Michael Pasternack

Retarded Growth and Deficits in the Enteric and Parasympathetic Nervous System in Mice Lacking GFRα2, a Functional Neurturin Receptor

Glial cell line-derived neurotrophic factor (GDNF) and a related protein, neurturin (NTN), require a GPI-linked coreceptor, either GFR alpha1 or GFR alpha2, for signaling via the transmembrane Ret tyrosine kinase. We show that mice lacking functional GFR alpha2 coreceptor (Gfra2-/-) are viable and fertile but have dry eyes and grow poorly after weaning, presumably due to malnutrition. While the sympathetic innervation appeared normal, the parasympathetic cholinergic innervation was almost absent in the lacrimal and salivary glands and severely reduced in the small bowel. Neurite outgrowth and trophic effects of NTN at low concentrations were lacking in Gfra2-/- trigeminal neurons in vitro, whereas responses to GDNF were similar between the genotypes. Thus, GFR alpha2 is a physiological NTN receptor, essential for the development of specific postganglionic parasympathetic neurons.

Distribution of GABA receptor ρ subunit transcripts in the rat brain

The γ‐aminobutyric acid (GABA) receptor ρ subunits recently cloned from rat and human retina are thought to form GABA receptor channels belonging to a pharmacologically distinct receptor class, termed GABAC. In this work we have examined the distribution of ρ1, ρ2 and ρ3 subunits, and found expression of all three transcripts in several regions of the rat nervous system.

A founder mutation of the potassium channel KCNQ1 in long QT syndrome ☆: Implications for estimation of disease prevalence and molecular diagnostics

OBJECTIVES We took advantage of the genetic isolate of Finns to characterize a common long QT syndrome (LQTS) mutation, and to estimate the prevalence of LQTS. BACKGROUND The LQTS is caused by mutations in different ion channel genes, which vary in their molecular nature from family to family. METHODS The potassium channel gene KCNQ1 was sequenced in two unrelated Finnish patients with Jervell and Lange-Nielsen syndrome (JLNS), followed by genotyping of 114 LQTS probands and their available family members. The functional properties of the mutation were studied using a whole-cell patch-damp technique. RESULTS We identified a novel missense mutation (G589D or KCNQ1-Fin) in the C-terminus of the KCNQ1 subunit. The voltage threshold of activation for the KCNQ1-Fin channel was markedly increased compared to the wild-type channel. This mutation was present in homozygous form in two siblings with JLNS, and in heterozygous form in 34 of 114 probands with Romano-Ward syndrome (RWS) and 282 family members. The mean (+/- SD) rate-corrected QT intervals of the heterozygous subjects (n = 316) and noncarriers (n = 423) were 460 +/- 40 ms and 410 +/- 20 ms (p < 0.001), respectively. CONCLUSIONS A single missense mutation of the KCNQ1 gene accounts for 30% of Finnish cases with LQTS, and it may be associated with both the RWS and JLNS phenotypes of the syndrome. The relative enrichment of this mutation most likely represents a founder gene effect. These circumstances provide an excellent opportunity to examine how genetic and nongenetic factors modify the LQTS phenotype.

Influence of GABA‐gated bicarbonate conductance on potential, current and intracellular chloride in crayfish muscle fibres.

