Hemoglycin visible fluorescence induced by x rays.

Hemoglycin, a 1494 Da polymer composed of iron and glycine, has been detected in several carbonaceous meteorites. Iron atoms close out the ends of a 5 nm anti-parallel glycine beta sheet and contribute visible and near infrared absorptions that are not present with glycine alone. The 483 nm absorption of hemoglycin was discovered in theory and then observed on beamline I24 at Diamond Light Source. Light absorption in a molecule involves a coupled lower set of states receiving light energy that causes a transition into an upper set of states. In the reverse process, some energy source, such as an x-ray beam, populates the upper set of molecular states, which then radiates light as it returns to the lower "ground" set of states. We report on visible light re-emission during x-ray irradiation of a hemoglycin crystal. The emission is dominated by bands centered at 489 and 551 nm.

Topology of the mammalian cell via cryo-FIB etching.

Mapping of a mammalian cell down to a feature size of 20-30 nm in 3D is a goal that will answer many questions concerning the connectivity (topology) of a Eukaryotic cell's traffic routes. These routes are defined and separated from one another by the protein-impregnated lipid membrane barrier of the endoplasmic reticulum (ER). We trace the routes from outside a live flash frozen buccal epithelial cell via gold (Au) labelled pores in the plasma membrane to the ER below and then through the cell as isosurfaces in 3D maps. The outer tubular ER with three-way branching changes to a sheet-like ER nearer the nucleus, and the cytoplasmic space between the ER membranes continues as a volume into the nuclear interior via the nuclear pores. We find some evidence that the last layer of the cytoplasmic ER membrane, also termed the outer nuclear membrane, has discrete gaps, so the ER lumen in these areas is continuous with the nuclear luminal domain and further, the inner nuclear membrane has small protrusions into the nucleus. The routes were established in live, unstained, unfixed, cells etched with a pAmp current of a focused ion beam (cryo-FIB) dual beam electron microscope, at -150 degrees C, 1e-4Pa, and confirmed at 37 degrees C in lipid-dye stained cells. The cryo-FIB etch of a cuboid of 2D planes, and its reconstruction into many 3D maps, takes only hours, facilitating the execution of experiments with comparative conditions in a few days.

Batten disease and the control of the Fo subunit c pore by cGMP and calcium.

Subunit c of ATP synthase functions as a high conductance ion channel, tightly regulated by calcium. We have suggested that the pathogenesis of Batten syndromes involving overaccumulation of subunit c are linked to the protein's ion channel function. In normal electrically excitable tissue the channel could act as a pacer setting nodal voltage via control of cation entry. The channel conductance is controlled by voltage, calcium, cyclic nucleotides and polyamines. We discuss the pathogenic role that subunit c could play in the electrically excitable tissues of retina, brain and heart where Batten neurodegeneration is seen. Focus is given to potential links between subunit c and the known mutant gene products in the Batten diseases, the process of apoptosis, and the requirement of the growing brain for gradients of cGMP, a ligand of the subunit c channel.

Opposing actions of cGMP and calcium on the conductance of the F(0) subunit c pore.

Subunit c of ATP synthase can be purified from neuronal plasma membrane and from the inner mitochondrial membrane. In the latter location the hydrophobic 75 amino acid protein is one component of the F(1) F(0) ATP synthase complex but in the former it is alone as a pore that is capable of generating spontaneous electrical oscillations. Pure mammalian subunit c when reconstituted in lipid bilayers and voltage clamped, yields a voltage sensitive pore that conducts a cation current regulated by calcium. The current is here found to be activated by cGMP with a K(M) ranging from 14 nM to 19 microM depending on calcium and temperature. It is sensitively inhibited by a number of ligands. The K(I) for calcium ranges from 100 nM to 100 microM depending on cGMP and temperature. DCCD inhibits with a K(app) of 100 nM. The polyamine nicotine inhibits at 84 nM. The pore has properties that would allow it to deliver sodium or calcium through the cell membrane in a controlled manner while maintaining membrane polarization.

Biological-to-electronic interface with pores of ATP synthase subunit C in silicon nitride barrier.

