Neurosteroids as positive and negative allosteric modulators of ligand-gated ion channels: P2X receptor perspective.

Neurosteroids are steroids synthesized de novo in the brain from cholesterol in an independent manner from peripheral steroid sources. The term "neuroactive steroid" includes all steroids independent of their origin, and newly synthesized analogs of neurosteroids that modify neuronal activities. In vivo application of neuroactive steroids induces potent anxiolytic, antidepressant, anticonvulsant, sedative, analgesic and amnesic effects, mainly through interaction with the γ-aminobutyric acid type-A receptor (GABAAR). However, neuroactive steroids also act as positive or negative allosteric regulators on several ligand-gated channels including N-methyl-d-aspartate receptors (NMDARs), nicotinic acetylcholine receptors (nAChRs) and ATP-gated purinergic P2X receptors. Seven different P2X subunits (P2X1-7) can assemble to form homotrimeric or heterotrimeric ion channels permeable for monovalent cations and calcium. Among them, P2X2, P2X4, and P2X7 are the most abundant within the brain and can be regulated by neurosteroids. Transmembrane domains are necessary for neurosteroid binding, however, no generic motif of amino acids can accurately predict the neurosteroid binding site for any of the ligand-gated ion channels including P2X. Here, we will review what is currently known about the modulation of rat and human P2X by neuroactive steroids and the possible structural determinants underlying neurosteroid-induced potentiation and inhibition of the P2X2 and P2X4 receptors. This article is part of the Special Issue on "Purinergic Signaling: 50 years".

Identification of residues in the first transmembrane domain of the P2X7 that regulates receptor trafficking, sensitization, and dye uptake function.

P2X receptors (P2X1-7) are trimeric ion channels activated by extracellular ATP. Each P2X subunit contains two transmembrane helices (TM1 and TM2). We substituted all residues in TM1 of rat P2X7 with alanine or leucine one by one, expressed mutants in HEK293T cells, and examined the pore permeability by recording both membrane currents and fluorescent dye uptake in response to agonist application. Alanine substitution of G27, K30, H34, Y40, F43, L45, M46, and D48 inhibited agonist-stimulated membrane current and dye uptake, and all but one substitution, D48A, prevented surface expression. Mutation V41A partially reduced both membrane current and dye uptake, while W31A and A44L showed reduced dye uptake not accompanied by reduced membrane current. Mutations T28A, I29A, and L33A showed small changes in agonist sensitivity, but they had no or small impact on dye uptake function. Replacing charged residues with residues of the same charge (K30R, H34K, and D48E) rescued receptor function, while replacement with residues of opposite charge inhibited (K30E and H34E) or potentiated (D48K) receptor function. Prolonged stimulation with agonist-induced current facilitation and a leftward shift in the dose-response curve in the P2X7 wild-type and most functional mutants, but sensitization was absent in the W31A, L33A, and A44L. Detailed analysis of the decay of responses revealed two kinetically distinct mechanisms of P2X7 deactivation: fast represents agonist unbinding, and slow might represent resetting of the receptor to the resting closed state. These results indicate that conserved and receptor-specific TM1 residues control surface expression of the P2X7 protein, non-polar residues control receptor sensitization, and D48 regulates intrinsic channel properties.

Purinergic P2 Receptors: Structure and Function 2.0.

This Special Issue of the International Journal of Molecular Sciences (IJMS) is a direct continuation of the previous Special Issue of this journal, entitled "Purinergic P2 Receptors: Structure and Function" https://www [...].

Role of Conserved Residues and F322 in the Extracellular Vestibule of the Rat P2X7 Receptor in Its Expression, Function and Dye Uptake Ability.

Activation of the P2X7 receptor results in the opening of a large pore that plays a role in immune responses, apoptosis, and many other physiological and pathological processes. Here, we investigated the role of conserved and unique residues in the extracellular vestibule connecting the agonist-binding domain with the transmembrane domain of rat P2X7 receptor. We found that all residues that are conserved among the P2X receptor subtypes respond to alanine mutagenesis with an inhibition (Y51, Q52, and G323) or a significant decrease (K49, G326, K327, and F328) of 2',3'-O-(benzoyl-4-benzoyl)-ATP (BzATP)-induced current and permeability to ethidium bromide, while the nonconserved residue (F322), which is also present in P2X4 receptor, responds with a 10-fold higher sensitivity to BzATP, much slower deactivation kinetics, and a higher propensity to form the large dye-permeable pore. We examined the membrane expression of conserved mutants and found that Y51, Q52, G323, and F328 play a role in the trafficking of the receptor to the plasma membrane, while K49 controls receptor responsiveness to agonists. Finally, we studied the importance of the physicochemical properties of these residues and observed that the K49R, F322Y, F322W, and F322L mutants significantly reversed the receptor function, indicating that positively charged and large hydrophobic residues are important at positions 49 and 322, respectively. These results show that clusters of conserved residues above the transmembrane domain 1 (K49-Y51-Q52) and transmembrane domain 2 (G326-K327-F328) are important for receptor structure, membrane expression, and channel gating and that the nonconserved residue (F322) at the top of the extracellular vestibule is involved in hydrophobic inter-subunit interaction which stabilizes the closed state of the P2X7 receptor channel.

