Hana Zemkova

Biophysical basis of pituitary cell type-specific Ca2+ signaling–secretion coupling

All secretory pituitary cells exhibit spontaneous and extracellular Ca2+-dependent electrical activity. Somatotrophs and lactotrophs fire plateau-bursting action potentials, which generate Ca2+ signals of sufficient amplitude to trigger hormone release. Gonadotrophs also fire action potentials spontaneously, but as single, high-amplitude spikes with limited ability to promote Ca2+ influx and secretion. However, Ca2+ mobilization in gonadotrophs transforms single spiking into plateau-bursting-type electrical activity and triggers secretion. Patch clamp analysis revealed that somatotrophs and lactotrophs, but not gonadotrophs, express BK (big)-type Ca2+-controlled K+ channels, activation of which is closely associated with voltage-gated Ca2+ influx. Conversely, pituitary gonadotrophs express SK (small)-type Ca2+-activated K+ channels that are colocalized with intracellular Ca2+ release sites. Activation of both channels is crucial for plateau-bursting-type rhythmic electrical activity and secretion.

COPPER MODULATION OF NMDA RESPONSES IN MOUSE AND RAT CULTURED HIPPOCAMPAL NEURONS

The effect of Cu2+ on NMDA receptors was studied in cultured mouse and rat hippocampal neurons using whole‐cell patch‐clamp and a fast perfusion system. Analysis of the Cu2+ concentration‐response curve for inhibition of NMDA‐induced currents suggests that free Cu2+ directly inhibits NMDA receptors with an IC50 of 0.27 μM. Cu2+ was ineffective in blocking NMDA receptor activity when complexed with NMDA or glycine; NMDA‐Cu2+ and glycine‐Cu2+ complexes acted as agonists of similar potency to the free amino acids. The inhibition by Cu2+ (10–100 μM) of responses to 10 μM NMDA was essentially voltage‐independent. The onset of inhibition by 100 μM Cu2+ of responses to 2 FM glutamate acting at NMDA receptors was significantly faster than NMDA receptor deactivation evoked by a sudden decrease in the concentration of glycine or glutamate, or of both agonists. This suggests that CU2+ acts as a non‐competitive antagonist, and does not directly interfere with the binding of glutamate or glycine to their recognition sites on the NMDA receptor complex. In the absence of NMDA the apparent association rate constant for binding of Cu2+ to NMDA receptors, calculated from the rate of onset of block by Cu2+ of test responses to NMDA, was 19 times slower than in the presence of 30 μM NMDA, suggesting that Cuz+ interacts preferentially with agonist‐bound receptors. Our results show that Cu2+ is a potent inhibitor of NMDA receptor‐mediated responses.

Effect of ivermectin on γ-aminobutyric acid-induced chloride currents in mouse hippocampal embryonic neurones

The effect of ivermectin on gamma-aminobutyric acid (GABA)-induced Cl- currents was studied in embryonicse hippocampal cells in culture. When 0.1 microM ivermectin was applied to the perfusion medium, the responses to 2 microM GABA were enhanced to 273% within 60 s, and the GABA EC50 was reduced from 8.2 to 3.2 microM. Half-maximal potentiation of GABA responses was found with 17.8 nM ivermectin. The potentiating effect of ivermectin diminished to 146% within 10 min but the GABA EC50 did not change any further. At the same time, the maximal GABA-induced Cl- current decreased to 64%. Both the fast and slow desensitization time constants of GABA-activated membrane currents were shortened after ivermectin application. The final effect of ivermectin was irreversible. Modulation of the GABA responses by ivermectin did not interfere with the potentiation induced by diazepam and pentobarbital or with the sensitivity to blockade by bicuculline, picrotoxin and Zn2+. These results support the view that ivermectin binds to a novel site on the GABAA receptor and allosterically enhances the affinity of the GABA binding site. The more slowly occurring conformational changes in the ivermectin-GABAA receptor complex apparently accelerate the desensitization of the GABAA receptor, reducing the amplitude of maximal GABA-induced currents.

Identification of P2X 4 receptor transmembrane residues contributing to channel gating and interaction with ivermectin

Ivermectin (IVM), a large macrocyclic lactone, specifically enhances P2X4 receptor-channel function by interacting with residues of transmembrane (TM) helices in the open conformation state. In this paper, we used cysteine-scanning mutagenesis of rat P2X4-TMs to identify and map residues of potential importance for channel gating and interaction with IVM. The receptor function was unchanged by mutations in 29 different residues, and among them, the IVM effects were altered in Gln36, Leu40, Val43, Val47, Trp50, Asn338, Gly342, Leu346, Ala349, and Ile356 mutants. The substitution-sensitive Arg33 and Cys353 mutants could also be considered as IVM-sensitive hits. The pattern of these 12 residues was consistent with helical topology of both TMs, with every third or fourth amino acid affected by substitution. These predominantly hydrophobic-nonpolar residues are also present in the IVM-sensitive Schistosoma mansoni P2X subunit. They lie on the same side of their helices and could face lipids in the open conformation state and provide the binding pocket for IVM. In contrast, the IVM-independent hits Met31, Tyr42, Gly45, Val49, Gly340, Leu343, Ala344, Gly347, Thr350, Asp354, and Val357 map on the opposite side of their helices, probably facing the pore of receptor or protein and playing important roles in gating.