An oscillator pore is identified that generates intermittent, large amplitude, ionic current in the plasma membrane. The pore is thought to be composed of 10-12 units of subunit c of ATP synthase. Pore opening and closing is a co-operative process, dependent on the release, or binding, of as many as six calcium ions. This mechanism suggests a more general method of co-operative threshold detection of chemical agents via protein modification, the output being directly amplified in a circuit. Here the authors describe steps in the development of a sensor of chemical agents. The subunit c pore in a lipid bilayer spans a nanometer-scale hole in a silicon nitride barrier. Either side of the barrier are electrolyte solutions and current through the pore is amplified by circuitry. The techniques of laser ablation, electron beam lithography and ion beam milling are used to make successively smaller holes to carry the lipid patch. Holes of diameter as small as 20 nm are engineered in a silicon nitride barrier and protein activity in lipid membranes spanning holes as small as 30 nm in diameter is measured. The signal-to-noise ratio of the ionic current is improved by various measures that reduce the effective capacitance of the barrier. Some limits to scale reduction are discussed.

The yeast Saccharomyces cerevisiae does not sequester chloride but can express a functional mammalian chloride channel.

Chloride uptake into yeast was measured as a function of pH. A small amount of uptake was seen at pH values of 3.0 and 4.0; at pH 6.0 chloride uptake was substantially less than the uptake of phosphate and rubidium. Because chloride uptake is inefficient, we expressed the putative mammalian chloride channel, pI(Cln), in yeast and observed a chloride-selective current when total membrane protein was reconstituted into lipid bilayers. The current was inhibited by a specific chloride channel blocker, 5-nitro-2-(3-phenylpropylamino)-benzoic acid. These results suggest that yeast may serve as a means to characterize chloride channels from other organisms.

Ion pores made of mitochondrial ATP synthase subunit c in the neuronal plasma membrane and Batten disease.

A hypothesis is outlined that the neurodegeneration of the Batten disease syndromes that involve an overaccumulation of subunit c is caused by a newly characterized function of the protein, its ability to assemble in the plasma membrane into ion pores (J. E. M. McGeoch and G. Guidotti, Brain Res 766: 188-194, 1997), rendering the cell liable to constant electrical excitability to a degree that causes cell death.

A 0.1-700 Hz current through a voltage-clamped pore: candidate protein for initiator of neural oscillations.

A protein of mass 7643 Da and sequence identical to that of subunit c, the pore part, of the mitochondrial adenosine triphosphate synthase complex, was co-purified with cholesterol in crystals formed from a chloroform/methanol extract of bovine brain gray matter plasma membranes. Reconstitution of the protein-containing crystals in phospholipid bilayers and assay of current by patch-clamp analysis, showed an oscillating cation current at constant voltage, typically of frequency 0.5-200 Hz. The ceroid-lipofuscinoses state in mammals and man (Batten disease), in which subunit c accumulates in lysosomes, affords a rich source of the protein. Pure subunit c from affected sheep liver (in the absence of cholesterol) was also assayed, the current displaying identical sodium oscillations to those of brain crystals. The results suggest that if a protein similar to subunit c resides in the plasma membrane of neural cells, it could be responsible for spontaneous oscillations in brain tissue. The relevance of these results to the pathogenesis of Batten disease is discussed.

Eye CNG channel is modulated by nicotine.

The cyclic-nucleotide gated channel (CNG channel) of the rod outer segment of the retina (ROS) has its closed conformation stabilized by nicotine. Calcium and cGMP influence the Ki of the channel current to nicotine. Calcium lowers the Ki and cGMP increases it, giving a range of Ki between 10(-11) and 10(-8) M.

An insulin-sensitive cation channel controls [Na+]i via [Ca2+]o-regulated Na+ and Ca2+ entry.

The insulin-stimulated cation channel previously identified in patch-clamped muscle preparations is here shown to be responsible for bulk Na+ entry into the cell. The mainly Na+ current of the channel was shown to be accompanied by an inhibitory Ca2+ component responsible for oscillations. Here, using quantitative fluorescence imaging of Fura-2- and SBFI-loaded soleus muscle, we measure changes in [Na+]i and [Ca2+]i related to channel function. Insulin increased [Na+]i and [Ca+]i in a transient spike of < 1-min duration. There was a momentary dip in [Na+]i related to inhibition of the channel by the Ca2+ spike, and changes in external Ca2+ were shown to alter [Na+]i via the cation channel, all effects being blocked by the specific channel inhibitor mu-conotoxin, but not by tetrodotoxin. The [Ca2+]i spike could also be induced by 8-bromo cyclic-guanosine 5'-monophosphate, an analogue of the channel-activator cyclic-guanosine 5'-monophosphate (cGMP). In addition it was noted that insulin reduced the [Ca2+]i rise upon subsequent muscle depolarization by a factor of 3.5. Insulin could be substituted with phorbol ester for the same effect and HA1004, a protein kinase inhibitor, blocked the reduction.