1. The effects of gamma‐aminobutyric acid (GABA) on membrane potential and conductance as well as on the intracellular Cl‐ activity (aiCl) and intracellular pH (pHi) were studied in crayfish muscle fibres using a three‐microelectrode voltage clamp and ion‐selective microelectrodes. In the presence of CO2‐HCO3‐, the intracellular HCO3‐ activity (aiHCO3) was estimated from pHi. 2. In a nominally HCO3(‐)‐free solution, a near‐saturating concentration of GABA (0.2 mM) produced a marked increase in membrane conductance but little change in potential. In a solution containing 30 mM‐HCO3‐ (equilibrated with 5% CO2 + 95% air; pH 7.4), the GABA‐induced increase in conductance was associated with a depolarization of about 15 mV, with an increase in aiCl and with a decrease in aiHCO3. All these effects were blocked by picrotoxin (PTX). The depolarizing action of GABA was augmented following depletion of extracellular and intracellular Cl‐. 3. The GABA‐induced increase in aiCl which took place in the presence of HCO3‐ was blocked by clamping the membrane potential at its resting level. This indicates that the increase in aiCl was due to passive redistribution of Cl‐. In both the presence and absence of HCO3‐, the GABA‐activated transmembrane flux of Cl‐ showed reversal at the level of the resting potential, which indicates that under steady‐state conditions the Cl‐ equilibrium potential (ECl) is identical to the resting potential. 4. In a Cl(‐)‐free, 30 mM‐HCO3(‐)‐containing solution, 0.5 mM‐GABA produced a PTX‐sensitive increase in conductance which amounted to 15% of the conductance activated in the presence of Cl‐. In the absence of both Cl‐ and HCO3‐, the respective figure was 2.8%. Assuming constant‐field conditions, the conductance data yielded a permeability ratio PHCO3/PCl of 0.42 for the GABA‐activated channels. 5. In a Cl(‐)‐containing, HCO3(‐)‐free solution, the reversal potential of the GABA‐activated current (EGABA) was, by about 1 mV, less negative than the resting membrane potential (RP). In a solution containing Cl‐ and 30 mM‐HCO3‐, EGABA‐RP was 12 mV. Simultaneous measurements of EGABA, aiCl and aiHCO3 (pHi) gave a PHCO3/PCl value of 0.33. 6. In a Cl(‐)‐free, HCO3(‐)‐containing solution EGABA was close to the HCO3‐ equilibrium potential (EHCO3) and an experimental acidosis which produced a negative shift in EHCO3 was associated with a similar shift in EGABA.(ABSTRACT TRUNCATED AT 400 WORDS)

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptor Channels Lacking the N-terminal Domain

Ionotropic glutamate receptor (iGluR) subunits contain a ∼400-residue extracellular N-terminal domain (“X domain”), which is sequence-related to bacterial amino acid-binding proteins and to class C G-protein-coupled receptors. The X domain has been implicated in the assembly, transport to the cell surface, allosteric ligand binding, and desensitization in various members of the iGluR family, but its actual role in these events is poorly characterized. We have studied the properties of homomeric α-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA)-selective GluR-D glutamate receptors carrying N-terminal deletions. Our analysis indicates that, surprisingly, transport to the cell surface, ligand binding properties, agonist-triggered channel activation, rapid desensitization, and allosteric potentiation by cyclothiazide can occur normally in the complete absence of the X domain (residues 22–402). The relatively intact ligand-gated channel function of a homomeric AMPA receptor in the absence of the X domain indirectly suggests more subtle roles for this domain in AMPA receptors, e.g. in the assembly of heteromeric receptors and in synaptic protein interactions.