Role of aromatic and charged ectodomain residues in the P2X4 receptor functions

The localization of ATP binding site(s) at P2X receptors and the molecular rearrangements associated with opening and closing of channels are still not well understood. At P2X4 receptor, substitution of the K67, F185, K190, F230, R278, D280, R295, and K313 ectodomain residues with alanine generated low or non‐responsive mutants, whereas the F294A mutant was functional. The loss of receptor function was also observed in K67R, R295K, and K313R mutants, but not in F185W, K190R, F230W, R278K, and D280E mutants. To examine whether the loss of function reflects decreased sensitivity of mutants for ATP, we treated cells with ivermectin, an antiparasitic agent that enhances responsiveness of P2X4R. In the presence of ivermectin, all low or non‐responsive mutants responded to ATP in a dose‐dependent manner, with the EC50 values for ATP of about 1, 2, 4, 20, 60, 125, 270, 420, 1000 and 2300 μmol/L at D280A, R278A, F185A, K190A, R295K, K313R, R295A, K313A, K67A and K67R mutants, respectively. These results indicate that lysines 67 and 313 and arginine 295 play a critical role in forming the proper three‐dimensional structure of P2X4R for agonist binding and/or channel gating.

Melatonin Receptors in the Spinal Cord

The pineal hormone, melatonin, plays an important role in the regulation of diurnal and seasonal rhythms in animals. In addition to the well established actions on the brain, the possibility of a direct melatonin action on the spinal cord has to be considered. In our laboratory, we have obtained data suggesting that melatonin receptors are present in the spinal cords of birds and mammals. Using radioreceptor binding and quantitative autoradiography assays with 2-[125I]iodomelatonin as the specific melatonin agonist, melatonin binding sites have been demonstrated in the rabbit and chicken spinal cords. These sites are saturable, reversible, specific, guanosine nucleotide-sensitive, of picomolar affinity and femtomolar density. The linearity of Scatchard plots of saturation data and the unity of Hill coefficients indicate that a single class of melatonin binding sites is present in the spinal cord membranes studied. The picomolar affinity of these sites is in line with the circulating levels of melatonin in these animals suggesting that these sites are physiologically relevant. Autoradiography studies in the rabbit spinal cord show that melatonin binding sites are localized in the central gray substance (lamina X). In the chicken spinal cord, these binding sites are localized in dorsal gray horns (laminae I-V) and lamina X. As lamina X and laminae I-II have similar functions, melatonin may have comparable roles in the chicken and rabbit spinal cords. Moreover, in the chicken spinal cord, the density of 2-[125I]iodomelatonin binding in the lumbar segment was significantly higher than those of the cervical and thoracic segments. The densities of these binding sites changed with environmental manipulations. When chickens were adapted to a 12L/12D photoperiod and sacrificed at mid-light and mid-dark, there was a significant diurnal variation in the density (maximum number of binding sites; Bmax) of melatonin binding sites in the spinal cord. After constant light treatment or pinealectomy, the Bmax of melatonin receptors in the chicken spinal cord increased significantly in the subjective mid-dark period. Moreover, there was an age-related decrease in the 2-[125I]iodomelatonin binding to the chicken spinal cord. Our results suggest that melatonin receptors in the chicken spinal cord are regulated by environmental lighting and change with development. These receptors may play an important role in the chronobiology of spinal cord function. The biological responses of melatonin on spinal cords have also been demonstrated in vitro. Melatonin decreased the forskolin-stimulated cAMP production in the chicken spinal cord explant. Preincubation with pertussis toxin blocked the melatonin effect. Our results suggest that melatonin receptors in the chicken spinal cord are linked to the adenylate cyclase via a pertussis toxin-sensitive G protein and that melatonin binding sites in spinal cords are melatonin receptors with biological functions. These receptors may be involved in the regulation of spinal cord functions related to sensory transmission, visceral reflexes and autonomic activities.

