The thermodynamic soliton theory of the nervous impulse and possible medical implications.

The textbook picture of nerve activity is that of a propagating voltage pulse driven by electrical currents through ion channel proteins, which are gated by changes in voltage, temperature, pressure or by drugs. All function is directly attributed to single molecules. We show that this leaves out many important thermodynamic couplings between different variables. A more recent alternative picture for the nerve pulse is of thermodynamic nature. It considers the nerve pulse as a soliton, i.e., a macroscopic excited region with properties that are influenced by thermodynamic variables including voltage, temperature, pressure and chemical potentials of membrane components. All thermodynamic variables are strictly coupled. We discuss the consequences for medical treatment in a view where one can compensate a maladjustment of one variable by adjusting another variable. For instance, one can explain why anesthesia can be counteracted by hydrostatic pressure and decrease in pH, suggest reasons why lithium over-dose may lead to tremor, and how tremor is related to alcohol intoxication. Lithium action as well as the effect of ethanol and the anesthetic ketamine in bipolar patients may fall in similar thermodynamic patterns. Such couplings remain obscure in a purely molecular picture. Other fields of application are the response of nerve activity to muscle stretching and the possibility of neural stimulation by ultrasound.

Calcium electroporation and electrochemotherapy for cancer treatment: Importance of cell membrane composition investigated by lipidomics, calorimetry and in vitro efficacy.

Calcium electroporation is a novel anti-cancer treatment investigated in clinical trials. We explored cell sensitivity to calcium electroporation and electroporation with bleomycin, using viability assays at different time and temperature points, as well as heat calorimetry, lipidomics, and flow cytometry. Three cell lines: HT29 (colon cancer), MDA-MB231 (breast cancer), and HDF-n (normal fibroblasts) were investigated for; (a) cell survival dependent on time of addition of drug relative to electroporation (1.2 kV/cm, 8 pulses, 99 µs, 1 Hz), at different temperatures (37 °C, 27 °C, 17 °C); (b) heat capacity profiles obtained by differential scanning calorimetry without added calcium; (c) lipid composition by mass spectrometry; (d) phosphatidylserine in the plasma membrane outer leaflet using flow cytometry. Temperature as well as time of drug administration affected treatment efficacy in HT29 and HDF-n cells, but not MDA-MB231 cells. Interestingly the HT29 cell line displayed a higher phase transition temperature (approximately 20 °C) versus 14 °C (HDF-n) and 15 °C (MDA-MB231). Furthermore the HT29 cell membranes had a higher ratio of ethers to esters, and a higher expression of phosphatidylserine in the outer leaflet. In conclusion, lipid composition and heat capacity of the membrane might influence permeabilisation of cells and thereby the effect of calcium electroporation and electrochemotherapy.

Characterization of the secondary structure and assembly of the transmembrane domains of trypsinized Na,K-ATPase by Fourier transform infrared spectroscopy.

Fourier transform infrared spectroscopy has been used to compare native Na,K-ATPase-containing membranes with those trypsinized in the presence of either Rb+ or Na+ ions to remove the extramembranous parts of the protein. The protein secondary structure content deduced from the amide I band is approximately 30-35% alpha-helix, 37-40% beta-structure, and 13-15% random coil for native membranes from shark rectal gland and from pig kidney, in both the Na- and K-forms. Trypsinization in either Rb+ (a K+ congener) or Na+ removes approximately 35% of the amide I band intensity of native membranes from shark rectal gland. The protein secondary structural content of the trypsinized membranes lies in the range of approximately 23-32% alpha-helix, 37-46% beta-structure, and 12-18% random coil for the shark and kidney enzymes. The distribution of intensity between the bands corresponding to protonated and deuterium-exchanged alpha-helices, and between the component bands attributed to beta-structure, changes considerably on trypsinization, in the direction of a greater proportion of protonated alpha-helix and a broader range of frequencies for beta-structure. The kinetics of deuteration of the slowly exchanging population of protein amide groups is also changed on trypsinization. The mean rate constant for deuteration of trypsinized membranes is approximately half that for native membranes, whereas the proportion of amides contributing to this population increases on trypsinization. The temperature dependence of the amide I band in the Fourier transform infrared spectra indicates that the onset of thermal denaturation occurs at 58 degrees C for native membranes (in either Na+ or K+) and for membranes trypsinized in Rb+, but the major denaturation event for membranes trypsinized in Na+ occurs at approximately 84 degrees C. These results correlate with the functional properties of the intramembranous section of the enzyme.