Lithocholic acid inhibits P2X2 and potentiates P2X4 receptor channel gating.

The family of ATP-gated purinergic P2X receptors comprises seven bunits (P2X1-7) that are unevenly distributed in the central and peripheral nervous systems as well as other organs. Endogenous modulators of P2X receptors are phospholipids, steroids and neurosteroids. Here, we analyzed whether bile acids, which are natural products derived from cholesterol, affect P2X receptor activity. We examined the effects of primary and secondary bile acids and newly synthesized derivatives of lithocholic acid on agonist-induced responses in HEK293T cells expressing rat P2X2, P2X4 and P2X7 receptors. Electrophysiology revealed that low micromolar concentrations of lithocholic acid and its structural analog 4-dafachronic acid strongly inhibit ATP-stimulated P2X2 but potentiate P2X4 responses, whereas primary bile acids and other secondary bile acids exhibit no or reduced effects only at higher concentrations. Agonist-stimulated P2X7 responses are significantly potentiated by lithocholic acid at moderate concentrations. Structural modifications of lithocholic acid at positions C-3, C-5 or C-17 abolish both inhibitory and potentiation effects to varying degrees, and the 3α-hydroxy group contributes to the ability of the molecule to switch between potentiation and inhibition. Lithocholic acid allosterically modulates P2X2 and P2X4 receptor sensitivity to ATP, reduces the rate of P2X4 receptor desensitization and antagonizes the effect of ivermectin on P2X4 receptor deactivation. Alanine-scanning mutagenesis of the upper halve of P2X4 transmembrane domain-1 revealed that residues Phe48, Val43 and Tyr42 are important for potentiating effect of lithocholic acid, indicating that modulatory sites for lithocholic acid and ivermectin partly overlap. Lithocholic acid also inhibits ATP-evoked currents in pituitary gonadotrophs expressing native P2X2, and potentiates ATP currents in nonidentified pituitary cells expressing P2X4 receptors. These results indicate that lithocholic acid is a bioactive steroid that may help to further unveil the importance of the P2X2, and P2X4 receptors in many physiological processes.

Special Issue of International Journal of Molecular Sciences (IJMS) "Purinergic P2 Receptors: Structure and Function".

This Special Issue of International Journal of Molecular Sciences (IJMS) contains 7 reviews and 12 original research papers written by a panel of experts who highlight recent advances in molecular structure and cellular function of purinergic P2 receptors[...].

P2X2 Receptor Expression and Function Is Upregulated in the Rat Supraoptic Nucleus Stimulated Through Refeeding After Fasting.

Magnocellular neurons in the supraoptic nucleus (SON), which synthesize and release arginine vasopressin (AVP) and oxytocin (OT), express several subtypes of ATP-stimulated purinergic P2X receptors (P2XR) that modulate neuronal activity as well as neurotransmitter and hormone release. However, the physiological impact of this modulation is not well understood. Here, we tested a hypothesis that P2XRs play a role in the sustained release of hormones from SON neurons stimulated through fasting/refeeding. We studied the effect of 2 h of refeeding after 48 h of fasting on P2XR and P2YR mRNA expression and ATP-induced presynaptic and postsynaptic responses in the SON of 30-day-old rats. Quantitative real-time PCR revealed that the expression of P2X2R and AVP mRNA was upregulated, whereas P2X4R, P2X7R, P2Y2R, and OT mRNA levels were not significantly changed and P2Y1R mRNA expression was decreased. Whole-cell patch clamp recordings performed on isolated rat brain slices showed that the amplitude of the ATP-stimulated somatic current and the ATP-induced increases in the frequency of spontaneous GABAergic inhibitory postsynaptic currents were significantly higher in SON neurons from fasted/refed rats than in SON neurons from normally fed rats. No evidence was found for changes in the presynaptic effect of ATP in SON neurons not expressing somatic P2XRs. These results suggest that the increased activity of SON neurons synthesizing AVP is associated with enhanced expression of P2X2Rs on neuronal cell bodies and their GABAergic presynaptic nerve terminals.