Inhibitory Effect of Melatonin on Gonadotropin-Releasing Hormone-Induced Ca2+ Oscillations in Pituitary Cells of Newborn Rats

The effect of melatonin on the gonadotropin-releasing-hormone (GnRH)-induced oscillatory rises in intracellular calcium concentration, [Ca2+]i, was studied in cultured cells from the anterior pituitary gland of 6- to 8-day-old rats. GnRH-induced [Ca2+]i oscillations were recorded indirectly by monitoring the activity of apamin-sensitive Ca(2+)-activated K+ channels using the perforated patch-clamp technique and fast microperfusion system. Melatonin (1 nM) inhibited the initiation or attenuated the amplitude of oscillatory current responses induced by 10 nM GnRH in 72% of GnRH-sensitive cells. Analysis of the melatonin dose-inhibition relationship showed that melatonin inhibited the initiation of [Ca2+]i oscillations with IC50 = 0.35 nM. In partially inhibited cells, melatonin reduced the GnRH-induced current amplitude by 55% on the average, prolonged the delay in onset of response to GnRH and decreased the frequency of oscillations. Once initiated by GnRH, the amplitude and frequency of oscillatory currents was inhibited by melatonin after a latency of 10-30 s. These effects of melatonin were fully reversible. After pretreatment of neonatal gonadotropes with pertussis toxin, no inhibition by melatonin was observed. The inhibitory effect of melatonin on initiation, amplitude and frequency of GnRH-induced oscillatory current persisted in the absence of external Ca2+. Melatonin alone did not induce any transmembrane current or membrane potential changes. These observations suggest that melatonin reduces GnRH-induced calcium mobilization from intracellular stores.

Molecular dissection of purinergic P2X receptor channels.

Abstract: The P2X receptors (P2XRs) are a family of ATP‐gated channels expressed in the plasma membrane of numerous excitable and nonexcitable cells and play important roles in control of cellular functions, such as neurotransmission, hormone secretion, transcriptional regulation, and protein synthesis. P2XRs are homomeric or heteromeric proteins, formed by assembly of at least three of seven subunits named P2X1‐P2X7. All subunits possess intracellular N‐ and C‐termini, two transmembrane domains, and a relatively large extracellular ligand‐binding loop. ATP binds to still an unidentified extracellular domain, leading to a sequence of conformational transitions between closed, open, and desensitized states. Removal of extracellular ATP leads to deactivation and resensitization of receptors. Activated P2XRs generate inward currents caused by Na+ and Ca2+ influx through the pore of channels, and thus mediate membrane depolarization and facilitation of voltage‐gated calcium entry in excitable cells. No crystal structures are available for P2XRs and these receptors have no obvious similarity to other ion channels or ATP binding proteins, which limits the progress in understanding the relationship between molecular structure and conformational transitions of receptor in the presence of agonist and after its washout. We summarize here the alternative approaches in studies on molecular properties of P2XRs, including heteromerization, chimerization, mutagenesis, and biochemical studies.

Dual Gating Mechanism and Function of P2X7 Receptor Channels

The ATP-gated P2X7 receptor channel (P2X7R) operates as a cytolytic and apoptotic receptor but also controls sustained cellular responses, including cell growth and proliferation. However, it has not been clarified how the same receptor mediates such opposing effects. To address this question, we have combined electrophysiological, imaging, and mathematical studies using wild-type and mutant rat P2X7Rs. Activation of naïve (not previously stimulated) receptors by low agonist concentrations caused monophasic slow desensitizing currents and internalization of receptors without other changes in the cellular morphology, much like other P2XRs. In contrast, saturating agonist concentrations induced high-amplitude biphasic currents, reflecting pore dilation and causing rapid cell swelling and lysis. The existence of these two signaling patterns was accounted for using a revised Markov-state model that included, in addition to naïve and sensitized states, desensitized states. Occupancy of one or two ATP-binding sites of naïve receptors favored a slow transition to desensitized states, whereas occupancy of the third binding site favored a transition to sensitized/dilated states. Consistent with model predictions, nondilating P2X7R mutants always generated desensitizing currents. These results suggest that the level of saturation of the ligand binding sites determines the nature of the P2X7R gating and cellular actions.

Differences in Gonadotropin-Releasing Hormone-Induced Calcium Signaling between Melatonin-Sensitive and Melatonin-Insensitive Neonatal Rat Gonadotrophs

The sensitivity of GnRH-stimulated calcium signaling to melatonin, in a subpopulation of neonatal gonadotrophs, is supposed to be attributable to melatonin receptors. However, it is not yet known whether the intracellular pathway for GnRH action in melatonin-sensitive cells is the same as in melatonin-insensitive cells. By monitoring intracellular Ca2+ changes as an outward current carried through apamin-sensitive Ca2+-activated K+ channels, we compared GnRH-induced calcium responses in these two subpopulations of neonatal gonadotrophs. GnRH induced various oscillatory, as well as nonoscillatory, responses in both cell types that was not related to melatonin sensitivity. Melatonin-sensitive GnRH-induced responses could be clearly distinguished according to the pharmacological properties of their latency. The latency increased in zero extracellular Ca2+ or with the addition of nifedipine, staurosporine, and ryanodine. This effect was only rarely observed in melatonin-insensitive cells. This indicates that ...