Activators of cation channels: potential in treatment of channelopathies

ABSTRACT Cation channels are membrane proteins that provide controlled pathways for ion passage through cellular membranes. They play important roles in physiological processes such as secretory transduction, control of ion homeostasis, cell volume, vesicle cycling, and electrical control of excitable tissues. In a variety of channelopathies, ion channel function is reduced, and activators of cation channels are promising candidates to regain channel function in acquired or inherited channelopathies. Shortage in cation channel activators prevents testing of efficiency of activators in a variety of indications. This shortage might result from the relative incapability of modern drug screening methods, but increasing knowledge about cation channel activator binding and action might enable us in the future to use in silico-guided drug design of channel modulators. New compounds such as the HERG channel activator (3R,4R)-4-[3-(6-methoxy-quinolin-4-yl)-3-oxo-propyl]-1-[3-(2,3,5-trifluoro-phenyl)-prop-2-ynyl]-piperidine-3-carboxylic acid (RPR260243) will enable us to increase our understanding in cation channel modulation and to test the concept of channel activation as a clinically relevant principle in treatment of channelopathies. Ion channels are integral membrane proteins that provide controlled pathways for ion passage through cellular membranes. Cation selective channels play important roles in physiological processes such as secretory transduction, control of ion homeostasis, cell volume, vesicle cycling, and electrical control of excitable tissues. The importance of cation channels is also amplified by the fact that many therapeutic drugs mediate their effects by targeting these proteins. Potassium-selective channels are the most genetically diverse of all cation channels. Starting with the first cloned potassium selective ion channel from Drosophila melanogaster, Shaker, more than 100 potassium channels have been identified. The number of functionally distinct channels in native tissues is further increased by heteromultimeric assembly of potassium channel ␣-subunits with other ␣-and ␤-subunits and other modifications such as alternative splicing of mRNAs, glycosylation, and phosphorylation. In light of the broad range of physiological roles of cation channels, it is not surprising that channel impairment results in a variety of pathophysiological conditions. Channels might lose or gain function as a result of mutations in the promotor or codin

Untersuchung der spannungsabhängigen Kaliumkanäle KCNQ1 und IKs unter dem Einfluss des Chromanols 293B und die Bedeutung des IKs bei Arrhythmien

In der vorliegenden Arbeit wurden die pharmakologischen und elektrophysiologischen Eigenschaften des Kaliumkanals KCNQ1 und des KCNQ1/MinK (IKs) untersucht. Hierzu wurden die Kanäle in Xenopus-laevis-Oozyten und der KCNQ1/MinK (IKs) in CHO-Zellen exprimiert und in Voltage-Clamp-Experimenten untersucht. Die Blockwirkung des Chromanol-293B-Razemates und die der beiden Enantiomere wurden am KCNQ1- und KCNQ1/MinK-Kanal untersucht. Eine Enantiomerenselektivität der Chromanol 293B-Enantiomere wurde nachgewiesen. Beide Enantiomere wirken abhängig vom Zustand der KCNQ1- und IKs-Kanäle. Das 3S,4R-293B blockierte nur geschlossene Kanäle, während das andere Enantiomer 3R,4S-293B auf geschlossene und auf geöffnete Kanäle wirkte. Als Grundlage dieser Aussagen wurden kinetische Analysen der Kanalkinetiken ohne Blocker und bei partieller Blockade der Kanäle durchgeführt. Die Inhibiton des IK

The Potential of Human Induced Pluripotent Stem Cells (hiPSCs) for the Study of Channelopathies: Advances and Future Directions

Human induced pluripotent stem cells (hiPSCs) have revolutionized research on ion channels and channelopathies. Channelopathies are a group of genetic disorders characterized by dysfunctional ion channels, which are responsible for the regulation of ion flow across cell membranes. These disorders can affect various organ systems, leading to a wide range of symptoms and clinical manifestations. Differentiating pluripotent stem cells into various cell types results in the possibility of creating tissue- and disease-specific cell models. These models offer the possibility to investigate the underlying mechanisms of channelopathies and develop potential therapies. Using hiPSC-derived cells has allowed crucial insights into diseases like epilepsy, long QT syndrome, and periodic paralysis. However, the full potential of hiPSCs in this field is still to be exploited. The research will most likely focus on developing more complex cell models to further investigate channel dysfunction and its pathological consequences. In addition, hiPSCs will be increasingly used in drug screening and developing personalized therapies for various diseases. This chapter outlines the past and present achievements of hiPSCs in the field of channelopathies as well as provides an outlook on future possibilities

