1,720,991 research outputs found
Structural and functional modularity of voltage-gated potassium channels
No full textSequence similarity among known potassium channels indicates the voltage-gated potassium channels consist of two modules: the N-terminal portion of the channel up to and including transmembrane segment S4, called in this paper the 'sensor' module, and the C-terminal portion from transmembrane segment S5 onwards, called the 'pore' module. We investigated the functional role of these modules by constructing chimeric channels which combine the 'sensor' from one native voltage-gated channel, mKv1.1, with the 'pore' from another, Shaker H4, and vice versa. Functional studies of the wild type and chimeric channels show that these modules can operate outside their native context. Each channel has a unique conductance-voltage relation. Channels incorporating the mKv1.1 sensor module have similar rates of activation while channels having the Shaker pore module show similar rates of deactivation. This observation suggests the mKv1.1 sensor module limits activation and the Shaker pore module determines deactivation. We propose a model that explains the observed equilibrium and kinetic properties of the chimeric constructs in terms of the characteristics of the native modules and a novel type of intrasubunit cooperativity. The properties ascribed to the modules are the same whether the modules function in their native context or have been assembled into a chimera
First Joint Italian-Spanish Summer School in Biophysics and Molecular Biology of Ion Channels, Bertinoro, Italia, 27-30 settembre 2007.
No full textNell’ambito della collaborazione italo-spagnola fra i gruppi di ricerca dell'Universita' di Bologna e della Miguel Hernandez di Elche (Spagna), nell’anno 2007 il Dott. Caprini e’ stato co-organizzatore della Scuola Estiva Italo-Spagnola ́ ́Biofisica e Biologia Molecolare dei canali ionici ́ ́ rivolta a studenti di dottorato e post-dottorato
Expression of a genomic clone encoding a brain potassium channel in mammalian cells using lipofection
No full textA genomic clone encoding a mouse brain K+ channel (MBK1) was isolated, characterized and expressed in COS cells using the lipofection technique. Transfected COS cells expressed voltage-dependent K+ currents that activated within 20 ms at 0 mV and showed less than 10% inactivation during 250 ms depolarizing pulses at 60 mV. Expressed K+ currents were reversibly blocked by 4-aminopyridine and tetraethylammonium, and were moderately sensitive to dendrotoxin, but insensitive to charybdotoxin. Thus MBK1, expressed transiently in a mammalian cell line, exhibits features characteristic of non-inactivating K+ channels with a conspicuous insensitivity to charybdotoxin. Lipofection is, therefore, a valuable strategy for expression of channel proteins in mammalian cells. © 1992 Springer-Verlag
Second Hispano-Italian workshop on the Molecular Biology and Biophysics and of Ion Channels (5th-8th November 2009, Xorret de Cati', Alicante, Spagna)
No full textThe 2nd Hispanic – Italian Workshop on the Biophysics and molecular biology of ion channels is a biannual initiative proposed in 2006 by a group of Italian and Spanish researchers that led to the organization of the I Workshop in September 2007 in Bertinoro, Italy. The success of this event, along with the attendees enthusiasm, has fostered the organization this second edition in Spain. The goal of this initiative is to increase the internationalization by incorporating other European countries in order to consolidate a forum to discuss the last advances and results in the field of the ion channels research and to thrust the internationalization of Spanish groups stimulating them to collaborate with European partners. This initiative also tries to promote the training of young researchers in this exciting field which will warranty the research excellence in this area
GAP43 stimulates inositol trisphosphate-mediated calcium release in response to hypotonicity
No full textThe identification of osmo/mechanosensory proteins in mammalian sensory neurons is still elusive. We have used an expression cloning approach to screen a human dorsal root ganglion CDNA library to look for proteins that respond to hypotonicity by raising the intracellular Ca2+ concentration ([Ca2+]i). We report the unexpected identification of GAP43 (also known as neuromodulin of B50), a membrane-anchored neuronal protein implicated in axonal growth and synaptic plasticity, as an osmosensory protein that augments [Ca2+]i in response to hypotonicity. Palmitoylation of GAP43 plays an important role in the protein osmosensitivity. Depletion of intracellular stores or inhibition of phospholipase C (PLC) activity abrogates hypotonicity-evoked, GAP43-mediated [Ca2+]i elevations. Notably, hypotonicity promoted the selective association of GAP43 with the PLC-δ1 isoform, and a concomitant increase in inositol-1,4,5-trisphosphate (IP3) formation. Collectively, these findings indicate that hypo-osmotic activation of GAP43 induces Ca2+ release from IP3-sensitive intracellular stores. The osmosensitivity of GAP43 furnishes a mechanistic framework that links axon elongation with phosphoinositide metabolism, spontaneous triggering of cytosolic Ca2+ transients and the regulation of actin dynamics and motility at the growth cone in response to temporal and local mechanical forces
