Alvaro Villarroel
Spanish National Research Council
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Featured researches published by Alvaro Villarroel.
Journal of Biological Chemistry | 2002
Eva Yus-Nájera; Irene Santana-Castro; Alvaro Villarroel
We show here that in a yeast two-hybrid assay calmodulin (CaM) interacts with the intracellular C-terminal region of several members of the KCNQ family of potassium channels. CaM co-immunoprecipitates with KCNQ2, KCNQ3, or KCNQ5 subunits better in the absence than in the presence of Ca2+. Moreover, in two-hybrid assays where it is possible to detect interactions with apo-CaM but not with Ca2+-bound calmodulin, we localized the CaM-binding site to a region that is predicted to contain two α-helices (A and B). These two helices encompass ∼85 amino acids, and in KCNQ2 they are separated by a dispensable stretch of ∼130 amino acids. Within this CaM-binding domain, we found an IQ-like CaM-binding motif in helix A and two overlapping consensus 1–5-10 CaM-binding motifs in helix B. Point mutations in helix A or B were capable of abolishing CaM binding in the two-hybrid assay. Moreover, glutathione S-transferase fusion proteins containing helices A and B were capable of binding to CaM, indicating that the interaction with KCNQ channels is direct. Full-length CaM (both N and C lobes) and a functional EF-1 hand were required for these interactions to occur. These observations suggest that apo-CaM is bound to neuronal KCNQ channels at low resting Ca2+ levels and that this interaction is disturbed when the [Ca2+] is raised. Thus, we propose that CaM acts as a mediator in the Ca2+-dependent modulation of KCNQ channels.
British Journal of Pharmacology | 1999
Gabriel Olmos; Nuria DeGregorio-Rocasolano; M.Paz Regalado; Teresa Gasull; M Assumpció Boronat; Ramón Trullas; Alvaro Villarroel; Juan Lerma; Jesús A. García-Sevilla
This study was designed to assess the potential neuroprotective effect of several imidazol(ine) drugs and agmatine on glutamate‐induced necrosis and on apoptosis induced by low extracellular K+ in cultured cerebellar granule cells. Exposure (30u2003min) of energy deprived cells to L‐glutamate (1–100u2003μM) caused a concentration‐dependent neurotoxicity, as determined 24u2003h later by a decrease in the ability of the cells to metabolize 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazoliumbromide (MTT) into a reduced formazan product. L‐glutamate‐induced neurotoxicity (EC50=5u2003μM) was blocked by the specific NMDA receptor antagonist MK‐801 (dizocilpine). Imidazol(ine) drugs and agmatine fully prevented neurotoxicity induced by 20u2003μM (EC100) L‐glutamate with the rank order (EC50 in μM): antazoline (13)>cirazoline (44)>LSLu200361122 [2‐styryl‐2‐imidazoline] (54)>LSLu200360101 [2‐(2‐benzofuranyl) imidazole] (75)>idazoxan (90)>LSLu200360129 [2‐(1,4‐benzodioxan‐6‐yl)‐4,5‐dihydroimidazole] (101)>RX821002 (2‐methoxy idazoxan) (106)>agmatine (196). No neuroprotective effect of these drugs was observed in a model of apoptotic neuronal cell death (reduction of extracellular K+) which does not involve stimulation of NMDA receptors. Imidazol(ine) drugs and agmatine fully inhibited [3H]‐(+)‐MK‐801 binding to the phencyclidine site of NMDA receptors in rat brain. The profile of drug potency protecting against L‐glutamate neurotoxicity correlated well (r=0.90) with the potency of the same compounds competing against [3H]‐(+)‐MK‐801 binding. In HEK‐293 cells transfected to express the NR1‐1a and NR2C subunits of the NMDA receptor, antazoline and agmatine produced a voltage‐ and concentration‐dependent block of glutamate‐induced currents. Analysis of the voltage dependence of the block was consistent with the presence of a binding site for antazoline located within the NMDA channel pore with an IC50 of 10–12u2003μM at 0u2003mV. It is concluded that imidazol(ine) drugs and agmatine are neuroprotective against glutamate‐induced necrotic neuronal cell death in vitro and that this effect is mediated through NMDA receptor blockade by interacting with a site located within the NMDA channel pore.
