Karla Kretschmannova

Carboxyl-Terminal Splicing Enhances Physical Interactions between the Cytoplasmic Tails of Purinergic P2X Receptors

Purinergic P2X receptors are ion-conducting channels composed of three subunits, each having two transmembrane domains and intracellular amino (N) and carboxyl (C) termini. Although alternative splicing extensively modifies the C-terminal sequences of P2X subunits, the direct influence of such post-transcriptional modifications on receptor architecture and function remains poorly understood. In this study, we focused on mouse pituitary P2X2 receptors. In this tissue, progressive splicing of the P2X2a C terminus generated two functional subunit variants, P2X2b and P2X2e, which exhibited accelerated desensitization rates and attenuated calcium signals when the receptors were in homomeric states. To measure the intersubunit interaction in living cells, the efficient transfer of bioluminescent resonance energy between luciferase and fluorescent proteins attached to the N- or C-subunit termini of these subunits was used. The constitutive interactions between the full-length C termini of P2X2a receptor were detected by a significant increase in fluorescence/luminescence intensity ratio compared with negative controls. Moreover, interactions between C termini and between C- and N termini of adjacent subunits were significantly enhanced in homomeric and heteromeric receptors containing P2X2b or P2X2e subunits. Finally, deletion of two amino acids at the splicing junction, but not at the C-terminal end of the P2X2b receptor, resulted in the enhancement of channel desensitization and luminescence resonance energy transfer. These results indicate that C-terminal structure plays a critical role in the cytoplasmic intersubunit interactions and suggest that the extent of subunit interactions before ATP application could contribute to the subsequent channel activity and conformation changes associated with agonist-dependent desensitization.

Role of nonselective cation channels in spontaneous and protein kinase A-stimulated calcium signaling in pituitary cells.

Several receptors linked to the adenylyl cyclase signaling pathway stimulate electrical activity and calcium influx in endocrine pituitary cells, and a role for an unidentified sodium-conducting channel in this process has been proposed. Here we show that forskolin dose-dependently increases cAMP production and facilitates calcium influx in about 30% of rat and mouse pituitary cells at its maximal concentration. The stimulatory effect of forskolin on calcium influx was lost in cells with inhibited PKA (cAMP-dependent protein kinase) and in cells that were haploinsufficient for the main PKA regulatory subunit but was preserved in cells that were also haploinsufficient for the main PKA catalytic subunit. Spontaneous and forskolin-stimulated calcium influx was present in cells with inhibited voltage-gated sodium and hyperpolarization-activated cation channels but not in cells bathed in medium, in which sodium was replaced with organic cations. Consistent with the role of sodium-conducting nonselective cation channels in PKA-stimulated Ca(2+) influx, cAMP induced a slowly developing current with a reversal potential of about 0 mV. Two TRP (transient receptor potential) channel blockers, SKF96365 and 2-APB, as well as flufenamic acid, an inhibitor of nonselective cation channels, also inhibited spontaneous and forskolin-stimulated electrical activity and calcium influx. Quantitative RT-PCR analysis indicated the expression of mRNA transcripts for TRPC1 >> TRPC6 > TRPC4 > TRPC5 > TRPC3 in rat pituitary cells. These experiments suggest that in pituitary cells constitutively active cation channels are stimulated further by PKA and contribute to calcium signaling indirectly by controlling the pacemaking depolarization in a sodium-dependent manner and directly by conducting calcium.

The Expression and Role of Hyperpolarization-Activated and Cyclic Nucleotide-Gated Channels in Endocrine Anterior Pituitary Cells

Pituitary cells fire action potentials independently of external stimuli, and such spontaneous electrical activity is modulated by a large variety of hypothalamic and intrapituitary agonists. Here, we focused on the potential role of hyperpolarization-activated and cyclic nucleotide-gated (HCN) channels in electrical activity of cultured rat anterior pituitary cells. Quantitative RT-PCR analysis showed higher level of expression of mRNA transcripts for HCN2 and HCN3 subunits and lower expression of HCN1 and HCN4 subunits in these cells. Western immunoblot analysis of lysates from normal and GH(3) immortalized pituitary cells showed bands with appropriate molecular weights for HCN2, HCN3, and HCN4. Electrophysiological experiments showed the presence of a slowly developing hyperpolarization-activated inward current, which was blocked by Cs(+) and ZD7288, in gonadotrophs, thyrotrophs, somatotrophs, and a fraction of lactotrophs, as well as in other unidentified pituitary cell types. Stimulation of adenylyl cyclase and addition of 8-Br-cAMP enhanced this current and depolarized the cell membrane, whereas 8-Br-cGMP did not alter the current and hyperpolarized the cell membrane. Both inhibition of basal adenylyl cyclase activity and stimulation of phospholipase C signaling pathway inhibited this current. Inhibition of HCN channels affected the frequency of firing but did not abolish spontaneous electrical activity. These experiments indicate that cAMP and cGMP have opposite effects on the excitability of endocrine pituitary cells, that basal cAMP production in cultured cells is sufficient to integrate the majority of HCN channels in electrical activity, and that depletion of phosphatidylinositol 4,5-bisphosphate caused by activation of phospholipase C silences them.

