Guiscard Seebohm

GLUT1 mutations are a cause of paroxysmal exertion-induced dyskinesias and induce hemolytic anemia by a cation leak

Paroxysmal dyskinesias are episodic movement disorders that can be inherited or are sporadic in nature. The pathophysiology underlying these disorders remains largely unknown but may involve disrupted ion homeostasis due to defects in cell-surface channels or nutrient transporters. In this study, we describe a family with paroxysmal exertion-induced dyskinesia (PED) over 3 generations. Their PED was accompanied by epilepsy, mild developmental delay, reduced CSF glucose levels, hemolytic anemia with echinocytosis, and altered erythrocyte ion concentrations. Using a candidate gene approach, we identified a causative deletion of 4 highly conserved amino acids (Q282_S285del) in the pore region of the glucose transporter 1 (GLUT1). Functional studies in Xenopus oocytes and human erythrocytes revealed that this mutation decreased glucose transport and caused a cation leak that alters intracellular concentrations of sodium, potassium, and calcium. We screened 4 additional families, in which PED is combined with epilepsy, developmental delay, or migraine, but not with hemolysis or echinocytosis, and identified 2 additional GLUT1 mutations (A275T, G314S) that decreased glucose transport but did not affect cation permeability. Combining these data with brain imaging studies, we propose that the dyskinesias result from an exertion-induced energy deficit that may cause episodic dysfunction of the basal ganglia, and that the hemolysis with echinocytosis may result from alterations in intracellular electrolytes caused by a cation leak through mutant GLUT1.

Position of aromatic residues in the S6 domain, not inactivation, dictates cisapride sensitivity of HERG and eag potassium channels

Unintended block of HERG K+ channels is a side effect of many common medications and is the most common cause of acquired long QT syndrome associated with increased risk of life-threatening arrhythmias. The molecular mechanism of high-affinity HERG block by structurally diverse compounds has been attributed to π-stacking and cation-π interactions of a drug (e.g., cisapride) with specific aromatic amino acid residues (Tyr-652 and Phe-656) in the S6 α-helical domain that face the central cavity of the channel. It also has been proposed that strong C-type inactivation of HERG facilitates or is the primary determinant of high-affinity drug binding. The structurally related, but noninactivating eag channel is insensitive to HERG blockers unless inactivation is induced by specific amino acid mutations [Ficker, E., Jarolimek, W. & Brown, A. M. (2001) Mol. Pharmacol. 60, 1343–1348]. Here we examine the relative importance of inactivation vs. positioning of S6 aromatic residues in determining sensitivity of HERG and eag channels to block by cisapride. The repositioning of Tyr-652 or Phe-656 along the S6 α-helical domain of HERG reduced sensitivity of channels to block by cisapride. Moreover, independent of inactivation, repositioning of the equivalent aromatic residues in Drosophila eag channels induced sensitivity to block by cisapride. These findings suggest that positioning of S6 aromatic residues relative to the central cavity of the channel, not inactivation per se determines drug block of HERG or eag channels.

Comparison of cloned Kir2 channels with native inward rectifier K+ channels from guinea-pig cardiomyocytes.

