Structure of the KcsA potassium channel from Streptomyces lividans: a site-directed spin labeling study of the second transmembrane segment.

KcsA is a prokaryotic potassium channel. The present study employs cysteine scanning mutagenesis and site-directed spin labeling to investigate the structure of the second transmembrane segment (residues 82-120) in functional tetrameric channels reconstituted in lipid bilayers. Spin-spin interactions are observed between nitroxide side chains at symmetry-related sites close to the 4-fold axis of symmetry. To aid in quantitative analysis of these interactions, a new diamagnetic analogue of the nitroxide side chain is used to prepare magnetically dilute samples with constant structure. Using constraints imposed by the spin-spin interactions, a packing model for this segment is deduced that is in excellent agreement with the recently reported crystal structure [Doyle, D., et al. (1998) Science 280, 69-77]. The relatively immobilized state of the nitroxide side chains suggests that the channel is rigid on the electron paramagnetic resonance time scale. Moreover, the poor sulfhydryl reactivity of the cysteine at many locations indicates that the channel is not subject to the low-frequency fluctuations that permit reaction of buried cysteines. At sites expected to be located in the pore, the accessibility of the side chains to collision with O(2) or nickel(II) ethylenediaminediacetate is low. This inaccessibility, together with the generally low mobility of the side chains throughout the sequence, makes it difficult to detect the presence of the pore based on these measurements. However, the presence of a solvated pore can be directly demonstrated using a polarity parameter deduced from the EPR spectra recorded at low temperature. These measurements also reveal the presence of a polarity gradient in the phospholipid bilayer.     

Hydrocarbon-metabolizing activity of various mammalian cells in culture

Summary Two methods for determining the hydrocarbon-metabolizing enzyme activity of cultured mammalian cells were compared. The method designed to measure benzo[a]an-thracene-induced aryl hydrocarbon hydroxylase activity could detect and quantify enzyme activities in low passage rodent cells, but could not reproducibly detect levels in intermediate or high passage mouse, rat, or human cells. The method designed to measure the ability of a cell to convert benzo[a]pyrene from an organic-soluble to an aqueous acetone-soluble form proved to be more reproducible. This technique, when modified, was demonstrated to be an effective screening test for the detection of those lines with higher levels of hydrocarbon-metabolizing enzymes. Supported by the Council for Tobacco Research and Contract NIH 70-2068 within the Virus Cancer Program, National Cancer Institute, National Institutes of Health.     

Plasticity of monkey triceps muscle fibers in microgravity conditions.

We examined the changes in functional properties of triceps brachii skinned fibers from monkeys flown aboard the BION 11 satellite for 14 days and after ground-based arm immobilization. The composition of myosin heavy chain (MHC) isoforms allowed the identification of pure fibers containing type I (slow) or type IIa (fast) MHC isoforms or hybrid fibers coexpressing predominantly slow (hybrid slow; HS) or fast (hybrid fast) MHC isoforms. The ratio of HS fibers to the whole slow population was higher after flight (28%) than in the control population (7%), and the number of fast fibers was increased (up to 86% in flight vs. 12% in control). Diameters and maximal tensions of slow fibers were decreased after flight. The tension-pCa curves of slow and fast fibers were modified, with a decrease in pCa threshold and an increase in steepness. The proper effect of microgravity was distinguishable from that of immobilization, which induced less marked slow-to-fast transitions (only 59% of fast fibers) and changed the tension-pCa relationships.