Neuron | 2000
Amir Fayyazuddin; Alvaro Villarroel; Anne Le Goff; Juan Lerma; Jacques Neyton
NMDA receptors are allosterically inhibited by Zn2+ ions in a voltage-independent manner. The apparent affinity for Zn2+ of the heteromeric NMDA receptors is determined by the subtype of NR2 subunit expressed, with NR2A-containing receptors being the most sensitive (IC50, approximately 20 nM) and NR2C-containing receptors being the least sensitive (IC50, approximately 30 microM). Using chimeras constructed from these two NR2 subtypes, we show that the N-terminal LIVBP-like domain of the NR2A subunit controls the high-affinity Zn2+ inhibition. Mutations at four residues in this domain markedly reduce Zn2+ affinity (by up to >500-fold) without affecting either receptor activation by glutamate and glycine or inhibition by extracellular protons and Ni2+ ions, indicating that these residues most likely participate in high-affinity Zn2+ binding.
Neuron | 1998
Alvaro Villarroel; M.Paz Regalado; Juan Lerma
In studying chimeras of NR2A and NR2C subunits of the NMDA receptor, we have found that glycine-independent desensitization depends on two regions of the extracellular N-terminal domain. One corresponds to a stretch of approximately 190 amino acids preceding the glutamate-binding domain S1. The other localizes at the interface between the N-terminal segment and the first transmembrane domain of NR2A subunits and involves A555 and S556. Both regions support desensitization in the absence of the other with different time courses. Desensitization did not develop with time in receptors containing the entire N-terminal region of NR2C. The introduction of A555 into the corresponding position of NR2C subunits enabled the receptors to manifest time-dependent increase in desensitization. Thus, this determinant behaves as an allosteric effector for glycine-independent desensitization.
The FASEB Journal | 2008
Ainhoa Etxeberría; Paloma Aivar; Jose Angel Rodriguez-Alfaro; Alessandro Alaimo; Patricia Villacé; Juan Camilo Gómez-Posada; Pilar Areso; Alvaro Villarroel
Voltage‐dependent potassium KCNQ2 (Kv7.2) channels play a prominent role in the control of neuronal excitability. These channels must associate with calmodulin to function correctly and, indeed, a mutation (R353G) that impairs this association provokes the onset of a form of human neonatal epilepsy known as benign familial neonatal convulsions (BFNC). We show here that perturbation of calmodulin binding leads to endoplasmic reticulum (ER) retention of KCNQ2, reducing the number of channels that reach the plasma membrane. Interestingly, elevating the expression of calmodulin in the BFNC mutant partially restores the intracellular distribution of the KCNQ channel. In contrast, overexpression of a Ca2+‐binding incompetent calmodulin or sequestering of calmodulin promotes the retention of wild‐type channels in the ER. Thus, a direct interaction with Ca2+‐calmodulin appears to be critical for the correct activity of KCNQ2 potassium channels as it controls the channels’ exit from the ER. Etxeberria A., Aivar, P., Rodriguez‐Alfaro, J. A., Alaimo, A., Villace, P., Gómez‐Posada J. C., Areso, P., Villarroel A. Calmodulin regulates the trafficking of KCNQ2 potassium channels. FASEB J. 22, 1135–1143 (2008)
Neuron | 2001
M.Paz Regalado; Alvaro Villarroel; Juan Lerma
Opening of the NMDA receptor channel requires simultaneous binding of glutamate and glycine. Although the binding sites for each agonist are in different subunits, the presence of one agonist influences the binding of the other. We have localized regions in the S1 binding domain of both subunits required for the transmission of allosteric signals from the glutamate binding NR2A subunit to the glycine binding NR1 subunit. Three-dimensional modeling indicates that these segments are not directly involved in ligand binding, but likely form solvent-accessible loops protruding out of the binding pocket, making them suitable to relay interactions between adjacent subunits. Thus, these segments mediate negative allosteric coupling between the two subunit types that form the NMDA receptor.
