Membrane impermeability

Several compounds that inhibit vesicular glutamate transport have been identified These include the dyes Evans Blue and Rose Bengal. In addition, the stilbene derivative 4,4 -diisothiocyanatostilbene-2,2 -disulfonic acid (DEDS), a compound commonly used as a specific inhibitor of anion channels, inhibits vesicular glutamate transport. Most known inhibitors have limited use as they are membrane impermeant, with the exception of Rose Bengal. 

Like other cells, a neuron has a nucleus with genetic DNA, although nerve cells cannot divide (replicate) after maturity, and a prominent nucleolus for ribosome synthesis. There are also mitochondria for energy supply as well as a smooth and a rough endoplasmic reticulum for lipid and protein synthesis, and a Golgi apparatus. These are all in a fluid cytosol (cytoplasm), containing enzymes for cell metabolism and NT synthesis and which is surrounded by a phospholipid plasma membrane, impermeable to ions and water-soluble substances. In order to cross the membrane, substances either have to be very lipid soluble or transported by special carrier proteins. It is also the site for NT receptors and the various ion channels important in the control of neuronal excitability. 

The conformationally sensitive sulphydryl group modified by membrane-impermeant reagents is the only exofacial sulphydryl, and as described in section 4.2.1, has been tentatively identified as Cys429 (Fig. 3). It is the most reactive sulphydryl in the... 

FIG. 14 A model for the uptake of weakly basic compounds into lipid bilayer membrane (inside acidic) in response to the pH difference. For compounds with appropriate pki values, a neutral outside pH results in a mixture of both the protonated form AH (membrane impermeable) and unprotonated form A (membrane permeable) of the compound. The unprotonated form diffuse across the membrane until the inside and outside concentrations are equal. Inside the membrane an acidic interior results in protonation of the neutral unprotonated form, thereby driving continued uptake of the compound. Depending on the quantity of the outside weak base and the buffering capacity of the inside compartment, essentially complete uptake can usually be accomplished. The ratio between inside and outside concentrations of the weakly basic compound at equilibrum should equal the residual pH gradient. 

A Donnan membrane, i.e., a membrane impermeable to certain kinds of ions, which results in the occurrence of the Donnan potential. 

While both paracellular and passive transcellular pathways are available to a solute, the relative contribution of each to the observed transport will depend on the properties of the solute and the membrane in question. Generally, polar membrane-impermeant molecules diffuse through the paracellular route, which is dominated by tight junctions (Section III.A). Exceptions include molecules that are actively transported across one or both membrane domains of a polarized cell (Fig. 2). The tight junction provides a rate-limiting barrier for many ions, small molecules, and macromolecules depending on the shape, size, and charge of the solute and the selectivity and dimensions of the pathway. 

Hu Y, Litwin T, Nagaraja AR et al (2007) Cytosolic delivery of membrane-impermeable molecules in dendritic cells using pH-responsive core-shell nanoparticles. Nano Lett 7 3056-3064... 

Beth, A.H., Conturo, T.E., Venkataramu, S.D., and Staros, J.V. (1986) Dynamics and interactions of the anion channel in intact human erythrocytes An electron paramagnetic resonance spectroscopic study employing a new membrane-impermeant bifunctional spin-label. Biochemistry 25, 3824-3832. 

Staros, J.V. (1982) N-hydroxysulfosuccinimide active esters Bw(N-hydroxysulfosuccinimide) esters of two dicarboxylic acids are hydrophilic, membrane impermeant, protein cross-linkers. Biochemistry 21, 3950-3955. 

Staros, J.V. (1988) Membrane-impermeant cross-linking reagents Probes of the structure and dynamics of membrane proteins. Acc. Chem. Res. 21, 435-441. 

Staros, J.V., Lee, W.T., and Conrad, D.H. (1987) Membrane-impermeant cross-linking reagents Application to the study of the cell surface receptor for IgE. In Methods in Enzymology, (G. Di Sabato, ed.), Vol. 150, pp. 503-512. Academic Press, Orlando, FL. 

Allen MC, Gale PA, Hunter AC, Lloyd A, Hardy SP (2000) Membrane impermeant antioestrogens discriminate between ligand- and voltage-gated cation channels in NG108-15 cells. Biochim Biophys Acta 1509(1—2) 229—236... 

