Stanko S. Stojilkovic

Activation and Regulation of Purinergic P2X Receptor Channels

Mammalian ATP-gated nonselective cation channels (P2XRs) can be composed of seven possible subunits, denoted P2X1 to P2X7. Each subunit contains a large ectodomain, two transmembrane domains, and intracellular N and C termini. Functional P2XRs are organized as homomeric and heteromeric trimers. This review focuses on the binding sites involved in the activation (orthosteric) and regulation (allosteric) of P2XRs. The ectodomains contain three ATP binding sites, presumably located between neighboring subunits and formed by highly conserved residues. The detection and coordination of three ATP phosphate residues by positively charged amino acids are likely to play a dominant role in determining agonist potency, whereas an AsnPheArg motif may contribute to binding by coordinating the adenine ring. Nonconserved ectodomain histidines provide the binding sites for trace metals, divalent cations, and protons. The transmembrane domains account not only for the formation of the channel pore but also for the binding of ivermectin (a specific P2X4R allosteric regulator) and alcohols. The N- and C- domains provide the structures that determine the kinetics of receptor desensitization and/or pore dilation and are critical for the regulation of receptor functions by intracellular messengers, kinases, reactive oxygen species and mercury. The recent publication of the crystal structure of the zebrafish P2X4.1R in a closed state provides a major advance in the understanding of this family of receptor channels. We will discuss data obtained from numerous site-directed mutagenesis experiments accumulated during the last 15 years with reference to the crystal structure, allowing a structural interpretation of the molecular basis of orthosteric and allosteric ligand actions.

Expression and signal transduction pathways of gonadotropin-releasing hormone receptors.

Publisher Summary Gonadotropin-releasing hormone (GnRH)—the primary regulator of mammalian reproductive function—is produced in hypothalamic neurons and secreted episodically into the hypothalamic portal system of the median eminence. The distinctive distributions of GnRH-producing cells in the brain reflect their specific functions because GnRH acts as a neurohormone, neurotransmitter, or neuromodulator. In mammals, the majority of the GnRH neurons are located in the hypothalamus and are functionally coupled to form a pulse generator that determines the frequency of pulsatile GnRH release. This chapter discusses the major physiological action of GnRH, which is related to the control of gonadotropin secretion and is expressed through activation of GnRH receptors (GnRH-R) in the plasma membrane of pituitary gonadotrophs. Activation of GnRH receptors during agonist stimulation initiates a series of steps that lead to a cascade of intracellular responses. The first step is G protein-mediated activation of phospholipase C, leading to hydrolysis of phosphoinositides and the formation of InsP3 and DAG. These initial changes determine the second step in signalling. The signal molecules of the phospholipase C-dependent pathway also exert positive and negative controls on the signal transduction mechanism, including the activities of phospholipase D and A2 . These feedback mechanisms provide an additional degree of complexity in signaling that is important in the amplification, maintenance, and termination of specific aspects of the cellular activation pathways.

The P2X7 Receptor Channel Pore Dilates under Physiological Ion Conditions

Activation of the purinergic P2X7 receptor leads to the rapid opening of an integral ion channel that is permeable to small cations. This is followed by a gradual increase in permeability to fluorescent dyes by integrating the actions of the pannexin-1 channel. Here, we show that during the prolonged agonist application a rapid current that peaked within 200 ms was accompanied with a slower current that required tens of seconds to reach its peak. The secondary rise in current was observed under different ionic conditions and temporally coincided with the development of conductivity to larger organic cations. The biphasic response was also observed in cells with blocked pannexin channels and in cells not expressing these channels endogenously. The biphasic current was preserved in N-terminal T15A, T15S, and T15V mutants that have low or no permeability to organic cations, reflecting enhanced permeability to inorganic cations. In contrast, the T15E, T15K, and T15W mutants, and the Δ18 mutant with deleted P2X7 receptor–specific 18–amino acid C-terminal segment, were instantaneously permeable to organic cations and generated high amplitude monophasic currents. These results indicate that the P2X7 receptor channel dilates under physiological ion conditions, leading to generation of biphasic current, and that this process is controlled by residues near the intracellular side of the channel pore.

Ion Channels and Signaling in the Pituitary Gland

Endocrine pituitary cells are neuronlike; they express numerous voltage-gated sodium, calcium, potassium, and chloride channels and fire action potentials spontaneously, accompanied by a rise in intracellular calcium. In some cells, spontaneous electrical activity is sufficient to drive the intracellular calcium concentration above the threshold for stimulus-secretion and stimulus-transcription coupling. In others, the function of these action potentials is to maintain the cells in a responsive state with cytosolic calcium near, but below, the threshold level. Some pituitary cells also express gap junction channels, which could be used for intercellular Ca(2+) signaling in these cells. Endocrine cells also express extracellular ligand-gated ion channels, and their activation by hypothalamic and intrapituitary hormones leads to amplification of the pacemaking activity and facilitation of calcium influx and hormone release. These cells also express numerous G protein-coupled receptors, which can stimulate or silence electrical activity and action potential-dependent calcium influx and hormone release. Other members of this receptor family can activate calcium channels in the endoplasmic reticulum, leading to a cell type-specific modulation of electrical activity. This review summarizes recent findings in this field and our current understanding of the complex relationship between voltage-gated ion channels, ligand-gated ion channels, gap junction channels, and G protein-coupled receptors in pituitary cells.