1 The aim of the study was to compare the properties of cloned Kir2 channels with the properties of native rectifier channels in guinea‐pig (gp) cardiac muscle. The cDNAs of gpKir2.1, gpKir2.2, gpKir2.3 and gpKir2.4 were obtained by screening a cDNA library from guinea‐pig cardiac ventricle. 2 A partial genomic structure of all gpKir2 genes was deduced by comparison of the cDNAs with the nucleotide sequences derived from a guinea‐pig genomic library. 3 The cell‐specific expression of Kir2 channel subunits was studied in isolated cardiomyocytes using a multi‐cell RT‐PCR approach. It was found that gpKir2.1, gpKir2.2 and gpKir2.3, but not gpKir2.4, are expressed in cardiomyocytes. 4 Immunocytochemical analysis with polyclonal antibodies showed that expression of Kir2.4 is restricted to neuronal cells in the heart. 5 After transfection in human embryonic kidney cells (HEK293) the mean single‐channel conductance with symmetrical K+ was found to be 30.6 pS for gpKir2.1, 40.0 pS for gpKir2.2 and 14.2 pS for Kir2.3. 6 Cell‐attached measurements in isolated guinea‐pig cardiomyocytes (n= 351) revealed three populations of inwardly rectifying K+ channels with mean conductances of 34.0, 23.8 and 10.7 pS. 7 Expression of the gpKir2 subunits in Xenopus oocytes showed inwardly rectifying currents. The Ba2+ concentrations required for half‐maximum block at ‐100 mV were 3.24 μm for gpKir2.1, 0.51 μm for gpKir2.2, 10.26 μm for gpKir2.3 and 235 μm for gpKir2.4. 8 Ba2+ block of inward rectifier channels of cardiomyocytes was studied in cell‐attached recordings. The concentration and voltage dependence of Ba2+ block of the large‐conductance inward rectifier channels was virtually identical to that of gpKir2.2 expressed in Xenopus oocytes. 9 Our results suggest that the large‐conductance inward rectifier channels found in guinea‐pig cardiomyocytes (34.0 pS) correspond to gpKir2.2. The intermediate‐conductance (23.8 pS) and low‐conductance (10.7 pS) channels described here may correspond to gpKir2.1 and gpKir2.3, respectively.

Regulation of Endocytic Recycling of KCNQ1/KCNE1 Potassium Channels

Stress-dependent regulation of cardiac action potential duration is mediated by the sympathetic nervous system and the hypothalamic-pituitary-adrenal axis. It is accompanied by an increased magnitude of the slow outward potassium ion current, IKs. KCNQ1 and KCNE1 subunits coassemble to form the IKs channel. Mutations in either subunit cause long QT syndrome, an inherited cardiac arrhythmia associated with an increased risk of sudden cardiac death. Here we demonstrate that exocytosis of KCNQ1 proteins to the plasma membrane requires the small GTPase RAB11, whereas endocytosis is dependent on RAB5. We further demonstrate that RAB-dependent KCNQ1/KCNE1 exocytosis is enhanced by the serum- and glucocorticoid-inducible kinase 1, and requires phosphorylation and activation of phosphoinositide 3-phosphate 5-kinase and the generation of PI(3,5)P2. Identification of KCNQ1/KCNE1 recycling and its modulation by serum- and glucocorticoid-inducible kinase 1-phosphoinositide 3-phosphate 5-kinase -PI(3,5)P2 provides a mechanistic insight into stress-induced acceleration of cardiac repolarization.

Refinement of the binding site and mode of action of the anticonvulsant retigabine on KCNQ K + channels

The discovery of retigabine has provided access to alternative anticonvulsant compounds with a novel mode of action. Acting as potassium channel opener, retigabine exclusively activates neuronal KCNQ-type K+ channels, mainly by shifting the voltage-dependence of channel activation to hyperpolarizing potentials. So far, only parts of the retigabine-binding site have been described, including Trp-265 and Gly-340 (according to KCNQ3 numbering) within transmembrane segments S5 and S6, respectively. Using a refined chimeric strategy, we additionally identified a Leu-314 within the pore region of KCNQ3 as crucial for the retigabine effect. Both Trp-265 and Leu-314 are likely to interact with the retigabine molecule, representing the upper and lower margins of the putative binding site. Guided by a structural model of KCNQ3, which was constructed based on the Kv1.2 crystal structure, further residues affecting retigabine-binding could be proposed and were experimentally verified as mediators for the action of the compound. These results strongly suggest that, besides Trp-265 and Leu-314, it is highly likely that another S5 residue, Leu-272, which is conserved in all KCNQ subunits, contributes to the binding site in KCNQ3. More importantly, Leu-338, extending from S6 of the neighboring subunit is also apparently involved in lining the hydrophobic binding pocket for the drug. This pocket, which is formed at the interface of two adjacent subunits, may be present only in the open state of the channel, consistent with the idea that retigabine stabilizes an open-channel conformation.