Dependence of Hyperpolarisation‐Activated Cyclic Nucleotide‐Gated Channel Activity on Basal Cyclic Adenosine Monophosphate Production in Spontaneously Firing GH3 Cells

The hyperpolarisation‐activated cyclic nucleotide‐gated (HCN) channels play a distinct role in the control of membrane excitability in spontaneously active cardiac and neuronal cells. Here, we studied the expression and role of HCN channels in pacemaking activity, Ca2+ signalling, and prolactin secretion in GH3 immortalised pituitary cells. Reverse transcriptase‐polymerase chain reaction analysis revealed the presence of mRNA transcripts for HCN2, HCN3 and HCN4 subunits in these cells. A hyperpolarisation of the membrane potential below − 60 mV elicited a slowly activating voltage‐dependent inward current (Ih) in the majority of tested cells, with a half‐maximal activation voltage of −89.9 ± 4.2 mV and with a time constant of 1.4 ± 0.2 s at −120 mV. The bath application of 1 mM Cs+, a commonly used inorganic blocker of Ih, and 100 µM ZD7288, a specific organic blocker of Ih, inhibited Ih by 90 ± 4.1% and 84.3 ± 1.8%, respectively. Receptor‐ and nonreceptor‐mediated activation of adenylyl and soluble guanylyl cyclase and the addition of a membrane permeable cyclic adenosine monophosphate (cAMP) analogue, 8‐Br‐cAMP, did not affect Ih. Inhibition of basal adenylyl cyclase activity, but not basal soluble guanylyl cyclase activity, led to a reduction in the peak amplitude and a leftward shift in the activation curve of Ih by 23.7 mV. The inhibition of the current was reversed by stimulation of adenylyl cyclase with forskolin and by the addition of 8‐Br‐cAMP, but not 8‐Br‐cGMP. Application of Cs+ had no significant effect on the resting membrane potential or electrical activity, whereas ZD7288 exhibited complex and Ih‐independent effects on spontaneous electrical activity, Ca2+ signalling, and prolactin release. These results indicate that HCN channels in GH3 cells are under tonic activation by basal level of cAMP and are not critical for spontaneous firing of action potentials.

Dependence of multidrug resistance protein-mediated cyclic nucleotide efflux on the background sodium conductance

Anterior pituitary cells fire action potentials and release cyclic nucleotides both spontaneously and in response to agonist stimulation, but the relationship between electrical activity and cyclic nucleotide efflux has not been studied. In these cells, a tetrodotoxin-resistant background N+ conductance is critical for firing of action potentials, and multidrug resistance proteins (MRPs) MRP4 and MRP5 contribute to cyclic nucleotide efflux. Here, we show that abolition of the background Na+ conductance in rat pituitary cells by complete or partial replacement of extracellular Na+ with organic cations or sucrose induced a rapid and reversible hyperpolarization of cell membranes and inhibition of action potential firing, accompanied by a rapid inhibition of cyclic nucleotide efflux. Valinomycin-induced hyperpolarization of plasma membranes also inhibited cyclic nucleotide efflux, whereas depolarization of cell membranes induced by the inhibition of Ca2+ influx or stimulation of Na+ influx by gramicidin was accompanied by a facilitation of cyclic nucleotide efflux. In contrast, inhibition of cyclic nucleotide efflux by probenecid did not affect the background Na+ conductance. In human embryonic kidney 293 cells stably transfected with human MRP4 or MRP5, replacement of bath Na+ with organic cations also hyperpolarized the cell membranes and inhibited cyclic nucleotide efflux. In these cells, the Na+/H+ antiporter monensin did not affect the membrane potential and was practically ineffective in altering cyclic nucleotide efflux. In both pituitary and MRP4- and MRP5-expressing cells, 3-[[3-[2-(7-chloroquinolin-2-yl)vinyl]phenyl]-(2-dimethylcarbamoylethylsulfanyl)methylsulfanyl] propionic acid (MK571) inhibited cyclic nucleotide efflux. These results indicate that the MRP4/5-mediated cyclic nucleotide efflux can be rapidly modulated by membrane potential determined by the background Na+ conductance.

Circadian Rhythmicity in AVP Secretion and GABAergic Synaptic Transmission in the Rat Suprachiasmatic Nucleus

Abstract: A variety of physiological and behavioral functions exhibit circadian changes and these circadian rhythms are driven by oscillatory expression of clock genes in the suprachiasmatic nuclei (SCN). It is still unknown how this molecular clockwork is controlled by extracellular neurohormones and neurotransmitters and which membrane receptors undergo circadian modulation. Circadian rhythm can be measured as a secretion of arginine vasopressin (AVP) in organotypic SCN culture for several weeks. Melatonin applied directly to the SCN late in the day induces a phase advance, when applied late at night or at the beginning of the day melatonin causes a phase delay. The time window for phase advance corresponds with the highest level of melatonin receptors in the SCN but the mechanism of melatonin‐induced phase delay is unknown. The principal neurotransmitter on SCN synapses is γ‐aminobutyric acid (GABA), which acts at postsynaptic GABAA receptors. Spontaneous release of GABA from presynaptic nerve terminals, recorded as miniature inhibitory postsynaptic currents in the presence of TTX, does not change, but zinc sensitivity of exogenous GABA‐induced currents varies during the day and night, possibly due to changes in subunit composition of GABAA receptors. We conclude that there is daily variation in the postsynaptic, but not presynaptic, function in the SCN.

Dependence of the Excitability of Pituitary Cells on Cyclic Nucleotides

Cyclic 3′,5′‐adenosine monophosphate and cyclic 3′,5′‐guanosine monophosphate are intracellular (second) messengers that are produced from the nucleotide triphosphates by a family of enzymes consisting of adenylyl and guanylyl cyclases. These enzymes are involved in a broad array of signal transduction pathways mediated by the cyclic nucleotide monophosphates and their kinases, which control multiple aspects of cell function through the phosphorylation of protein substrates. We review the findings and working hypotheses on the role of the cyclic nucleotides and their kinases in the control of electrical activity of the endocrine pituitary cells and the plasma membrane channels involved in this process.