Measurement of Ensemble TRPV1 Ion Channel Currents Using Droplet Bilayers

Electrophysiological characterization of ion channels is useful for elucidation of channel function as well as quantitative assessment of pharmaceutical effects on ion channel conductance. We used droplet bilayers to measure ensemble ion channel currents from membrane preparations made from TRPV1-expressing HEK cells. Conductance measurements showed rectification, activation by acid and capsaicin, and inhibition by capsazepine, SB 452533, and JNJ 17293212. We also quantitatively measured concentration-dependent inhibition of channel conductance through determination of capsazepine IC₅₀ in agreement with previously published studies using patch clamp. These results, combined with the reduced apparatus and material requirements of droplet bilayers, indicate that this platform could be used for study of other physiologically relevant ion channels.     

Proton transport through influenza A virus M2 protein reconstituted in vesicles

Influenza A virus M2 protein is known to form acid-activated, proton-selective, amantadine-sensitive channels. We directly measured proton uptake in vesicles containing reconstituted M2 by monitoring external pH after addition of valinomycin to vesicles with 100-fold-diluted external [K⁺]. External pH typically increased by a few tenths of a pH unit over a few minutes after valinomycin addition, but proton uptake was not significantly altered by acidification. Under neutral conditions, external addition of 1 mM amantadine produced a reduction in flux consistent with randomly ordered channels; however, experimental variation is high with this method and the block was not statistically significant. Amantadine block was reduced at pH 5.4. In accord with Lin and Schroeder's study of reconstituted M2 using a pH-sensitive dye to monitor intravesicular pH, we conclude that bath pH weakly affects or does not significantly affect proton flow in the pH range 5.4-7.0 for the reconstituted system, contrary to results from electrophysiological studies. Theoretical analysis of the relaxation to Donnan equilibrium utilized for such vesicle uptake assays illuminates the appropriate timescale of the initial slope and an important limitation that must be placed on inferences about channel ion selectivity. The rise in pH over 10 s after ionophore addition yielded time-averaged single-channel conductances of 0.35 ± 0.20 aS and 0.72 ± 0.42 aS at pH 5.4 and 7.0, respectively, an order of magnitude lower than previously reported in vesicles. Assuming complete membrane incorporation and tetramerization of the reconstituted protein, such a low time-averaged conductance in the face of previously observed single-channel conductance (6 pS at pH 3) implies an open channel probability of 10⁻⁶-10⁻⁴. Based on leakage of potassium from M2-containing vesicles, compared to protein-free vesicles, we conclude that M2 exhibits ∼10⁷ selectivity for hydrogen over potassium.     

Proton conductance of influenza virus M2 protein in planar lipid bilayers

Purified M2 protein from the Udorn strain of influenza virus was reconstituted into planar lipid bilayers from liposomes. In 1 mM HCl, the single-channel conductance was measured as 6 pS with open probability of < or =0.03. The current voltage curve is linear over the achievable voltage range. The current amplitude is amantadine sensitive. In HCl solutions, the single-channel current was essentially invariant with changes in [Cl(-)], [Na(+)], and [tetraethylammonium] ([TEA(+)]), but dependent on [H(+)]. The reversal potential, determined with asymmetrical hydrogen chloride solution, is very close to the equilibrium potential of hydrogen. This appears to be the first report of single-channel proton currents with the full-length M2 protein.     

High-level expression of rabbit 15-lipoxygenase induces collapse of the mitochondrial pH gradient in cell culture

A critical step in the development of mammalian erythroblasts into mature red blood cells is the extrusion of the nucleus, followed by intracellular degradation of the remaining organelles. It has been hypothesized that the breakdown of cellular organelles in rabbit reticulocytes is initiated by 15-lipoxygenase. In vitro, the purified rabbit reticulocyte 15-lipoxygenase binds and permeabilizes organellar membranes, thereby releasing the lumenal contents of the organelle. Here, we demonstrate that ectopic expression of 15-lipoxygenase leads to the collapse of the mitochondrial pH gradient in nonerythroid cells, using a novel reporter of mitochondrial pH, mito-pHluorin. No change in mitochondrial pH was observed with a mutant of 15-lipoxygenase that lacks enzymatic activity. These data demonstrate that 15-lipoxygenase is capable of disrupting the pH gradient maintained by mitochondria in living cells without additional factors specific for red blood cell development.     