Journal of Molecular Biology | 2014
Alvaro Villarroel; Maurizio Taglialatela; Ganeko Bernardo-Seisdedos; Alessandro Alaimo; Jon Agirre; Araitz Alberdi; Carolina Gomis-Perez; Maria Virginia Soldovieri; Paolo Ambrosino; Covadonga Malo; Pilar Areso
The exceptional versatility of calmodulin (CaM) three-dimensional arrangement is reflected in the growing number of structural models of CaM/protein complexes currently available in the Protein Data Bank (PDB) database, revealing a great diversity of conformations, domain organization, and structural responses to Ca(2+). Understanding CaM binding is complicated by the diversity of target proteins sequences. Data mining of the structures shows that one face of each of the eight CaM helices can contribute to binding, with little overall difference between the Ca(2+) loaded N- and C-lobes and a clear prevalence of the C-lobe low Ca(2+) conditions. The structures reveal a remarkable variety of configurations where CaM binds its targets in a preferred orientation that can be reversed and where CaM rotates upon Ca(2+) binding, suggesting a highly dynamic metastable relation between CaM and its targets. Recent advances in structure-function studies and the discovery of CaM mutations being responsible for human diseases, besides expanding the role of CaM in human pathophysiology, are opening new exciting avenues for the understanding of the how CaM decodes Ca(2+)-dependent and Ca(2+)-independent signals.
Human Genetics | 2008
Ángeles Mencía; Daniel González-Nieto; Silvia Modamio-Høybjør; Ainhoa Etxeberría; Gracia Aránguez; Nieves Salvador; Ignacio del Castillo; Alvaro Villarroel; Felipe Moreno; Luis Barrio; Miguel A. Moreno-Pelayo
Mutations in the potassium channel gene KCNQ4 underlie DFNA2, a subtype of autosomal dominant progressive, high-frequency hearing loss. Based on a phenotype-guided mutational screening we have identified a novel mutation c.886G>A, leading to the p.G296S substitution in the pore region of KCNQ4 channel. The possible impact of this mutation on total KCNQ4 protein expression, relative surface expression and channel function was investigated. When the G296S mutant was expressed in Xenopus oocytes, electrophysiological recordings did not show voltage-activated K+ currents. The p.G296S mutation impaired KCNQ4 channel activity in two manners. It greatly reduced surface expression and, secondarily, abolished channel function. The deficient expression at the cell surface membrane was further confirmed in non-permeabilized NIH-3T3 cells transfected with the mutant KCNQ4 tagged with the hemagglutinin epitope in the extracellular S1–S2 linker. Co-expression of mutant and wild type KCNQ4 in oocytes was performed to mimic the heterozygous condition of the p.G296S mutation in the patients. The results showed that the G296S mutant exerts a strong dominant-negative effect on potassium currents by reducing the wild type KCNQ4 channel expression at the cell surface. This is the first study to identify a trafficking-dependent dominant mechanism for the loss of KCNQ4 channel function in DFNA2.
Organic and Biomolecular Chemistry | 2014
M. Angeles Bonache; Alessandro Alaimo; Covadonga Malo; Oscar Millet; Alvaro Villarroel; Rosario González-Muñiz
Biophysical Journal | 2015
Carolina Gomis-Perez; Maria Virginia Soldovieri; Araitz Alberdi; Paolo Ambrosino; Michela Di Maria; Alessandro Alaimo; Ganeko Bernardo-Seisdedos; Covadonga Malo; Pilar Areso; Maurizio Taglialatela; Alvaro Villarroel