Integration of a K+ channel-associated peptide in a lipid bilayer: conformation, lipid-protein interactions, and rotational diffusion.

The 26-residue peptide of sequence KEALYILMVLGFFGFFTLGIMLSYIR, which contains the single putative transmembrane domain of a small protein that is associated with slow voltage-gated K+ channels, has been incorporated in bilayers of dimyristoylphosphatidylcholine by dialysis from 2-chloroethanol to form complexes of homogeneous lipid/peptide ratio. Fourier transform infrared spectroscopy indicates that the peptide is integrated in the lipid bilayer wholly in a beta-sheet conformation. The electron spin resonance spectra of spin-labeled lipids in the lipid/peptide complexes contain a component corresponding to lipids whose chains are motionally restricted in a manner similar to those of lipids at the hydrophobic surface of integral transmembrane proteins. From the dependence of the lipid spin label spectra on the lipid/peptide ratio of the complexes, it is found that ca. 2.5 lipids per peptide monomer, independent of the species of spin-labeled lipid, are motionally restricted by direct interaction with the peptide in the bilayer. This value would be consistent with, e.g., a beta-barrel structure for the peptide in which the beta-strands either are strongly tilted or have a reverse turn at their center. A preferential selectivity of interaction with the peptide is observed for the negatively charged spin-labeled lipids phosphatidic acid, stearic acid, and phosphatidylserine, which indicates close proximity of the positively charged residues at the peptide termini to the lipid headgroups. The saturation-transfer electron spin resonance spectra of the peptide spin-labeled at a cysteine residue replacing Leu18 evidence rather slow rotational diffusion in the lipid complexes.(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence for a common structure for a class of membrane channels.

Electron microscopic analysis of gap-junction-like structures isolated from an anthropod (Nephrops norvegicus) and composed of a 16-kDa polypeptide, show the functional unit to be a star-shaped hexamer of protein arranged around a central channel which runs perpendicular to the plane of the membrane. Estimations of the molecular volume carried out on an averaged projection are consistent with a subunit mass of 16-18 kDa. Fourier transform infrared spectroscopy indicates a high alpha-helical content for the protein, supporting secondary-structure predictions of four transmembrane alpha helices/monomer. The averaged projection shows a close resemblance to a hexamer of the 16-kDa protein built on the basis of a four alpha-helical bundle [Finbow, M. E., Eliopoulos, E. E., Jackson, P. J., Keen, J. N., Meagher, L., Thompson, P., Jones, P. C. & Findlay, J. B. C. (1992) Protein Eng. 5, 7-15]. The reconstructed image is also similar to that obtained for gap-junction-like channels isolated from a related arthropod [Homarus americanus; Sikerwar, S. S., Downing, K. H. & Glaeser, R. M. (1991) J. Struct. Biol. 106, 255-263] whose protein content was unknown but which we demonstrate may be composed of a related 16-kDa protein. Previous studies have shown a high sequence identity of the Nephrops 16-kDa protein with the 16-kDa proteolipid subunit c of the vascular H(+)-ATPase, both of which in turn bear similarity to the 8-kDa proteolipid subunit of the F1F0-ATP synthase. Expression of cDNA coding for the Nephrops 16-kDa protein in Saccharomyces cerevisiae, in which the endogenous gene coding for the V-ATPase proteolipid has been inactivated, restores V-ATPase activity and cell growth.