Dependence of STIM1/Orai1-mediated Calcium Entry on Plasma Membrane Phosphoinositides

Recent studies identified two main components of store-operated calcium entry (SOCE): the endoplasmic reticulum-localized Ca2+ sensor protein, STIM1, and the plasma membrane (PM)-localized Ca2+ channel, Orai1/CRACM1. In the present study, we investigated the phosphoinositide dependence of Orai1 channel activation in the PM and of STIM1 movements from the tubular to PM-adjacent endoplasmic reticulum regions during Ca2+ store depletion. Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) levels were changed either with agonist stimulation or by chemically induced recruitment of a phosphoinositide 5-phosphatase domain to the PM, whereas PtdIns4P levels were decreased by inhibition or down-regulation of phosphatidylinositol 4-kinases (PI4Ks). Agonist-induced phospholipase C activation and PI4K inhibition, but not isolated PtdIns(4,5)P2 depletion, substantially reduced endogenous or STIM1/Orai1-mediated SOCE without preventing STIM1 movements toward the PM upon Ca2+ store depletion. Patch clamp analysis of cells overexpressing STIM1 and Orai1 proteins confirmed that phospholipase C activation or PI4K inhibition greatly reduced ICRAC currents. These results suggest an inositide requirement of Orai1 activation but not STIM1 movements and indicate that PtdIns4P rather than PtdIns(4,5)P2 is a likely determinant of Orai1 channel activity.

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.

Novel aspects of GnRH-induced intracellular signaling and secretion in pituitary gonadotrophs.

The activation of phospholipase C by gonadotropin-releasing hormone (GnRH) in cultured pituitary cells is mediated by pertussis toxin-insensitive G proteins, specifically G, and/or G, , (1-5). GnRH receptors (GnRH-R) in aT3-1 gonadotrophs also interact functionally with both G,, and G,,, in a non-selective manner (6). GnRH induces rapid and prominent phosphoinositide turnover in pituitary and aT3-1 cells (7-12), primarily by stimulating the hydrolysis of phosphatidylinositol 43bisphosphate (PIP,) (13, 14). The rapid Ca2+ signaling observed in these cells in response to agonist stimulation is also consistent with the coupling of GnRH-R to the phospholipase C pathway (15-21). In addition to pituitary cells, GTl neuronal cells, gonadal cells, and transfected cells expressing cloned mouse, rat, sheep, and human GnRH-R exhibit an extracellular Ca2+-independent rise in cytoplasmic calcium concentration ( [Ca”+Ii) during GnRH stimulation (22-29). The functional roles of the inositol ( 1, 4, 5)-trisphosphate (InsP,) and CaZt pathways in pituitary gonadotrophs include the control of plasma membrane channel activity, luteinizing hormone (LH) release and synthesis, and gene expression (reviewed in (30, 31)). In addition to phospholipase C, activation of phospholipase D has been implicated in GnRH action in rat pituitary cells, aT3-1 gonadotrophs, and ovarian granulosa cells (32-34). In GnRHstimulated cells, and in several other cells operated by G proteincoupled receptors, rapid hydrolysis of phosphatidylcholine (PC ) by phospholipase D leads to the formation of phosphatidic acid (PA) which is further metabolized to diacylglycerol (DAG) by PA phosphohydrolase. However, the mechanism of activation of phospholipase D is not well defined. It has been proposed to be a G-protein-mediated process (35-37), and also to result from receptor-tyrosine kinase-mediated phosphorylation (38). It may also occur in a protein kinase C-dependent manner, either resulting from or independent of activation of phospholipase C (39, 40). In GnRH-stimulated cells, both protein kinase C-dependent and -independent activation of this enzyme have been proposed (32, 34). Not only the mechanism of activation of phospholipase D but also the cellular functions of this signaling pathway are uncertain, and the products of this pathway that act as intracellular messengers are not fully defined. Because the turnover rate of DAG is extremely rapid, the integrated activities of phospholipase C and phospholipase D could serve to provide sustained production of DAG and maintenance of protein kinase C activity. In turn, activation of the protein kinase C system is an important component of long-term cellular responses such as growth, differentiation, and division (41). The sustained DAG production and activation of protein kinase C by phospholipase D could provide a positive feedback mechanism for selective stimulation of enzymes of the protein kinase family. It is noteworthly that PC contains mostly oleic and linoleic acids whereas phosphoinositides are relatively enriched in stearic and arachidonic acids (42). Furthermore, there is a shift in DAG composition from tetraenoic to more saturated fatty acids during the sustained phase of TRH stimulation in GH, pituitary cells (43). Thus, it is possible that phospholipase D-dependent production of specific molecular species of DAG activates a different set of protein kinase C enzymes than those responsive to DAG released from phosphoinositides by phospholipase C. Furthermore, other molecules produced via the phospholipase D pathway may also act as second messengers in the control of cellular functions (44-46), further complicating the integration of the phospholipase D pathway into phospholipase C-mediated signaling transduction. In the present manuscript, we review the dependence of agonist action on phospholipase C and phospholipase D activation in cells expressing GnRH-R. The kinetic aspects of activation of phospholipases C and D by GnRH-R, the mechanism of integration of phospholipase D into phospholipase C-dependent pathway, and the physiological importance of activation of these two enzymes, are discussed. In addition, the importance of InsP, in Ca2+ signaling and plasma membrane electrical activity, and the developmental and physiological aspects of CaZ + signaling, have been addressed.