Synthetic testosterone derivatives modulate rat P2X2 and P2X4 receptor channel gating.

P2X receptors (P2XRs) are ATP-gated cationic channels that are allosterically modulated by numerous compounds, including steroids and neurosteroids. These compounds may both inhibit and potentiate the activity of P2XRs, but sex steroids such as 17ß-estradiol or progesterone are reported to be inactive. Here, we tested a hypothesis that testosterone, another sex hormone, modulates activity of P2XRs. We examined actions of native testosterone and a series of testosterone derivatives on the gating of recombinant P2X2R, P2X4R and P2X7R and native channels expressed in pituitary cells and hypothalamic neurons. The 17ß-ester derivatives of testosterone rapidly and positively modulate the 1 µM ATP-evoked currents in P2X2R- and P2X4R-expressing cells, but not agonist-evoked currents in P2X7R-expressing cells. In general, most of the tested testosterone derivatives are more potent modulators than endogenous testosterone. The comparison of chemical structures and whole-cell recordings revealed that their interactions with P2XRs depend on the lipophilicity and length of the alkyl chain at position C-17. Pre-treatment with testosterone butyrate or valerate increases the sensitivity of P2X2R and P2X4R to ATP by several fold, reduces the rate of P2X4R desensitization, accelerates resensitization, and enhances ethidium uptake by P2X4R. Native channels are also potentiated by testosterone derivatives, while endogenously expressed GABA receptors type A are inhibited. The effect of ivermectin, a P2X4R-specific allosteric modulator, on deactivation is antagonized by testosterone derivatives in a concentration-dependent manner. Together, our results provide evidence for potentiation of particular subtypes of P2XRs by testosterone derivatives and suggest a potential role of ivermectin binding site for steroid-induced modulation. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. The complete Open Science Disclosure form for this article can be found at the end of the article. More information about the Open Practices badges can be found at https://cos.io/our-services/open-science-badges/.

Circadian ATP Release in Organotypic Cultures of the Rat Suprachiasmatic Nucleus Is Dependent on P2X7 and P2Y Receptors.

The circadian rhythms in physiological and behavioral functions are driven by a pacemaker located in the suprachiasmatic nucleus (SCN). The rhythms continue in constant darkness and depend on cell-cell communication between neurons and glia. The SCN astrocytes generate also a circadian rhythm in extracellular adenosine 5'-triphosphate (ATP) accumulation, but molecular mechanisms that regulate ATP release are poorly understood. Here, we tested the hypothesis that ATP is released via the plasma membrane purinergic P2X7 receptors (P2X7Rs) and P2Y receptors (P2YRs) which have been previously shown to be expressed in the SCN tissue at transcriptional level. We have investigated this hypothesis using SCN organotypic cultures, primary cultures of SCN astrocytes, ATP bioluminescent assays, immunohistochemistry, patch-clamping, and calcium imaging. We found that extracellular ATP accumulation in organotypic cultures followed a circadian rhythm, with a peak between 24:00 and 04:00 h, and the trough at ~12:00 h. ATP rhythm was inhibited by application of AZ10606120, A438079, and BBG, specific blockers of P2X7R, and potentiated by GW791343, a positive allosteric modulator of this receptor. Double-immunohistochemical staining revealed high expression of the P2X7R protein in astrocytes of SCN slices. PPADS, a non-specific P2 antagonist, and MRS2179, specific P2Y1R antagonist, also abolished ATP rhythm, whereas the specific P2X4R blocker 5-BDBD was not effective. The pannexin-1 hemichannel blocker carbenoxolone displayed a partial inhibitory effect. The P2Y1R agonist MRS2365, and the P2Y2R agonist MRS2768 potentiated ATP release in organotypic cultures and increase intracellular Ca2+ level in cultured astrocytes. Thus, SCN utilizes multiple purinergic receptor systems and pannexin-1 hemichannels to release ATP.

Neurotransmitter receptors as signaling platforms in anterior pituitary cells.

The functions of anterior pituitary cells are controlled by two major groups of hypothalamic and intrapituitary ligands: one exclusively acts on G protein-coupled receptors and the other activates both G protein-coupled receptors and ligand-gated receptor channels. The second group of ligands operates as neurotransmitters in neuronal cells and their receptors are termed as neurotransmitter receptors. Most information about pituitary neurotransmitter receptors was obtained from secretory studies, RT-PCR analyses of mRNA expression and immunohistochemical and biochemical analyses, all of which were performed using a mixed population of pituitary cells. However, recent electrophysiological and imaging experiments have characterized γ-aminobutyric acid-, acetylcholine-, and ATP-activated receptors and channels in single pituitary cell types, expanding this picture and revealing surprising differences in their expression between subtypes of secretory cells and between native and immortalized pituitary cells. The main focus of this review is on the electrophysiological and pharmacological properties of these receptors and their roles in calcium signaling and calcium-controlled hormone secretion.

