Julie E.M. McGeoch

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.

The α-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 (0.08-5μM) inhibited mainly the high affinity α2 isomer. In axolemma all the (Na,K)ATPase had high ouabain affinity and calcium inhibited 40–50% of the activity with a K i of 1.9±0.9×10 −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 K i of 1.3±0.3×10 −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 K i of 6.5±6×10 −4 M for the kidney and 5.9±4×10 −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.

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.

Entrapment of Water by Subunit C of ATP Synthase

We consider an ancient protein, and water as a smooth surface, and show that the interaction of the two allows the protein to change its hydrogen bonding to encapsulate the water. This property could have made a three-dimensional microenvironment, 3–4 Gyr ago, for the evolution of subsequent complex water-based chemistry. Proteolipid, subunit c of ATP synthase, when presented with a water surface, changes its hydrogen bonding from an α-helix to β-sheet-like configuration and moves away from its previous association with lipid to interact with water surface molecules. Protein sheets with an intra-sheet backbone spacing of 3.7 Å and inter-sheet spacing of 6.0 Å hydrogen bond into long ribbons or continuous surfaces to completely encapsulate a water droplet. The resulting morphology is a spherical vesicle or a hexagonal crystal of water ice, encased by a skin of subunit c. Electron diffraction shows the crystals to be highly ordered and compressed and the protein skin to resemble β-sheets. The protein skin can retain the entrapped water over a temperature rise from 123 to 223 K at 1×10−4 Pa, whereas free water starts to sublime significantly at 153 K.

Regulation of the Na+,K+-pump by Insulin

Insulin administration reduces the serum glucose and K+ concentrations (6) by promoting their entry into skeletal muscle (20). The acute function of insulin, thus, is to clear from the blood two of the major nutrients ingested during a meal. Clausen and Kohn (13) and Resh et al. (33) showed that insulin stimulates the activity of the Na+,K+-pump in rat skeletal muscle and adipocytes, respectively, by a direct effect on the pump rather than by an increase in the intracellular Na+ concentration. On the other hand, there are instances in which insulin does stimulate a Na+/H+ exchanger resulting in an increase in intracellular Na+ and increased activity of the (Na+,K+)-pump through an increase in the substrate concentration (15,26,34). The central question here is the mechanism of the direct activation of the (Na+,K+)-pump by insulin.

Specific Insulin Binding to Purified Na,K-ATPase Associated with Rapid Activation of the Enzyme

Publisher Summary This chapter presents an experimental study on specific insulin binding to purified Na, K–ATPase associated with rapid activation of the enzyme. The first observations of insulin interaction with highly purified Na, K–ATPase is reported in the chapter. The enzyme was prepared by the method of Jorgensen from dog renal outer medulla. The chapter shows the initial stimulation of a function of insulin concentration. Analysis of 66 test experiments with 100 controls showed a highly significant stimulation of the specific activity, although the sensitivity to insulin was rather variable. Specific insulin binding to the Na, K–ATPase preparations was measured by the techniques of filtration and centrifugation.

Polymer amide as an early topology.

Hydrophobic polymer amide (HPA) could have been one of the first normal density materials to accrete in space. We present ab initio calculations of the energetics of amino acid polymerization via gas phase collisions. The initial hydrogen-bonded di-peptide is sufficiently stable to proceed in many cases via a transition state into a di-peptide with an associated bound water molecule of condensation. The energetics of polymerization are only favorable when the water remains bound. Further polymerization leads to a hydrophobic surface that is phase-separated from, but hydrogen bonded to, a small bulk water complex. The kinetics of the collision and subsequent polymerization are discussed for the low-density conditions of a molecular cloud. This polymer in the gas phase has the properties to make a topology, viz. hydrophobicity allowing phase separation from bulk water, capability to withstand large temperature ranges, versatility of form and charge separation. Its flexible tetrahedral carbon atoms that alternate with more rigid amide groups allow it to deform and reform in hazardous conditions and its density of hydrogen bonds provides adhesion that would support accretion to it of silicon and metal elements to form a stellar dust material.