Experimental Characterization and Mathematical Modeling of P2X7 Receptor Channel Gating

The P2X7 receptor is a trimeric channel with three binding sites for ATP, but how the occupancy of these sites affects gating is still not understood. Here we show that naive receptors activated and deactivated monophasically at low and biphasically at higher agonist concentrations. Both phases of response were abolished by application of Az10606120, a P2X7R-specific antagonist. The slow secondary growth of current in the biphasic response coincided temporally with pore dilation. Repetitive stimulation with the same agonist concentration caused sensitization of receptors, which manifested as a progressive increase in the current amplitude, accompanied by a slower deactivation rate. Once a steady level of the secondary current was reached, responses at high agonist concentrations were no longer biphasic but monophasic. Sensitization of receptors was independent of Na+ and Ca2+ influx and ∼30 min washout was needed to reestablish the initial gating properties. T15E- and T15K-P2X7 mutants showed increased sensitivity for agonists, responded with monophasic currents at all agonist concentrations, activated immediately with dilated pores, and deactivated slowly. The complex pattern of gating exhibited by wild-type channels can be accounted for by a Markov state model that includes negative cooperativity of agonist binding to unsensitized receptors caused by the occupancy of one or two binding sites, opening of the channel pore to a low conductance state when two sites are bound, and sensitization with pore dilation to a high conductance state when three sites are occupied.

Kisspeptin-10 facilitates a plasma membrane-driven calcium oscillator in gonadotropin-releasing hormone-1 neurons.

Kisspeptins, the natural ligands of the G-protein-coupled receptor (GPR)-54, are the most potent stimulators of GnRH-1 secretion and as such are critical to reproductive function. However, the mechanism by which kisspeptins enhance calcium-regulated neuropeptide secretion is not clear. In the present study, we used GnRH-1 neurons maintained in mice nasal explants to examine the expression and signaling of GPR54. Under basal conditions, GnRH-1 cells exhibited spontaneous baseline oscillations in intracellular calcium concentration ([Ca(2+)](i)), which were critically dependent on the operation of voltage-gated, tetrodotoxin (TTX)-sensitive sodium channels and were not coupled to calcium release from intracellular pools. Activation of native GPR54 by kisspeptin-10 initiated [Ca(2+)](i) oscillations in quiescent GnRH-1 cells, increased the frequency of calcium spiking in oscillating cells that led to summation of individual spikes into plateau-bursting type of calcium signals in a subset of active cells. These changes predominantly reflected the stimulatory effect of GPR54 activation on the plasma membrane oscillator activity via coupling of this receptor to phospholipase C signaling pathways. Both components of this pathway, inositol 1,3,4-trisphosphate and protein kinase C, contributed to the receptor-mediated modulation of baseline [Ca(2+)](i) oscillations. TTX and 2-aminoethyl diphenylborinate together abolished agonist-induced elevation in [Ca(2+)](i) in almost all cells, whereas flufenamic acid was less effective. Together these results indicate that a plasma membrane calcium oscillator is spontaneously operative in the majority of prenatal GnRH-1 neurons and is facilitated by kisspeptin-10 through phosphatidyl inositol diphosphate hydrolysis and depolarization of neurons by activating TTX-sensitive sodium channels and nonselective cationic channels.

Ca2+-regulated exocytosis and SNARE function

Secretory vesicles formed at the trans-Golgi network of neuroendocrine and endocrine cells must undergo several steps, such as translocation, docking and priming, before they are ready to fuse with the plasma membrane and deliver their cargo into the extracellular space. This process is called regulated exocytosis and is controlled by Ca(2+) (using synaptotagmin) and mediated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) proteins. Recent studies from three leading laboratories reveal novel details about the mechanism by which Ca(2+) and SNAREs regulate this complex process. These findings highlight the roles of both SNAP25 (synaptosome-associated protein of 25kD), one of the SNARE proteins, and CAPS (Ca(2+)-dependent activator protein for secretion), a Ca(2+)-sensor protein, in vesicle priming, depriming and fusion.