Structural compatibility between the putative voltage sensor of voltage-gated K+ channels and the prokaryotic KcsA channel.

Sequence 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

VRAC: molecular identification as LRRC8 heteromers with differential functions

Abstract A major player of vertebrate cell volume regulation is the volume-regulated anion channel (VRAC), which conducts halide ions and organic osmolytes to counteract osmotic imbalances. The molecular entity of this channel was unknown until very recently, although its biophysical characteristics and diverse physiological roles have been extensively studied over the last 30 years. On the road to the molecular identification of VRAC, experimental difficulties led to the proposal of a variety of false candidates. In 2014, in a final breakthrough, two groups independently identified LRRC8A as indispensable component of VRAC. LRRC8A is part of the leucine-rich repeat containing 8 family, which is comprised of five members (LRRC8A-E). Of those, LRRC8A is an obligatory subunit of VRAC but it needs at least one of the other family members to mediate the swelling-induced Cl − current I Cl,vol . This review discusses the remarkable journey which led to the molecular identification of VRAC, evidence for LRRC8 proteins forming the VRAC pore and their heteromeric assembly. Furthermore, first major insights on the role of LRRC8 proteins in cancer drug resistance and apoptosis and the role of LRRC8D in cisplatin and taurine transport will be summarized

Role of the transient receptor potential vanilloid 1 in inflammation and sepsis

The transient receptor potential vanilloid 1 (TRPV1) is a thermoreceptor that responds to noxious temperatures, as well as to chemical agonists, such as vanilloids and protons. In addition, its channel activity is notably potentiated by proinflammatory mediators released upon tissue damage. The TRPV1 contribution to sensory neuron sensitization by proalgesic agents has signaled this receptor as a prime target for analgesic and anti-inflammatory drug intervention. However, TRPV1 antagonists have notably failed in clinical and preclinical studies because of their unwanted side effects. Recent reports have unveiled previously unrecognized anti-inflammatory and protective functions of TRPV1 in several diseases. For instance, this channel has been suggested to play an anti-inflammatory role in sepsis. Therefore, the use of potent TRPV1 antagonists as a general strategy to treat inflammation must be cautiously considered, given the deleterious effects that may arise from inhibiting the population of channels that have a protective function. The use of TRPV1 antagonists may be limited to treating those pathologies where enhanced receptor activity contributes to the inflamed state. Alternatively, therapeutic paradigms, such as reduction of inflammatory-mediated increase of receptor expression in the cell surface, may be a better strategy to prevent abrogation of the TRPV1 subpopulation involved in anti-inflammatory and protective processes

Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration

CLC chloride/proton exchangers may support acidification of endolysosomes and raise their luminal Cl− concentration. Disruption of endosomal ClC‐3 causes severe neurodegeneration. To assess the importance of ClC‐3 Cl−/H+ exchange, we now generate Clcn3unc/unc mice in which ClC‐3 is converted into a Cl− channel. Unlike Clcn3−/− mice, Clcn3unc/unc mice appear normal owing to compensation by ClC‐4 with which ClC‐3 forms heteromers. ClC‐4 protein levels are strongly reduced in Clcn3−/−, but not in Clcn3unc/unc mice because ClC‐3unc binds and stabilizes ClC‐4 like wild‐type ClC‐3. Although mice lacking ClC‐4 appear healthy, its absence in Clcn3unc/unc/Clcn4−/− mice entails even stronger neurodegeneration than observed in Clcn3−/− mice. A fraction of ClC‐3 is found on synaptic vesicles, but miniature postsynaptic currents and synaptic vesicle acidification are not affected in Clcn3unc/unc or Clcn3−/− mice before neurodegeneration sets in. Both, Cl−/H+‐exchange activity and the stabilizing effect on ClC‐4, are central to the biological function of ClC‐3

Differentiation of Human Embryonic Stem Cells to Regional Specific Neural Precursors in Chemically Defined Medium Conditions