Dick GM, Hunter AC, Sanders KM (2002) Ethylbromide tamoxifen, a membrane-impermeant antiestrogen, activates smooth muscle calcium-activated large-conductance potassium channels from the extracellular side. Mol Pharmacol 61 (5) 1105—1113... 

Controls for Membrane Impermeance. Some fluoro-chromes can enter protoplasts via probenecid-sensitive anion channels (19) and control experiments must establish membrane impermeance. Soak the tissues in the fluorochrome at the concentration used for up to 12 h, rinsing and examining the tissues for fluorescence at hourly intervals. Cell viability is assessed by rate of cytoplasmic streaming, or by chloroplast autofluorescence, which declines in dead cells. 

Sretcher, M.S., 1972b, Phosphatidyl-ethanolamine differential labelling in intact ceUs and cell ghosts of human erythrocytes by a membrane-impermeable reagent J. Mol. Biol., 71 523-528. 

Fixation procedures are necessary when biohazardous samples are analyzed, and are sometimes used to allow access of membrane impermeant fluorochromes, or to stabilize samples for short-term storage. Optimal fixatives are those that have low autofluorescence and do not significantly affect staining. Paraformaldehyde, at concentrations of 0.5-2%, and ethanol (70%, 4 C) are widely used fixatives for flow cytometry. Combinations of paraformaldehyde with Triton X-100 or saponin have been employed in procedures that fix and permeabilize cells. 

The analysis of cell-cycle progression was one of the earliest applications of flow cytometry (for review, see Darzynkiewicz et al., 2004). In this assay, fluorescence signals from cells stained with DNA-binding fluorochromes are plotted as DNA content histograms that may be analyzed by using histogram deconvolution software to quantify cell-cycle phase distributions (Rabinovitch 1994). Fluorochromes that are useful for this purpose are the plasma membrane-impermeant DNA stains, propidium iodide (PI),... 

MTSES carries a net negative charge (Karlin and Akabas, 1998) and is membrane impermeant (Holmgren et al., 1996 Seal et al., 1998). The half-life of MTSES in buffer (pH 7.5, 22 C) is 20min (Karlin and Akabas, 1998). The length of MTSES that covalently links to the cysteine sulfhydryl is 4.8A (Holden and Czajkowski, 2002). 

AT-hiotinylaminoethylmethanethiosulfonate (MTSEA-biotin) is a relatively membrane-impermeant MTS derivative to which a hiotin moiety is conjugated (Seal et al., 1998 Boileau et al., 2002). It is composed of two structural domains a flexible tail 12-14A in length, 2.5A in diameter, and 4 x 5A planar head group (Teissere and Czajkowski, 2001 Wagner and Czajkowski, 2001). The reactive disulfide is near the end of the tail 12A from the head group. 

Use of the pH-sensitive membrane-impermeable flurophore pyranine based on the ratiometric method, which determines directly level of dissociation of pyranine from the ratio between the charged (nnprotonated) pyranine and total pyranine in the intraliposome aqneons phase Addition of impermeable DPX, which acts as a quencher to pyranine fluorescence, into the liposome external medium ensures lack of contribution of extraliposome medium pyranine fluorescence (18,22). This method is considered invasive as the pyranine has to be added in the hydration medium prior to liposome preparation and cannot be used for pH determination of intraliposome aqueous phase... 

Isoniazid is bactericidal against growing M. tuberculosis. Its mechanism of action remains unclear. (In the bacterium it is converted to isonicotinic acid, which is membrane impermeable, hence likely to accumulate intracellu-larly.) Isoniazid is rapidly absorbed after oral administration. In the liver, it is inactivated by acetylation, the rate of which is genetically controlled and shows a characteristic distribution in different ethnic groups (fast vs. slow acetylators). Notable adverse effects are peripheral neuropathy, optic neuritis preventable by administration of vitamin Be (pyridoxine) hepatitis, jaundice. 

Oxygen consumption provides an index of function that incorporates several parameters, including irmer membrane impermeability and OXPHOS integrity. 

Acetylcholine is an ester of choline and acetic acid, the prototype for a small family of choline ester compounds. The choline moiety of ACh contains a quaternary ammonium group that gives ACh a permanent positive charge, making it very hydrophilic and membrane impermeant. 

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