Residues at the tip of the pore loop of NR3B-containing NMDA receptors determine Ca2+ permeability and Mg2+ block

<p>Abstract</p> <p>Background</p> <p>Members of the complex N-methyl-D-aspartate receptor (NMDAR) subfamily of ionotropic glutamate receptors (iGluRs) conventionally assemble from NR1 and NR2 subunits, the composition of which determines receptor properties. Hallmark features of conventional NMDARs include the requirement for a coagonist, voltage-dependent block by Mg²⁺, and high permeability for Ca²⁺. Both Mg²⁺sensitivity and Ca²⁺permeability are critically dependent on the amino acids at the N and N+1 positions of NR1 and NR2. The recently discovered NR3 subunits feature an unprecedented glycine-arginine combination at those critical sites within the pore. Diheteromers assembled from NR1 and NR3 are not blocked by Mg²⁺and are not permeable for Ca²⁺.</p> <p>Results</p> <p>Employing site-directed mutagenesis of receptor subunits, electrophysiological characterization of mutants in a heterologous expression system, and molecular modeling of the NMDAR pore region, we have investigated the contribution of the unusual NR3 N and N+1 site residues to the unique functional characteristics of receptors containing these subunits. Contrary to previous studies, we provide evidence that both the NR3 N and N+1 site amino acids are critically involved in mediating the unique pore properties. Ca²⁺permeability could be rescued by mutating the NR3 N site glycine to the NR1-like asparagine. Voltage-dependent Mg²⁺block could be established by providing an Mg²⁺coordination site at either the NR3 N or N+1 positions. Conversely, "conventional" receptors assembled from NR1 and NR2 could be made Mg²⁺insensitive and Ca²⁺impermeable by equipping either subunit with the NR3-like glycine at their N positions, with a stronger contribution of the NR1 subunit.</p> <p>Conclusions</p> <p>This study sheds light on the structure-function relationship of the least characterized member of the NMDAR subfamily. Contrary to previous reports, we provide evidence for a critical functional involvement of the NR3 N and N+1 site amino acids, and propose them to be the essential determinants for the unique pore properties mediated by this subunit.</p

Bioelectric-calcineurin signaling module regulates allometric growth and size of the zebrafish fin

AbstractThe establishment of relative size of organs and structures is paramount for attaining final form and function of an organism. Importantly, variation in the proportions of structures frequently underlies adaptive change in morphology in evolution and maybe a common mechanism underlying selection. However, the mechanism by which growth is integrated within tissues during development to achieve proper proportionality is poorly understood. We have shown that signaling by potassium channels mediates coordinated size regulation in zebrafish fins. Recently, calcineurin inhibitors were shown to elicit changes in zebrafish fin allometry as well. Here, we identify the potassium channelkcnk5bas a key player in integrating calcineurin’s growth effects, in part through regulation of the cytoplasmic C-terminus of the channel. We propose that the interaction between Kcnk5b and calcineurin acts as a signaling node to regulate allometric growth. Importantly, we find that this regulation is epistatic to inherent mechanisms instructing overall size as inhibition of calcineurin is able to bypass genetic instruction of size as seen insofand wild-type fins, however, it is not sufficient to re-specify positional memory of size of the fin. These findings integrate classic signaling mediators such as calcineurin with ion channel function in the regulation of size and proportion during growth.</jats:p

Can Unlikely Neanderthal Chloride Channel CLC-2 Gene Variants Provide Insights in Modern Human Infertility?

