Browsing by Subject "Vesicles"

Now showing 1 - 3 of 3
  • Thumbnail Image
    Publication
    Restricted
    Influence of C-terminal protein domains and protein-lipid interactions on tetramerization and stability of the potassium channel KcsA
    (American Chemical Society, 2004-11-05) Molina Gallego, María Luisa; Encinar Hidalgo, José Antonio; Barrera Olivares, Francisco Nicolás; Fernández Ballester, Gregorio; Riquelme Pino, Gloria; González Ros, José Manuel; Bioquímica y Biología Molecular B e Inmunología
    KcsA is a prokaryotic potassium channel formed by the assembly of four identical subunits around a central aqueous pore. Although the high-resolution X-ray structure of the transmembrane portion of KcsA is known [Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science280, 69−77], the identification of the molecular determinant(s) involved in promoting subunit tetramerization remains to be determined. Here, C-terminal deletion channel mutants, KcsA Δ125−160 and Δ120−160, as well as 1−125 KcsA obtained from chymotrypsin cleavage of full-length 1−160 KcsA, have been used to evaluate the role of the C-terminal segment on the stability and tetrameric assembly of the channel protein. We found that the lack of the cytoplasmic C-terminal domain of KcsA, and most critically the 120−124 sequence stretch, impairs tetrameric assembly of channel subunits in a heterologous E. coli expression system. Molecular modeling of KcsA predicts that, indeed, such sequence stretch provides intersubunit interaction sites by hydrogen bonding to amino acid residues in N- and C-terminal segments of adjacent subunits. However, once the KcsA tetramer is assembled, its remarkable in vitro stability to detergent or to heat-induced dissociation into subunits is not greatly influenced by whether the entire C-terminal domain continues being part of the protein. Finally and most interestingly, it is observed that, even in the absence of the C-terminal domain involved in tetramerization, reconstitution into membrane lipids promotes in vitro KcsA tetramerization very efficiently, an event which is likely mediated by allowing proper hydrophobic interactions involving intramembrane protein domains.
  • Thumbnail Image
    Publication
    Open Access
    Physiopathologic dynamics of vesicle traffic in astrocytes
    (Murcia: F. Hernández, 2011) Potokar, Maja; Stenovec, Matjaž; Kreft, Marko; Gabrijel, Mateja; Zorec, Robert
    . The view of how astrocytes, a type of glial cells, contribute to the functioning of the central nervous system (CNS) has changed greatly in the last decade. Although glial cells outnumber neurons in the mammalian brain, it was considered for over a century that they played a subservient role to neurons. This view changed. Functions thought to be exclusively present in neurons, i.e. excitability mediated release of chemical messengers, has also been demonstrated in astrocytes. In this process, following an increase in cytosolic calcium activity, membrane bound vesicles, storing chemical messengers (gliotransmitters), fuse with the plasma membrane, a process known as exocytosis, permitting the exit of vesicle cargo into the extracellular space. Vesicles are delivered to and are removed from the site of exocytosis by an amazingly complex set of processes that we have only started to learn about recently. In this paper we review vesicle traffic, which is subject to physiological regulation and may be changed under pathological conditions.
  • Thumbnail Image
    Publication
    Restricted
    Probing the channel-bound shaker B inactivating peptide by stereoisomeric substitution at a strategic tyrosine residue
    (American Chemical Society, 2003-07-01) Encinar Hidalgo, José Antonio; Fernández Carvajal, Asia María; Poveda Larrosa, José Antonio; Molina Gallego, María Luisa; Albar, J.P.; Gavilanes Franco, Francisco; González Ros, José Manuel; Bioquímica y Biología Molecular B e Inmunología
    A synthetic peptide patterned after the sequence of the inactivating ball domain of the Shaker B K+ channel, the ShB peptide, fully restores fast inactivation in the deletion Shaker BΔ6−46 K+ channel, which lacks the constitutive ball domains. On the contrary, a similar peptide in which tyrosine 8 is substituted by the secondary structure-disrupting d-tyrosine stereoisomer does not. This suggests that the stereoisomeric substitution prevents the peptide from adopting a structured conformation when bound to the channel during inactivation. Moreover, characteristic in vitro features of the wild-type ShB peptide such as the marked propensity to adopt an intramolecular β-hairpin structure when challenged by anionic phospholipid vesicles, a model target mimicking features of the inactivation site in the channel protein, or to insert into their hydrophobic bilayers, are lost in the d-tyrosine-containing peptide, whose behavior is practically identical to that of noninactivating peptide mutants. In the absence of high resolution crystallographic data on the inactivated channel/peptide complex, these latter findings suggest that the structured conformation required for the peptide to promote channel inactivation, as referred to above, is likely to be β-hairpin.