Monovalent and divalent cation permeation in acetylcholine receptor channels. Ion transport related to structure

Single channel patch-clamp techniques were used to study nicotinic acetylcholine receptors in cultured rat myotubes. The single channel conductance in pure cesium and sodium levels off at high concentrations, as if a binding site within the channel were saturating. The conductances at very low concentrations, however, are larger than predicted by the simplest one-site transport model fitted to the high-concentration data. At low concentrations, the current-voltage relations are inwardly rectifying, but they become more ohmic if a small amount of divalent cations is added externally. Magnesium and barium are good permeants that have rather high affinities for the channel. Upon adding low millimolar concentrations of these divalent cations externally to a membrane bathed in pure cesium, the inward current carried by cesium is decreased. As more divalent cations are added, the inward-going currents continued to decrease and the divalent cation replaces cesium as the main current carrier. The ion transport data are described by considering the size, shape, and possible net charge of the channel. In that way, even the complex features of transport are explained in a realistic physical framework. The results are consistent with the channel having long, wide, multiply occupied vestibules that serve as transition zones to the short, selective, singly occupied narrow region of the channel. A small amount of net negative charge within the pore could produce concentration-dependent potentials that provide a simple explanation for the more complicated aspects of the permeation properties.     

A second c-myb protein is translated from an alternatively spliced mRNA expressed from normal and 5''-disrupted myb loci.

The major protein encoded by the c-myb oncogene in many species has been identified as an unstable, nuclear DNA-binding protein with an apparent molecular mass of 75 to 80 kilodaltons (p75c-myb). Recently, an alternatively spliced form of c-myb-encoded mRNA has been identified in murine cells containing either normal or rearranged c-myb genes. This mRNA includes a new exon, termed E6A, formed through use of cryptic splice sites located in the large intron between c-myb exons vE6 and vE7. E6A is predicted to contribute an internal 121-residue in-frame insertion into a region C terminal of the DNA-binding domain the c-myb-encoded protein. Here we report the identification of an 85-kilodalton (p85c-myb-E6A) protein as the translation product of the alternatively spliced E6A c-myb mRNA. This protein as well as p75c-myb were precipitated with anti-Myb antibodies raised against the conserved DNA-binding region of c-Myb. Proteolytic mapping studies showed that the two proteins are highly related but not identical. However, only the p85 protein reacted with an antiserum prepared against the E6A region expressed in bacteria, demonstrating that p85 but not p75 contains E6A sequences. In addition, the mobilities of both p85 and p75 were increased in myeloid tumor cell lines containing proviral integrations upstream of the 5' coding exons of v-myb, indicating that both proteins are truncated forms of c-Myb expressed from the same disrupted allele. p75c-myb and p85c-myb-E6A were indistinguishable with respect to nuclear localization and protein half-life. Furthermore, both forms of Myb were synthesized continuously throughout the cell cycle in 70Z ore-B cells. The contribution of the E6A domain to c-myb function remains to be elucidated.     

Phylogenetic diversification of immunoglobulin genes and the antibody repertoire

Immunoglobulins are encoded by a large multigene system that undergoes somatic rearrangement and additional genetic change during the development of immunoglobulin-producing cells. Inducible antibody and antibody-like responses are found in all vertebrates. However, immunoglobulin possessing disulfide-bonded heavy and light chains and domain-type organization has been described only in representatives of the jawed vertebrates. High degrees of nucleotide and predicted amino acid sequence identity are evident when the segmental elements that constitute the immunoglobulin gene loci in phylogenetically divergent vertebrates are compared. However, the organization of gene loci and the manner in which the independent elements recombine (and diversify) vary markedly among different taxa. One striking pattern of gene organization is the "cluster type" that appears to be restricted to the chondrichthyes (cartilaginous fishes) and limits segmental rearrangement to closely linked elements. This type of gene organization is associated with both heavy- and light-chain gene loci. In some cases, the clusters are "joined" or "partially joined" in the germ line, in effect predetermining or partially predetermining, respectively, the encoded specificities (the assumption being that these are expressed) of the individual loci. By relating the sequences of transcribed gene products to their respective germ-line genes, it is evident that, in some cases, joined-type genes are expressed. This raises a question about the existence and/or nature of allelic exclusion in these species. The extensive variation in gene organization found throughout the vertebrate species may relate directly to the role of intersegmental (V<==>D<==>J) distances in the commitment of the individual antibody-producing cell to a particular genetic specificity. Thus, the evolution of this locus, perhaps more so than that of others, may reflect the interrelationships between genetic organization and function.      

Action Spectra for Conversions of Phycochrome c from Nostoc muscorum

The reversibly photochromic pigment, phycochrome c, was extracted from the blue-green alga Nostoc muscorum strain A. Action spectra were determined for in vitro conversions of the pigment from the short wavelength to the long wavelength form and vice versa. The action peak for the absorbance decrease at 650 nm is at 630 nm. During this decrease there is only a slight increase of the absorbance in the green region. Green and yellow light (maximum efficiency at 580 nm) completely restores absorbance at 650 nm. The observations are explained by the existence of three spectrally different forms of phycochrome c: P^c₆₃₀ and P^c₆₅₀ which equilibrate in darkness and P^c₅₈₀ which is reversibly photoconvertible to P^c₆₃₀. We have also measured the absorbance changes brought about by saturating irradiations with light of various wavelengths (“photostationary state spectrum”). Extreme photostationary states were obtained with about 650 nm and 500 nm light.     