PROTON MODULATION OF FUNCTIONALLY DISTINCT GABAA RECEPTORS IN ACUTELY ISOLATED PYRAMIDAL NEURONS OF RAT HIPPOCAMPUS

We have studied the effect of extracellular pH (pHo) on the GABAA receptor-mediated chloride conductance in acutely isolated pyramidal neurons from area CA1 of the rat hippocampus under whole-cell voltage clamp in bicarbonate-free solutions. The conductance evoked by saturating or near-saturating concentrations (200-1000 microM) of GABA showed a marked sensitivity to variations of pHo around 7.4. A decrease in pHo between 8.4 and 6.4 increased the GABAA receptor-mediated chloride conductance by about two-fold per pH unit. In contrast, when evoked by a low agonist concentration (1-10 microM) the conductance showed an equally marked decrease upon a decrease in pHo. The half-time for desensitization of the conductance induced by 500 microM GABA was around 900 ms at pHo 6.4 and 7.4, but decreased to 650 ms at pHo 8.4. A fall in pHo decreased the amount of desensitization of the conductance evoked by a 5 s application of 5 microM, but not of 500 microM, GABA. The concentration-response relationship of the GABA-induced conductance showed a local plateau between 50 and 100 microM of GABA, which was particularly evident at high pHo. Assuming two receptor populations with a high and a low affinity for GABA, the effect of H+ on the GABAA receptors could be explained as an increase in the EC50 of the high affinity receptor, and an apparently non-competitive potentiation of both the high and the low affinity receptors. The GABAA receptor-mediated conductance was markedly inhibited by 20-50 microM Zn2+. In addition, Zn2+ reverted the down-modulation by H+ observed at low GABA concentrations to up-modulation. Diazepam (1-10 microM) had only a marginal effect on the GABA-gated conductance. Taken together, the results suggest the coexistence in individual hippocampal neurons of two distinct GABAA receptor populations having differential sensitivities to H+. In the light of the inhibitory action of Zn2+ and the virtual absence of an effect of diazepam it is probable that a significant fraction of the GABAA receptors lack the gamma 2 subunit. The observation that an elevated pH has a strong suppressing effect on the conductance evoked by high concentrations of GABA may at least partly explain why an extracellular alkalosis leads to neuronal hyperexcitability.

Functional characterization of the common amino acid 897 polymorphism of the cardiac potassium channel KCNH2 (HERG)

OBJECTIVE To determine whether the amino acid 897 threonine (T) to lysine (K) polymorphism of the KCNH2 (HERG) potassium channel influences channel performance or patient phenotype. METHODS The phenotypic effects of this polymorphism were investigated in vitro by electrophysiological experiments in HEK-293 cells and in vivo by exercise electrocardiography in a group of LQTS patients carrying the same genetically proven KCNQ1 mutation. RESULTS When expressed in HEK-293 cells, the 897T isoform of the KCNH2 channel exhibited changes in inactivation and deactivation properties, and a smaller current density than the more common 897K isoform. Western blot experiments indicated that the decreased current density associated with 897T was caused by reduced channel expression. During a maximal exercise test in 39 LQT1 patients carrying an identical KCNQ1 mutation (G589D) and showing a prolonged QT interval (>440 ms), QT intervals were longer in patients carrying the 897T allele than in those homozygous for the 897K allele. CONCLUSIONS The K897T variation has an effect on channel function and clinical phenotype. Our data warrant further investigations into the significance of this polymorphism in drug-induced and inherited LQTS.

Homozygosity for a HERG potassium channel mutation causes a severe form of long QT syndrome: identification of an apparent founder mutation in the Finns☆

OBJECTIVES We studied the clinical characteristics and molecular background underlying a severe phenotype of long QT syndrome (LQTS). BACKGROUND Mutations of cardiac ion channel genes cause LQTS, manifesting as increased risk of ventricular tachycardia and sudden death. METHODS We studied two siblings showing prolonged QT intervals corrected for heart rate (QTc), their asymptomatic parents with only marginally prolonged QTc intervals and their family members. The potassium channel gene HERG was screened for mutations by deoxyribonucleic acid sequencing, and the electrophysiologic consequences of the mutation were studied in vitro using the whole-cell patch-clamp technique. RESULTS A novel missense mutation (L552S) in the HERG channel, present in the homozygous state in the affected siblings and in the heterozygous state in their parents, as well as in 38 additional subjects from six LQTS families, was identified. One of the homozygous siblings had 2:1 atrioventricular block immediately after birth, and died at the age of four years after experiencing unexplained hypoglycemia. The other sibling had an episode of torsade de pointes at the age of two years. The mean QTc interval differed significantly (p < 0.001) between heterozygous symptomatic mutation carriers (500 +/- 59 ms), asymptomatic mutation carriers (452 +/- 34 ms) and noncarriers (412 +/- 23 ms). When expressed in vitro, the HERG-L552S formed functional channels with increased activation and deactivation rates. CONCLUSIONS Our data demonstrate that homozygosity for a HERG mutation can cause a severe cardiac repolarization disorder without other phenotypic abnormalities. Absence of functional HERG channels appears to be one cause for intrauterine and neonatal bradycardia and 2:1 atrioventricular block.