Background: Human embryonic stem cells (hESC) provide a unique model to study early events in human development. The hESC-derived cells can potentially be used to replace or restore different tissues including neuronal that have been damaged by disease or injury. Methodology and Principal Findings: The cells of two different hESC lines were converted to neural rosettes using adherent and chemically defined conditions. The progenitor cells were exposed to retinoic acid (RA) or to human recombinant basic fibroblast growth factor (bFGF) in the late phase of the rosette formation. Exposing the progenitor cells to RA suppressed differentiation to rostral forebrain dopamine neural lineage and promoted that of spinal neural tissue including motor neurons. The functional characteristics of these differentiated neuronal precursors under both, rostral (bFGF) and caudalizing (RA) signals were confirmed by patch clamp analysis. Conclusions/Significance: These findings suggest that our differentiation protocol has the capacity to generate regionspecific and electrophysiologically active neurons under in vitro conditions without embryoid body formation, co-cultur

Physiology and pharmacology of the vanilloid receptor

15 pages, 7 figures.-- PMID: 18615132 [PubMed].-- PMCID: PMC2430674.The identification and cloning of the vanilloid receptor 1 (TRPV1) represented a significant step for the understanding of the molecular mechanisms underlying the transduction of noxious chemical and thermal stimuli by peripheral nociceptors. TRPV1 is a non-selective cation channel gated by noxious heat, vanilloids and extracellular protons. TRPV1 channel activity is remarkably potentiated by pro-inflammatory agents, a phenomenon that is thought to underlie the peripheral sensitisation of nociceptors that leads to thermal hyperalgesia. Cumulative evidence is building a strong case for the involvement of this receptor in the etiology of both peripheral and visceral inflammatory pain, such as inflammatory bowel disease, bladder inflammation and cancer pain. The validation of TRPV1 receptor as a key therapeutic target for pain management has thrust intensive drug discovery programs aimed at developing orally active antagonists of the receptor protein. Nonetheless, the real challenge of these drug discovery platforms is to develop antagonists that preserve the physiological activity of TRPV1 receptors while correcting over-active channels. This is a condition to ensure normal pro-prioceptive and nociceptive responses that represent a safety mechanism to prevent tissue injury. Recent and exciting advances in the function, dysfunction and modulation of this receptor will be the focus of this review.We thank all members of our group and colleagues of collaboration groups for their fundamental contribution to the results herein presented. We are indebted to the Financial support from the MEC, FIS, GVA, and Fundació La Caixa.Peer reviewe

Pharmacological Intervention at Ionotropic Glutamate Receptor Complexes

14 páginas.L-glutamate is considered the main excitatory neurotransmitter in the mammalian brain. Paradoxically, Lglutamate is also the most important excitotoxin pivotally involved in the aetiology of several neurodegenerative diseases such as stroke, Alzheimer, Parkinson, amyotropic lateral sclerosis, Huntington and neuropathic pain. L-glutamate signalling is transduced both presynaptically and postsynaptically by metabotropic and ionotropic receptors. Three types of glutamate-gated channels integrate the synaptic signal, namely AMPA, kainate and NMDA receptors. Sustained activation of these receptors, and especially of the NMDA receptor, is a casuistic phenomenon that leads to the neuronal death underlying neurodegeneration. Thus, pharmacological intervention at these neuronal receptors and their synaptic protein complexes is a valuable therapeutic strategy. The approval of memantine, a safe, well-tolerated uncompetitive NMDA antagonist for the treatment of moderate to severe Alzheimer dementia validates ionotropic glutamate receptors as key therapeutic targets of neurodegenerative diseases in humans. As a consequence, an enormous effort is being carried out to identify and develop safe and potent antagonists for the clinics. In this review, we will describe progress in this important arena of human health.Peer reviewe

Structural and functional modularity of voltage-gated potassium channels

AbstractSequence 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