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.

Understanding the physiology and pathology of an organ composed of a variety of cell populations depends critically on genome-wide information on each cell type. Here, we report single-cell transcriptome profiling of over 6,800 freshly dispersed anterior pituitary cells from postpubertal male and female rats. Six pituitary-specific cell types were identified based on known marker genes and characterized: folliculostellate cells and hormone-producing corticotrophs, gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. Also identified were endothelial and blood cells from the pituitary capillary network. The expression of numerous developmental and neuroendocrine marker genes in both folliculostellate and hormone-producing cells supports that they have a common origin. For several genes, the validity of transcriptome analysis was confirmed by qRT-PCR and single cell immunocytochemistry. Folliculostellate cells exhibit impressive transcriptome diversity, indicating their major roles in production of endogenous ligands and detoxification enzymes, and organization of extracellular matrix. Transcriptome profiles of hormone-producing cells also indicate contributions toward those functions, while also clearly demonstrating their endocrine function. This survey highlights many novel genetic markers contributing to pituitary cell type identity, sexual dimorphism, and function, and points to relationships between hormone-producing and folliculostellate cells.

Gonadotrophs are basophilic cells of the anterior pituitary gland specialized to secrete gonadotropins in response to elevation in intracellular calcium concentration. These cells fire action potentials spontaneously, coupled with voltage-gated calcium influx of insufficient amplitude to trigger gonadotropin release. The spontaneous excitability of gonadotrophs reflects the expression of voltage-gated sodium, calcium, potassium, nonselective cation-conducting, and chloride channels at their plasma membrane. These cells also express the hyperpolarization-activated and cyclic nucleotide-gated cation channels at the plasma membrane, as well as GABAA, nicotinic, and purinergic P2X channels gated by GABA, acetylcholine, and ATP, respectively. Activation of these channels leads to initiation or amplification of the pacemaking activity, facilitation of calcium influx, and activation of the exocytic pathway. Gonadotrophs also express calcium-conducting channels at the endoplasmic reticulum membranes gated by inositol trisphosphate and intracellular calcium. These channels are activated potently by hypothalamic gonadotropin-releasing hormone and less potently by several paracrine calcium-mobilizing agonists, including pituitary adenylate-activating peptides, endothelins, acetylcholine, vasopressin and oxytocin. Activation of these channels causes oscillatory calcium release and a rapid gonadotropin release, accompanied with a shift from tonic firing of single action potentials to periodic bursting type of electrical activity, which accounts for a sustained calcium signaling and gonadotropin secretion. This review summarizes our current understanding of ion channels as signaling molecules in gonadotrophs, the role of gonadotropin-releasing hormone and paracrine agonists in their gating, and the crosstalk among channels.

The most obvious functional differences between mammalian males and females are related to the control of reproductive physiology and include patterns of GnRH and gonadotropin release, the timing of puberty, sexual and social behavior, and the regulation of food intake and body weight. Using the rat as the best-studied mammalian model for maturation, we examined the expression of major anterior pituitary genes in five secretory cell types of developing males and females. Corticotrophs show comparable Pomc profiles in both sexes, with the highest expression occurring during the infantile period. Somatotrophs and lactotrophs also exhibit no difference in Gh1 and Prl profiles during embryonic-to-juvenile age but show the amplification of Prl expression in females and Gh1 expression in males during peripubertal and postpubertal ages. Gonadotrophs exhibit highly synchronized Lhb, Fshb, Cga, and Gnrhr expression in both sexes, but the peak of expression occurs during the infantile period in females and at the end of the juvenile period in males. Thyrotrophs also show different developmental Tshb profiles, which are synchronized with the expression of gonadotroph genes in males but not in females. These results indicate the lack of influence of sex on Pomc expression and the presence of two patterns of sexual dimorphism in the expression of other pituitary genes: a time shift in the peak expression during postnatal development, most likely reflecting the perinatal sex-specific brain differentiation, and modulation of the amplitude of expression during late development, which is secondary to the establishment of the hypothalamic-pituitary-gonadal and -thyroid axes.

The occupancy of the ATP binding sites of P2X receptor (P2XR) channels causes the rapid opening of a small cation-permeable pore, followed by a gradual dilation that renders the pore permeable to large organic cations. Electrophysiologically, this phenomenon was shown using whole-cell current recording on P2X2R-, P2X2/X5R-, P2X4R- and P2X7R-expressing cells that were bathed in N-methyl-D-glucamine-containing buffers in the presence and/or absence of small monovalent and divalent cations. The pore dilation of P2X4R and P2X7R caused a secondary current growth, whereas that of P2X2R showed a sustained kinetic coupling of dilation and desensitization, leading to receptor channel closure. The pore size of the P2X7R open and dilated states was estimated to be approximately 0.85 nm and greater than 1 nm, respectively. The P2XR pore dilation was also observed in intact cells by measurement of fluorescent dye uptake/release, application of polyethylene glycols of different sizes, and atomic force microscopy. However, pore dilation was not observed at the single channel level. Structural data describing the dilated state are not available, and the relevance of orthosteric and allosteric ligand interactions to pore dilation was not studied.