Generación y evaluación funcional de dominantes negativos para canales K2P

54 p.Los iones potasio difunden rápidamente a través de la membrana mediante proteínas llamadas canales de potasio. Este movimiento subraya muchos procesos biológicos fundamentales, incluyendo señalización eléctrica en el sistema nervioso. Estos canales muestran una alta selectividad a iones potasio, y la permeabilidad a iones más pequeños es extremadamente baja. Todos los canales de potasio sin excepción, presentan una secuencia aminoacídica crítica en el dominio formador de poro, conocida como la secuencia firma TVGYG en la cual las glicinas (G) se encuentran muy conservadas. La secuencia firma de los canales de potasio juega un rol importante en la selectividad a este ión, ya que su ordenamiento estructural específico, orientando los oxígenos de los grupos carbonilos del backbone hacia el poro, hace que estos actúen como reemplazo de la capa de solvatación del ión, logrando la conducción iónica, que se produce por una simple razón: la repulsión entre los iones es más fuerte que la atracción del ión hacia el sitio de unión dentro del filtro. La mutación de la primera glicina del segundo dominio formador de poro de los canales K2P9 y K2P10 por un ácido glutámico, genera cambios estructurales dentro del filtro de selectividad, haciendo que en primer lugar, los oxígenos de la cadena lateral del ácido glutámico se orienten hacia el interior del poro e interaccionen con el ión, y en segundo lugar, cambios en la orientación de los oxígenos de los grupos carbonilos del backbone de los aminoácidos del filtro de selectividad, teniendo como resultado canales no funcionales, que muestran una densidad de corriente nula en comparación con las corrientes de los canales Wild Type.Palabras claves: K2P./ABSTRACT: Potassium ions diffuse rapidly across membrane through proteins known as potassium channels. This movement highlight a lot of fundamental biological processes, including electric signaling in nervous system. These channels show a high selectivity to potassium ions, and a lower permeability to smaller ions. All potassium channels, without exception, have a critical amino acidic sequence in their pore domain, known as the signature sequence TVGYG with highly conserved glycines (G).The signature sequence of potassium channels plays an important role in the ion selectivity. Carbonyl oxygens from backbone of the filter act as hidratation shield substitutes, so the conduction is possible by a simple reason: the repulsion force between the ions is higher than the affinity between ion and binding site in the filter. Mutation of the first glycine in the second pore domain by a glutamic acid in K2P9 and K2P10 channels, produce structural changes in the selectivity filter: first, the oxygens from the lateral chain of glutamic acid is oriented to the pore and can interact with the K+, and second, changes the orientation of carbonyl oxygens from amino acids backbone in the selectivity filter. These changes result in non functional channels, showing a low density of current compared with the wild type currents. Key words: K2P

HCN Channels: New Therapeutic Targets for Pain Treatment

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are highly regulated proteins which respond to different cellular stimuli. The HCN currents (Ih) mediated by HCN1 and HCN2 drive the repetitive firing in nociceptive neurons. The role of HCN channels in pain has been widely investigated as targets for the development of new therapeutic drugs, but the comprehensive design of HCN channel modulators has been restricted due to the lack of crystallographic data. The three-dimensional structure of the human HCN1 channel was recently reported, opening new possibilities for the rational design of highly-selective HCN modulators. In this review, we discuss the structural and functional properties of HCN channels, their pharmacological inhibitors, and the potential strategies for designing new drugs to block the HCN channel function associated with pain perception

Correction: Concha, G., et al. The Insensitivity of TASK-3 K2P Channels to External Tetraethylammonium (TEA) Partially Depends on the Cap Structure. Int. J. Mol. Sci. 2018, 19, 2437

We would like to submit the following correction to our published paper [...

TASK-3 Downregulation Triggers Cellular Senescence and Growth Inhibition in Breast Cancer Cell Lines

TASK-3 potassium channels are believed to promote proliferation and survival of cancer cells, in part, by augmenting their resistance to both hypoxia and serum deprivation. While overexpression of TASK-3 is frequently observed in cancers, the understanding of its role and regulation during tumorigenesis remains incomplete. Here, we evaluated the effect of reducing the expression of TASK-3 in MDA-MB-231 and MCF-10F human mammary epithelial cell lines through small hairpin RNA (shRNA)-mediated knockdown. Our results show that knocking down TASK-3 in fully transformed MDA-MB-231 cells reduces proliferation, which was accompanied by an induction of cellular senescence and cell cycle arrest, with an upregulation of cyclin-dependent kinase (CDK) inhibitors p21 and p27. In non-tumorigenic MCF-10F cells, however, TASK-3 downregulation did not lead to senescence induction, although cell proliferation was impaired and an upregulation of CDK inhibitors was also evident. Our observations implicate TASK-3 as a critical factor in cell cycle progression and corroborate its potential as a therapeutic target in breast cancer treatment