Structural Compatibility between the Putative Voltage Sensor of Voltage-gated K+ Channels and the Prokaryotic KcsA Channel
No full textSequence similarity among and electrophysiological studies of known potassium channels, along with the three-dimensional structure of the Streptomyces lividans K+ channel (KcsA), support the tenet that voltage-gated K+ channels (Kv channels) consist of two distinct modules: the "voltage sensor" module comprising the N-terminal portion of the channel up to and including the S4 transmembrane segment and the "pore" module encompassing the C-terminal portion from the S5 transmembrane segment onward. To substantiate this modular design, we investigated whether the pore module of Kv channels may be replaced with the pore module of the prokaryotic KcsA channel. Biochemical and immunocytochemical studies showed that chimeric channels were expressed on the cell surface of Xenopus oocytes, demonstrating that they were properly synthesized, glycosylated, folded, assembled, and delivered to the plasma membrane. Unexpectedly, surface-expressed homomeric chimeras did not exhibit detectable voltage-dependent channel activity upon both hyperpolarization and depolarization regardless of the expression system used. Chimeras were, however, strongly dominant-negative when coexpressed with wild-type Kv channels, as evidenced by the complete suppression of wild-type channel activity. Notably, the dominant-negative phenotype correlated well with the formation of stable, glycosylated, nonfunctional, heteromeric channels. Collectively, these findings imply a structural compatibility between the prokaryotic pore module and the eukaryotic voltage sensor domain that leads to the biogenesis of non-responsive channels. Our results lend support to the notion that voltage-dependent channel gating depends on the precise coupling between both protein domains, probably through a localized interaction surface
Neuronal death and perinatal lethality in voltage-gated sodium channel α(II)-deficient mice
No full textNeural activity is crucial for cell survival and fine patterning of neuronal connectivity during neurodevelopment. To investigate the role in vivo of sodium channels (NaCh) in these processes, we generated knockout mice deficient in brain NaChα(II). NaChα(II)/(-/-) mice were morphologically and organogenically indistinguishable from their NaChα(+/-) littermates. Notwithstanding, NaChα(II)(-/-) mice died perinatally with severe hypoxia and massive neuronal apoptosis, notably in the brainstem. Sodium channel currents recorded from cultured neurons of NaChα(II)(-/-) mice were sharply attenuated. Death appears to arise from severe hypoxia consequent to the brainstem deficiency of NaChα(II). NaChα(II) expression is, therefore, redundant for embryonic development but essential for postnatal survival
Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration
Full text linkClC-7 is a chloride channel of late endosomes and lysosomes. In osteoclasts, it may cooperate with H(+)-ATPases in acidifying the resorption lacuna. In mice and man, loss of ClC-7 or the H(+)-ATPase a3 subunit causes osteopetrosis, a disease characterized by defective bone resorption. We show that ClC-7 knockout mice additionally display neurodegeneration and severe lysosomal storage disease despite unchanged lysosomal pH in cultured neurons. Rescuing their bone phenotype by transgenic expression of ClC-7 in osteoclasts moderately increased their lifespan and revealed a further progression of the central nervous system pathology. Histological analysis demonstrated an accumulation of electron-dense material in neurons, autofluorescent structures, microglial activation and astrogliosis. Like in human neuronal ceroid lipofuscinosis, there was a strong accumulation of subunit c of the mitochondrial ATP synthase and increased amounts of lysosomal enzymes. Such alterations were minor or absent in ClC-3 knockout mice, despite a massive neurodegeneration. Osteopetrotic oc/oc mice, lacking a functional H(+)-ATPase a3 subunit, showed no comparable retinal or neuronal degeneration. There are important medical implications as defects in the H(+)-ATPase and ClC-7 can underlie human osteopetrosis
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