Significance of SGK1 in the regulation of neuronal function

The present brief review highlights the putative role of the serum‐ and glucocorticoid‐inducible‐kinase‐1 (SGK1) in the regulation of neuronal function. SGK1 is genomically upregulated by cell shrinkage and by a variety of hormones including mineralocorticoids and glucocorticoids. The kinase is activated by insulin and growth factors via phosphatidylinositide‐3‐kinase (PI3‐kinase), phosphoinositide‐dependent kinase PDK1 and mammalian target of rapamycin mTORC2. SGK1 upregulates ion channels (e.g. SCN5A, ENaC, ASIC1, TRPV5,6, ROMK, Kv1.1–5, KCNEx/KCNQ1–5, GluR6, VSOAC, ClC2, CFTR), carriers (e.g. NHE3, NKCC2, NCC, NaPiIIb, SMIT, GLUT1,4, SGLT1, NaDC, EAAT1–5, SN1, ASCT2, 4F2/LAT, PepT2), and the Na+/K+‐ATPase. SGK1 regulates enzymes (e.g. glycogen‐synthase‐kinase‐3, ubiquitin‐ligase Nedd4‐2, phosphomannose‐mutase‐2), and transcription factors (e.g. forkhead transcription factor Foxo3a, β‐catenin, nuclear factor‐kappa‐B (NFκB)). SGK1 participates in the regulation of transport, hormone release, neuroexcitability, inflammation, coagulation, cell proliferation and apoptosis. SGK1 contributes to regulation of renal Na+ retention, renal K+ elimination, salt appetite, gastric acid secretion, intestinal Na+/H+ exchange and nutrient transport, insulin‐dependent salt sensitivity of blood pressure, salt sensitivity of peripheral glucose uptake, cardiac repolarization and memory consolidation. Presumably, SGK1 contributes to the regulation of diverse cerebral functions (e.g. memory consolidation, fear retention) and the pathophysiology of several cerebral diseases (e.g. Parkinsons disease, schizophrenia, depression, Alzheimers disease). Despite multiple SGK1 functions, the phenotype of the SGK1 knockout mouse is mild and becomes only apparent under challenging conditions.

Pharmacological Activation of Normal and Arrhythmia-Associated Mutant KCNQ1 Potassium Channels

Abstract— KCNQ1 &agr;-subunits coassemble with KCNE1 &bgr;-subunits to form channels that conduct the slow delayed rectifier K+ current (IKs) important for repolarization of the cardiac action potential. Mutations in KCNQ1 reduce IKs and cause long-QT syndrome, a disorder of ventricular repolarization that predisposes affected individuals to arrhythmia and sudden death. Current therapy for long-QT syndrome is inadequate. R-L3 is a benzodiazepine that activates IKs and has the potential to provide gene-specific therapy. In the present study, we characterize the molecular determinants of R-L3 interaction with KCNQ1 channels, use computer modeling to propose a mechanism for drug-induced changes in channel gating, and determine its effect on several long-QT syndrome–associated mutant KCNQ1 channels heterologously expressed in Xenopus oocytes. Scanning mutagenesis combined with voltage-clamp analysis indicated that R-L3 interacts with specific residues located in the 5th and 6th transmembrane domains of KCNQ1 subunits. Most KCNQ1 mutant channels responded to R-L3 similarly to wild-type channels, but one mutant channel (G306R) was insensitive to R-L3 possibly because it disrupted a key component of the drug-binding site.

Modulation of potassium channel function confers a hyperproliferative invasive phenotype on embryonic stem cells

Ion transporters, and the resulting voltage gradients and electric fields, have been implicated in embryonic development and regeneration. These biophysical signals are key physiological aspects of the microenvironment that epigenetically regulate stem and tumor cell behavior. Here, we identify a previously unrecognized function for KCNQ1, a potassium channel known to be involved in human Romano–Ward and Jervell–Lange–Nielsen syndromes when mutated. Misexpression of its modulatory wild-type β-subunit XKCNE1 in the Xenopus embryo resulted in a striking alteration of the behavior of one type of embryonic stem cell: the pigment cell lineage of the neural crest. Depolarization of embryonic cells by misexpression of KCNE1 non-cell-autonomously induced melanocytes to overproliferate, spread out, and become highly invasive of blood vessels, liver, gut, and neural tube, leading to a deeply hyperpigmented phenotype. This effect is mediated by the up-regulation of Sox10 and Slug genes, thus linking alterations in ion channel function to the control of migration, shape, and mitosis rates during embryonic morphogenesis. Taken together, these data identify a role for the KCNQ1 channel in regulating key cell behaviors and reveal the molecular identity of a biophysical switch, by means of which neoplastic-like properties can be conferred upon a specific embryonic stem cell subpopulation.

