it is possible to distinguish between the effeects of the various rates. For black lipid membranes made from glycerolmonooleate+ n- hexadecane , the potential energy barrier is high with a narrow top, but the rate of association still becomes increasingly limiting for Na + , K + and NH 4 + , in the order given. For bacterial phosphatidylethanolamine, with n- decane the barrier is much wider and no effect of the rate of association can be detected.

Functional and molecular characterization of a volume‐sensitive chloride current in rat brain endothelial cells

1 Volume‐activated chloride currents in cultured rat brain endothelial cells were investigated on a functional level using the whole‐cell voltage‐clamp technique and on a molecular level using the reverse transcriptase‐polymerase chain reaction (RT‐PCR). 2 Exposure to a hypotonic solution caused the activation of a large, outward rectifying current, which exhibited a slight time‐dependent decrease at strong depolarizing potentials. The anion permeability of the induced current was I− (1.7) > Br− (1.2) > Cl− (1.0) > F− (0.7) > gluconate (0.18). 3 The chloride channel blocker 5‐nitro‐2‐(3‐phenylpropylamino)‐benzoate (NPPB, 100 μM) rapidly and reversibly inhibited both inward and outward currents. The chloride transport blocker 4,4′‐diisothiocyanatostilbene‐2,2′‐disulphonic acid (DIDS, 100 μM) also blocked the hypotonicity‐induced current in a reversible manner. In this case, the outward current was more effectively suppressed than the inward current. The volume‐activated current was also inhibited by the antioestrogen tamoxifen (10 μM). 4 The current was dependent on intracellular ATP and independent of intracellular Ca2+. 5 Activation of protein kinase C by phorbol 12,13‐dibutyrate (PDBu, 100 nM) inhibited the increase in current normally observed following hypotonic challenge. 5 Extracellular ATP (10 mM) inhibited the current with a more pronounced effect on the outward than the inward current. 6 Verapamil (100 μM) decreased both the inward and the outward hypotonicity‐activated chloride current. 7 RT‐PCR analysis was used to determine possible molecular candidates for the volume‐sensitive current. Expression of the ClC‐2, ClC‐3 and ClC‐5 chloride channels, as well as pICln, could be shown at the mRNA level. 8 We conclude that rat brain endothelial cells express chloride channels which are activated by osmotic swelling. The biophysical and pharmacological properties of the current show strong similarities to those of ClC‐3 channel currents as described in other cell types.

Polarized distribution of nucleoside transporters in rat brain endothelial and choroid plexus epithelial cells

This study investigated mRNA expression and protein localization of equilibrative and concentrative nucleoside transporters (ENTs, CNTs) in primary cultures of rat brain endothelial cells (RBEC) and rat choroid plexus epithelial cells (RCPEC). Reverse transcriptase PCR analysis revealed that RBEC and RCPEC contained mRNA for rENT1, rENT2 and rCNT2 and for rENT1, rENT2, rCNT2 and rCNT3, respectively. Immunoblotting of membrane fractions of RBEC, fresh RCPEC and primary cultures of RCPEC revealed the presence of rENT1, rENT2 and rCNT2 proteins in all samples. Measurement of [14C]adenosine uptake into cells grown as monolayers on permeable plastic supports revealed a polarized distribution of Na+‐dependent adenosine uptake in that CNT activity was associated exclusively in membranes of RBEC facing the lower chamber (which corresponds to the surface facing the interstitial fluid in situ) and in membranes of RCPEC facing the upper chamber (which corresponds to the surface facing the cerebrospinal fluid in situ). In both RBEC and RCPEC, adenosine uptake from the opposite chambers was Na+‐independent and partially inhibited by nitrobenzylthioinosine, indicating the presence of the equilibrative sensitive transporter rENT1.

ABC transporters and drug resistance in parasitic protozoa

Parasitic protozoa are responsible for a wide spectrum of diseases in humans and domestic animals. The main line of defence available against these organisms is chemotherapy. However, the application of chemotherapeutic drugs has resulted in the development of resistance mechanisms, which limit the number of antiprotozoal drugs that are effective in the treatment and control of parasitic diseases. Knowledge about the resistance mechanisms involved may allow the development of new drugs that minimise or circumvent drug resistance or may identify new targets for drug development. This review focuses on the role of protozoal ATP-binding cassette (ABC) transporters in drug resistance. These membrane proteins mediate the ATP-dependent transport of a wide variety of chemotherapeutic drugs away from their targets inside the parasites. The genome sequence of Plasmodium falciparum and Plasmodium yoelii has recently been completed, and the sequencing of other parasitic genomes are now underway. As a result, many new membrane transporters belonging to the ABC superfamily are being discovered. We review the ABC transporters in major parasitic protozoa, including Plasmodium, Leishmania, Trypanosoma and Entamoeba species. Transporters with an established role in drug resistance have been emphasised, but newly discovered transporters with a significant amino acid sequence identity to established ABC drug transporters have also been included.