Synergistic effect between apolipoprotein E and apolipoprotein A1 gene polymorphisms in the risk for coronary artery disease

Alterations in lipid metabolism and genetic predisposition are major risk factors for coronary artery disease (CAD). Variations in genes involved in lipid metabolism may act synergistically to confer risk or protection against CAD. The objective of the present study was to determine such interactions in variants of apolipoprotein E and apolipoprotein A1 genes. One hundred and forty subjects with clinically confirmed CAD and 100 unrelated normal subjects participated in the study. Multiple regression analysis was used to relate lipid and apolipoprotein profiles with genotypes. Odd ratios were calculated for various combinations of ApoE and ApoA1 genotypes. Prevalence of ApoE 'E4' and ApoA1 'A' and 'T' alleles was significantly higher in patients than controls. Serum apolipoprotein E and apolipoprotein A1 levels were significantly lower in CAD patients than controls. When lipid parameters were related to genotypes, the polymorphisms associated to various markers were in agreement with previous reports. ApoE 2/4 genotype in combination with either ApoA1 heterozygous GA or CT genotype conferred higher risk of CAD. E3 allele in homozygous or heterozygous state in combination with ApoA1+83 CC genotype conferred highest protection (P < 0.05). Thus, it appears that ApoE and ApoA1 gene variants may act synergistically to associate with risk and protection against CAD.     

AFM visualization of mobile influenza A M2 molecules in planar bilayers

We report the observation of influenza A M2 (M2) incorporated in a dipalmitoylphosphatidylcholine (DPPC) supported planar bilayer on mica, formed by use of a modified vesicle fusion method from proteoliposomes and visualized with contact mode atomic force microscopy. Incubation of proteoliposomes in a hyperosmotic solution and increased DPPC/M2 weight ratios improved supported planar bilayer formation by M2/DPPC proteoliposomes. M2's extra-bilayer domains were observed as particles estimated to protrude 1-1.5 nm above the bilayer surface and <4 nm in diameter. Particle density was 5-18% of the nominal tetramer density. Movement of observable M2 particles was independent of the probe tip. The mean lateral diffusion coefficient (D) of M2 was 4.4 +/- 1.0 x 10(-14) cm(2)/s. Eighty-two percent of observable particles were mobile on the observable timescale (D > 6 x 10(-15) cm(2)/s). Protein-protein interactions were also observed directly.     

Bis[(benzo-15-crown-5)-15-yl methyl] pimelate forms ion channels in planar lipid bilayer: a novel model ion channel

Some crown ethers translocate cations across the liposomal membrane either by a carrier mechanism or by forming ion channels. We report formation of ion channels in lipid bilayer membranes by bis[(benzo-15-crown-5)-15-yl methyl] pimelate, a crown ether known to form ion inclusion complexes with alkali metal cations. The channels have characteristic long openings lasting several seconds and a low conductance (4 pS in 500 mM KCl and 2.5 pS in 500 mM NaCl). A model of the crown ether channel formed by stacking of four monomers is proposed. A large database of structural information on crown ethers and their ion inclusion complexes as well as large family of crown ethers with a variety of substitutions in the ring are commercially available. Thus the crown ether channel is an attractive model system to study the role of various chemical moieties in ion conduction which may provide deeper insight into understanding the mechanism(s) of selectivity, ion transport, etc. in biological ion channels.     

Collective behaviour of crown channels

Collective behaviour of a crown ether channel, bis[(benzo-15-crown-5)-15-yl methyl] pimelate, in a planar lipid bilayer membrane has been studied through electrophysiological methods. A characteristic feature of these channels is their sequential opening, indicated by a uniform stepwise increase in the multi-channel current. The experimental results show that there are three modes of relaxation, of which the slowest one is attributed to the channel–channel interaction. The latter varies with the number of channels incorporated in the bilayer membrane, leading to the interpretation that crown channels behave cooperatively.