Background/Aims: Neanderthals, although well adapted to local environments, were rapidly replaced by anatomically modern humans (AMH) for unknown reasons. Genetic information on Neanderthals is limited restricting applicability of standard population genetics. Methods: Here, we apply a novel combination of restricted genetic analyses on preselected physiological key players (ion channels), electrophysiological analyses of gene variants of unclear significance expressed in Xenopus laevis oocytes using two electrode voltage clamp and transfer of results to AMH genetics. Using genetic screening in infertile men identified a loss of CLC-2 associated with sperm deficiency. Results: Increased genetic variation caused functionally impaired Neanderthals CLC-2 channels. Conclusion: Increased genetic variation could reflect an adaptation to different local salt supplies at the cost of reduced sperm density. Interestingly and consistent with this hypothesis, lack of CLC-2 protein in a patient associates with high blood K+ concentration and azoospermia

Bioelectric Signaling Regulates Size in Zebrafish Fins

The scaling relationship between the size of an appendage or organ and that of the body as a whole is tightly regulated during animal development. If a structure grows at a different rate than the rest of the body, this process is termed allometric growth. The zebrafish another longfin (alf) mutant shows allometric growth resulting in proportionally enlarged fins and barbels. We took advantage of this mutant to study the regulation of size in vertebrates. Here, we show that alf mutants carry gain-of-function mutations in kcnk5b, a gene encoding a two-pore domain potassium (K+) channel. Electrophysiological analysis in Xenopus oocytes reveals that these mutations cause an increase in K+ conductance of the channel and lead to hyperpolarization of the cell. Further, somatic transgenesis experiments indicate that kcnk5b acts locally within the mesenchyme of fins and barbels to specify appendage size. Finally, we show that the channel requires the ability to conduct K+ ions to increase the size of these structures. Our results provide evidence for a role of bioelectric signaling through K+ channels in the regulation of allometric scaling and coordination of growth in the zebrafish

Up-regulation of amino acid transporter SLC6A19 activity and surface protein abundance by PKB/Akt and PIKfyve

Background: The amino acid transporter B0AT1 (SLC6A19) accomplishes concentrative cellular uptake of neutral amino acids. SLC6A19 is stimulated by serum- & glucocorticoid-inducible kinase (SGK) isoforms. SGKs are related to PKB/Akt isoforms, which also stimulate several amino acid transporters. PKB/Akt modulates glucose transport in part by phosphorylating and thus activating phosphatidylinositol-3-phosphate-5-kinase (PIKfyve), which fosters carrier protein insertion into the cell membrane. The present study explored whether PKB/Akt and/or PIKfyve stimulate SLC6A19. Methods: SLC6A19 was expressed in Xenopus oocytes with or without wild-type PKB/Akt or inactive T308A/S473APKB/Akt without or with additional expression of wild-type PIKfyve or PKB/Akt-resistant S318APIKfyve. Electrogenic amino acid transport was determined by dual electrode voltage clamping. Results: In SLC6A19-expressing oocytes but not in water-injected oocytes, the addition of the neutral amino acid L-leucine (2 mM) to the bath generated a current (Ile), which was significantly increased following coexpression of PKB/Akt, but not by coexpression of T308A/S473APKB/Akt. The effect of PKB/Akt was augmented by additional coexpression of PIKfyve but not of S318APIKfyve. Coexpression of PKB/Akt enhanced the maximal transport rate without significantly modifying the affinity of the carrier. The decline of Ile following inhibition of carrier insertion by brefeldin A (5 µM) was similar in the absence and presence of PKB/Akt indicating that PKB/Akt stimulated carrier insertion into rather than inhibiting carrier retrieval from the cell membrane. Conclusion: PKB/Akt up-regulates SLC6A19 activity, which may foster amino acid uptake into PKB/Akt-expressing epithelial and tumor cells

Cardiogenic programming of human pluripotent stem cells by dose-controlled activation of EOMES

Master cell fate determinants are thought to induce specific cell lineages in gastrulation by orchestrating entire gene programs. The T-box transcription factor EOMES (eomesodermin) is crucially required for the development of the heart—yet it is equally important for endoderm specification suggesting that it may act in a context-dependent manner. Here, we define an unrecognized interplay between EOMES and the WNT signaling pathway in controlling cardiac induction by using loss and gain-of-function approaches in human embryonic stem cells. Dose-dependent EOMES induction alone can fully replace a cocktail of signaling molecules otherwise essential for the specification of cardiogenic mesoderm. Highly efficient cardiomyocyte programming by EOMES mechanistically involves autocrine activation of canonical WNT signaling via the WNT3 ligand, which necessitates a shutdown of this axis at a subsequent stage. Our findings provide insights into human germ layer induction and bear biotechnological potential for the robust production of cardiomyocytes from engineered stem cells