Effects of Frost Hardening and Dehardening on Photosynthetic Electron Transport and Fluorescence Properties in Isolated Chloroplasts of Pinus silvestris

Photosynthetic electron transport and low-temperature fluorescence emission properties have been analyzed in isolated chloroplasts during the course of frost hardening and dehardening of Pinus silvestris L. Both the partial electron-transport reactions (H₂O DPIP and Asc./DPIP NADP) and the overall electron transport (H₂O — NAPD) showed decreasing capacities during the course of hardening. Upon exposing the plants to ?5°C and high irradiance a block in the electron-transport chain between the two photosystems developed, whereas the partial reactions still showed activities. The decrease in activity of PSl was accompanied by a decrease in P700 content, as determined by light oxidation of P700, which indicates a correlation between the two changes. Hardening also induced changes in the in vivo chlorophyll organization. During the course of hardening the fluorescence emission bands F692 and F726 decreased relative to F680. These changes were more pronounced if the plants were treated in high than in low irradiance. This suggests a greater destruction of the chlorophyll antennae in close association with the two photoreactions than in the so-called light-harvesting chlorophyll a/b antenna. During dehardening basically the reverse of the changes observed during hardening occurred. The recovery of secondary needles was complete, whereas primary needles only partly recovered.     

Phosphorylation of casein kinase II by p34cdc2 in vitro and at mitosis.

In human epidermal carcinoma A431 cells, the beta subunit of casein kinase II is phosphorylated at an autophosphorylation site and at serine 209 which can be phosphorylated in vitro by p34cdc2 (Litchfield, D. W., Lozeman, F. J., Cicirelli, M. F., Harrylock, M., Ericsson, L. H., Piening, C. J., and Krebs, E. G. (1991) J. Biol. Chem. 266, 20380-20389). Given the importance of p34cdc2 in the regulation of cell cycle events, we were interested in examining the phosphorylation of casein kinase II during different stages of the cell cycle. In this study it is demonstrated that the extent of phosphorylation of serine 209 in the beta subunit is significantly increased relative to phosphorylation of the autophosphorylation site when chicken bursal lymphoma BK3A cells are arrested at mitosis by nocodazole treatment. This result suggests that serine 209 is a likely physiological target for p34cdc2. In addition, the alpha subunit of casein kinase II also undergoes dramatic phosphorylation with an associated alteration in its electrophoretic mobility when BK3A cells or human Jurkat cells are arrested with nocodazole. Phosphopeptide mapping studies indicate that p34cdc2 can phosphorylate in vitro the same peptides on the alpha subunit that are phosphorylated in cells arrested at mitosis. These phosphorylation sites were localized to serine and threonine residues in the carboxyl-terminal domain of alpha. Taken together, the results of this study indicate that casein kinase II is a probable physiological substrate for p34cdc2 and suggest that its functional properties could be affected in a cell cycle-dependent manner.     

Control of cell volume in the J774 macrophage by microtubule disassembly and cyclic AMP

We have explored the possibilities that cell volume is regulated by the status of microtubule assembly and cyclic AMP metabolism and may be coordinated with shape change. Treatment of J774.2 mouse macrophages with colchicine caused rapid microtubule disassembly and was associated with a striking increase (from 15-20 to more than 90 percent) in the proportion of cells with a large protuberance at one pole. This provided a simple experimental system in which shape changes occurred in virtually an entire cell population in suspension. Parallel changes in cell volume could then be quantified by isotope dilution techniques. We found that the shape change caused by colchicine was accompanied by a decrease in cell volume of approximately 20 percent. Nocodozole, but not lumicolchicine, caused identical changes in both cell shape and cell volume. The volume loss was not due to cell lysis nor to inhibition of pinocytosis. The mechanism of volume loss was also examined. Colchicine induced a small but reproducible increase in activity of the ouabain-sensitive Na(+), K(+)-dependent ATPase. However, inhibition of this enzyme/transport system by ouabain did not change cell volume nor did it block the colchicines-induced decrease in volume. One the other hand, SITS (4’acetamido, 4-isothiocyano 2,2’ disulfonic acid stilbene), an inhibitor of anion transport, inhibited the effects of colchicines, thus suggesting a role for an anion transport system in cell volume regulation. Because colchicine is known to activate adenylate cyclase in several systems and because cell shape changes are often induced by hormones that elevate cyclic AMP, we also examined the effects of cyclic AMP on cell volume. Agents that act to increase syclic AMP (cholera toxin, which activates adenylate cyclase; IBMX, and inhibitor of phosphodiesterase; and dibutyryl cyclic AMP) all caused a volume decrease comparable to that of colchicine. To define the effective metabolic pathway, we studied two mutants of J774.2, one deficient in adenylate cyclase and the other exhibiting markedly reduced activity of cyclic AMP-dependent protein kinase. Cholera toxin did not produce a volume change in either mutant. Cyclic AMP produced a decrease in the cyclase-deficient line comparable to that in wild type, but did not cause a volume change in the kinase- deficient line. This analysis established separate roles for cyclic AMP and colchicine. The volume decrease induced by cyclic AMP requires the action of a cyclic AMP-dependent protein kinase. Colchicine, on the other hand, induced a comparable volume change in both mutants and wild type, and thus does not require the kinase.