Power spectra and cooperativity of a calcium-regulated cation channel.

In this article we show that a channel complex of cooperatively interacting subunits can produce a power law spectrum with the slope of the spectrum depending on the strength of the cooperative interaction. The effects of cooperativity are explored via a computational model of a calcium-regulated cation channel for which new data is presented. The results, which concern "flickering" conductances, are correlated with prior work on critical fluctuations in the Ising model of ferromagnetism.

An insulin-stimulated cation channel in skeletal muscle. Inhibition by calcium causes oscillation.

A cation channel has been identified in the plasma membrane of skeletal muscle that oscillates open and closed in a regular manner. In an experimental system of patch-clamped reconstituted plasma membrane in phospholipid bilayers, the oscillations are calcium-dependent and constitute regular closing events due to inhibition of the channel by calcium with a Ki of 2.2 +/- 1 x 10(-6) M, followed by reopening. There are 3.7 +/- 1 calcium binding sites/channel. With sodium as the current vehicle, conductance is increased by voltage, insulin (Km = 5 +/- 0.6 x 10(-9) M), and hydrolyzable guanine nucleotides. Cyclic GMP alone with increase the conductance with a Km of 3.7 +/- 0.6 x 10(-7) M. In the absence of calcium, the unitary conductance with insulin + GTP or cGMP at 150 mM NaCl is 153 picosiemens. Sodium current is insensitive to 10(-5) M tetrodotoxin but inhibited by mu-conotoxin (Ki = 5 x 10(-8) M). These findings in the reconstituted system were verified in patch-clamped whole muscle cells where an insulin and cGMP-dependent sodium current inhibited by mu-conotoxin could be demonstrated. In the whole cell experiments, slow calcium-dependent oscillations of the sodium current were also detected.

The alpha-2 isomer of the sodium pump is inhibited by calcium at physiological levels.

The inhibition of the (Na,K)ATPase by calcium was investigated in plasma membrane preparations of rat axolemma, skeletal muscle and kidney outer medulla. Ouabain titration curves demonstrated that physiological calcium (0.08-5 microM) inhibited mainly the high affinity alpha 2 isomer. In axolemma all the (Na,K)ATPase had high ouabain affinity and calcium inhibited 40-50% of the activity with a Ki of 1.9 +/- 0.9 x 10(-7) M. In skeletal muscle high and low ouabain affinity components were present in equal amounts and calcium inhibited only the high affinity component with a Ki of 1.3 +/- 0.3 x 10(-7) M. Kidney enzyme had a low affinity for ouabain and showed very little sensitivity to calcium in the physiological range. It was demonstrated that high calcium levels inhibit the enzyme in a general sense, irrespective of the isomer, with a Ki of 6.5 +/- 6 x 10(-4) M for the kidney and 5.9 +/- 4 x 10(-4) M for the axolemma enzymes. In axolemma, enzyme activity was studied as a function of sodium concentration. Physiological calcium reduced Vmax while not significantly changing K 0.5 for sodium binding.

Polycystic disease of kidney, liver and pancreas; a possible patholgenesis.

5,6,7,8-tetrahydrocarbzzole-3-acetic acid (AH 2835) given to maternal rats throughout their gestation produces an experimental model of the autosomally inherited human infantile polycystic disease Potter type I in the rat foetuses. The affected animals have cystic lesions in their kidneys, liver and pancreas like those seen in the human. Evidence is presented for the aetiology of the experimental lesion being related to the action of AH 2835 on the specific activity of the ouabain sensitive (Na+ + K+)-ATPase of absorptive epithelia. It is noted that two cystic kidney diseases, the ocngenital nephrotic syndrome and infantile polycystic disease Potter type I are both inherited as autosomal recessive traits and are therefore likely to be caused by enzyme abnormalities, and that the compound AH 2835 can be used to produce experimental models of both of these diseases.