Ceramide 1-phosphate enhances calcium entry through voltage-operated calcium channels by a protein kinase C-dependent mechanism in GH4C1 rat pituitary cells

Sphingomyelin derivatives modulate a multitude of cellular processes, including the regulation of [Ca2+]i (the intracellular free calcium concentration). Previous studies have shown that these metabolites often inhibit calcium entry through VOCCs (voltage-operated calcium channels). In the present study, we show that, in pituitary GH4C1 cells, C1P (C2-ceramide 1-phosphate) enhances calcium entry in a dose-dependent manner. The phospholipase C inhibitor U73122 attenuated the response. C1P invoked a small, but significant, increase in the formation of inositol phosphates. Pre-treatment of the cells with pertussis toxin was without an effect on the C1P-evoked increase in [Ca2+]i. The effect of C1P was critically dependent on extracellular calcium, since no increase in [Ca2+]i was observed when cells in a calcium-free buffer were stimulated with C1P. Furthermore, if the cells were retreated with 300 nM of the VOCC inhibitor nimodipine, the effect of C1P was almost totally abolished. In addition, ceramide C8-1-phosphate evoked an increase in [Ca2+]i, but the onset of the response was slow compared with that of C1P. In cells treated with 1 mM thapsigargin for 15 min, C1P still evoked an increase in [Ca2+]i. In patch-clamp experiments in the whole-cell mode, C1P enhanced calcium entry through the VOCCs compared with vehicle-treated cells. Dialysis of the cells with C1P did not enhance the calcium current. On-cell patch-clamp experiments showed an enhanced probability of the VOCCs being open (P(open)) in the presence of C1P. Inhibition of PKC (protein kinase C) with GF109203X and down-regulation of PKC with PMA attenuated the C1P-evoked increase in [Ca2+]i. Furthermore, down-regulation of PKC abolished the effect of C1P on P(open). This is the first report showing that a sphingomyelin derivative enhances calcium entry through VOCCs.

Effects of simple aromatic compounds and flavonoids on Ca2+fluxes in rat pituitary GH4C1 cells

The biological activity of phenolic compounds from plants is well documented in vitro, but little is known about the possible effect of simple aromatic compounds and flavonoids on voltage-operated Ca2+ channels (VOCCs). In pituitary cells, several intracellular pathways may regulate the activity of VOCCs. In this study, we investigated the effect of nine phenylpropanes and metanes, and 20 flavonoids on high K(+)-induced 45Ca2+ entry in clonal rat pituitary GH(4)C(1) cells. At the highest dose tested (20 microg/ml), flavone (a flavone) inhibited 45Ca2+ entry by 63.5%, naringenin (a flavanone) by 56.3% and genistein (an isoflavone) by 54.6%. The phenylmetane derivative octyl gallate was the most potent compound tested, with an IC(50) value of 15.0 microg/ml. The IC(50) value for the reference compound verapamil hydrochloride was 3.0 microg/ml. In sharp contrast to the above, the flavonols quercetin and morin potentiated 45Ca2+ entry. At 20 microg/ml, quercetin increased 45Ca2+ entry by 54.1% and morin by 48.0%. Quercetin increased the cellular cAMP content in a concentration-dependent manner. H 89, an inhibitor of protein kinase A, inhibited the effect of quercetin on 45Ca2+ entry. The results thus suggest that the effect of quercetin is the result of a protein kinase A-mediated activation of VOCCs. Quercetin induced a rapid and marked increase in both the transient (143.1+/-4.2%) and delayed (198.8+/-10.0%) Ca2+ currents, measured by the whole cell patch clamp technique. The onset of the inhibitory effect of octyl gallate was slow, but resulted in an almost complete inhibition of both Ca2+ currents.