Recessive cardiac phenotypes in induced pluripotent stem cell models of Jervell and Lange-Nielsen syndrome: disease mechanisms and pharmacological rescue.

Significance There are few laboratory models that recapitulate human cardiac disease. Here, we created human cell models for Jervell and Lange-Nielsen syndrome (JLNS) in vitro, based on human induced pluripotent stem cells (hiPSCs). JLNS is one of the most severe disorders of heart rhythm and can cause sudden death in young patients. JLNS is inherited recessively and is caused by homozygous mutations in the slow component of the delayed rectifier potassium current, IKs. Cardiomyocytes (CMs) from two independent sets of patient-derived and engineered hiPSCs showed electrophysiological defects that reflect the severity of the condition in patients. Our work allowed better understanding of the mechanisms of recessive inheritance. Furthermore, JLNS-CMs showed increased sensitivity to proarrhythmic drugs, which could be rescued pharmacologically, demonstrating the potential of hiPSC-CMs in drug testing. Jervell and Lange-Nielsen syndrome (JLNS) is one of the most severe life-threatening cardiac arrhythmias. Patients display delayed cardiac repolarization, associated high risk of sudden death due to ventricular tachycardia, and congenital bilateral deafness. In contrast to the autosomal dominant forms of long QT syndrome, JLNS is a recessive trait, resulting from homozygous (or compound heterozygous) mutations in KCNQ1 or KCNE1. These genes encode the α and β subunits, respectively, of the ion channel conducting the slow component of the delayed rectifier K+ current, IKs. We used complementary approaches, reprogramming patient cells and genetic engineering, to generate human induced pluripotent stem cell (hiPSC) models of JLNS, covering splice site (c.478-2A>T) and missense (c.1781G>A) mutations, the two major classes of JLNS-causing defects in KCNQ1. Electrophysiological comparison of hiPSC-derived cardiomyocytes (CMs) from homozygous JLNS, heterozygous, and wild-type lines recapitulated the typical and severe features of JLNS, including pronounced action and field potential prolongation and severe reduction or absence of IKs. We show that this phenotype had distinct underlying molecular mechanisms in the two sets of cell lines: the previously unidentified c.478-2A>T mutation was amorphic and gave rise to a strictly recessive phenotype in JLNS-CMs, whereas the missense c.1781G>A lesion caused a gene dosage-dependent channel reduction at the cell membrane. Moreover, adrenergic stimulation caused action potential prolongation specifically in JLNS-CMs. Furthermore, sensitivity to proarrhythmic drugs was strongly enhanced in JLNS-CMs but could be pharmacologically corrected. Our data provide mechanistic insight into distinct classes of JLNS-causing mutations and demonstrate the potential of hiPSC-CMs in drug evaluation.

K V 7 channelopathies

KV7 voltage-gated potassium channels, encoded by the KCNQ gene family, have caught increasing interest of the scientific community for their important physiological roles, which are emphasized by the fact that four of the five so far identified members are related to different hereditary diseases. Furthermore, these channels prove to be attractive pharmacological targets for treating diseases characterized by membrane hyperexcitability. KV7 channels are expressed in brain, heart, thyroid gland, pancreas, inner ear, muscle, stomach, and intestines. They give rise to functionally important potassium currents, reduction of which results in pathologies such as long QT syndrome, diabetes, neonatal epilepsy, neuromyotonia, or progressive deafness. Here, we summarize some key traits of KV7 channels and review how their molecular deficiencies could explain diverse disease phenotypes. We also assess the therapeutic potential of KV7 channels; in particular, how the activation of KV7 channels by the compounds retigabine and R-L3 may be useful for treatment of epilepsy or cardiac arrhythmia.