The Insensitivity of TASK-3 K2P Channels to External Tetraethylammonium (TEA) Partially Depends on the Cap Structure

Two-pore domain K+ channels (K2P) display a characteristic extracellular cap structure formed by two M1-P1 linkers, the functional role of which is poorly understood. It has been proposed that the presence of the cap explains the insensitivity of K2P channels to several K+ channel blockers including tetraethylammonium (TEA). We have explored this hypothesis using mutagenesis and functional analysis, followed by molecular simulations. Our results show that the deletion of the cap structure of TASK-3 (TWIK-related acid-sensitive K+ channel) generates a TEA-sensitive channel with an IC50 of 11.8 ± 0.4 mM. The enhanced sensitivity to TEA displayed by the cap-less channel is also explained by the presence of an extra tyrosine residue at position 99. These results were corroborated by molecular simulation analysis, which shows an increased stability in the binding of TEA to the cap-less channel when a ring of four tyrosine is present at the external entrance of the permeation pathway. Consistently, Y99A or Y205A single-residue mutants generated in a cap-less channel backbone resulted in TASK-3 channels with low affinity to external TEA

Elucidating the Structural Basis of the Intracellular pH Sensing Mechanism of TASK-2 K2P Channels

Two-pore domain potassium (K2P) channels maintain the cell’s background conductance by stabilizing the resting membrane potential. They assemble as dimers possessing four transmembrane helices in each subunit. K2P channels were crystallized in “up” and “down” states. The movements of the pore-lining transmembrane TM4 helix produce the aperture or closure of side fenestrations that connect the lipid membrane with the central cavity. When the TM4 helix is in the up-state, the fenestrations are closed, while they are open in the down-state. It is thought that the fenestration states are related to the activity of K2P channels and the opening of the channels preferentially occurs from the up-state. TASK-2, a member of the TALK subfamily of K2P channels, is opened by intracellular alkalization leading the deprotonation of the K245 residue at the end of the TM4 helix. This charge neutralization of K245 could be sensitive or coupled to the fenestration state. Here, we describe the relationship between the states of the intramembrane fenestrations and K245 residue in TASK-2 channel. By using molecular modeling and simulations, we show that the protonated state of K245 (K245+) favors the open fenestration state and, symmetrically, that the open fenestration state favors the protonated state of the lysine residue. We show that the channel can be completely blocked by Prozac, which is known to induce fenestration opening in TREK-2. K245 protonation and fenestration aperture have an additive effect on the conductance of the channel. The opening of the fenestrations with K245+ increases the entrance of lipids into the selectivity filter, blocking the channel. At the same time, the protonation of K245 introduces electrostatic potential energy barriers to ion entrance. We computed the free energy profiles of ion penetration into the channel in different fenestration and K245 protonation states, to show that the effects of the two transformations are summed up, leading to maximum channel blocking. Estimated rates of ion transport are in qualitative agreement with experimental results and support the hypothesis that the most important barrier for ion transport under K245+ and open fenestration conditions is the entrance of the ions into the channel

Discovery of Novel TASK-3 Channel Blockers Using a Pharmacophore-Based Virtual Screening

TASK-3 is a two-pore domain potassium (K2P) channel highly expressed in the hippocampus, cerebellum, and cortex. TASK-3 has been identified as an oncogenic potassium channel and it is overexpressed in different cancer types. For this reason, the development of new TASK-3 blockers could influence the pharmacological treatment of cancer and several neurological conditions. In the present work, we searched for novel TASK-3 blockers by using a virtual screening protocol that includes pharmacophore modeling, molecular docking, and free energy calculations. With this protocol, 19 potential TASK-3 blockers were identified. These molecules were tested in TASK-3 using patch clamp, and one blocker (DR16) was identified with an IC50 = 56.8 ± 3.9 μM. Using DR16 as a scaffold, we designed DR16.1, a novel TASK-3 inhibitor, with an IC50 = 14.2 ± 3.4 μM. Our finding takes on greater relevance considering that not many inhibitory TASK-3 modulators have been reported in the scientific literature until today. These two novel TASK-3 channel inhibitors (DR16 and DR16.1) are the first compounds found using a pharmacophore-based virtual screening and rational drug design protocol