Deciphering the regulation of P2X4 receptor channel gating by ivermectin using Markov models.

The P2X4 receptor (P2X4R) is a member of a family of purinergic channels activated by extracellular ATP through three orthosteric binding sites and allosterically regulated by ivermectin (IVM), a broad-spectrum antiparasitic agent. Treatment with IVM increases the efficacy of ATP to activate P2X4R, slows both receptor desensitization during sustained ATP application and receptor deactivation after ATP washout, and makes the receptor pore permeable to NMDG+, a large organic cation. Previously, we developed a Markov model based on the presence of one IVM binding site, which described some effects of IVM on rat P2X4R. Here we present two novel models, both with three IVM binding sites. The simpler one-layer model can reproduce many of the observed time series of evoked currents, but does not capture well the short time scales of activation, desensitization, and deactivation. A more complex two-layer model can reproduce the transient changes in desensitization observed upon IVM application, the significant increase in ATP-induced current amplitudes at low IVM concentrations, and the modest increase in the unitary conductance. In addition, the two-layer model suggests that this receptor can exist in a deeply inactivated state, not responsive to ATP, and that its desensitization rate can be altered by each of the three IVM binding sites. In summary, this study provides a detailed analysis of P2X4R kinetics and elucidates the orthosteric and allosteric mechanisms regulating its channel gating.

Ion Channels of Pituitary Gonadotrophs and Their Roles in Signaling and Secretion.

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 (APs) 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, non-selective cation-conducting, and chloride channels at their plasma membrane (PM). These cells also express the hyperpolarization-activated and cyclic nucleotide-gated cation channels at the PM, as well as GABAA, nicotinic, and purinergic P2X channels gated by γ-aminobutyric acid (GABA), acetylcholine (ACh), 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 (GnRH) and less potently by several paracrine calcium-mobilizing agonists, including pituitary adenylate cyclase-activating peptides, endothelins, ACh, 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 APs 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 GnRH and paracrine agonists in their gating, and the cross talk among channels.

Modeling the diversity of spontaneous and agonist-induced electrical activity in anterior pituitary corticotrophs.

Pituitary corticotrophs fire action potentials spontaneously and in response to stimulation with corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP), and such electrical activity is critical for calcium signaling and calcium-dependent adrenocorticotropic hormone secretion. These cells typically fire tall, sharp action potentials when spontaneously active, but a variety of other spontaneous patterns have also been reported, including various modes of bursting. There is variability in reports of the fraction of corticotrophs that are electrically active, as well as their patterns of activity, and the sources of this variation are not well understood. The ionic mechanisms responsible for CRH- and AVP-triggered electrical activity in corticotrophs are also poorly characterized. We use electrophysiological measurements and mathematical modeling to investigate possible sources of variability in patterns of spontaneous and agonist-induced corticotroph electrical activity. In the model, variation in as few as two parameters can give rise to many of the types of patterns observed in electrophysiological recordings of corticotrophs. We compare the known mechanisms for CRH, AVP, and glucocorticoid actions and find that different ionic mechanisms can contribute in different but complementary ways to generate the complex time courses of CRH and AVP responses. In summary, our modeling suggests that corticotrophs have several mechanisms at their disposal to achieve their primary function of pacemaking depolarization and increased electrical activity in response to CRH and AVP.NEW & NOTEWORTHY We and others recently demonstrated that the electrical activity and calcium dynamics of corticotrophs are strikingly diverse, both spontaneously and in response to the agonists CRH and AVP. Here we demonstrate this diversity with electrophysiological measurements and use mathematical modeling to investigate its possible sources. We compare the known mechanisms of agonist-induced activity in the model, showing how the context of ionic conductances dictates the effects of agonists even when their target is fixed.

Spontaneous and CRH-Induced Excitability and Calcium Signaling in Mice Corticotrophs Involves Sodium, Calcium, and Cation-Conducting Channels.

Transgenic mice expressing the tdimer2(12) form of Discosoma red fluorescent protein under control of the proopiomelanocortin gene's regulatory elements are a useful model for studying corticotrophs. Using these mice, we studied the ion channels and mechanisms controlling corticotroph excitability. Corticotrophs were either quiescent or electrically active, with a 22-mV difference in the resting membrane potential (RMP) between the 2 groups. In quiescent cells, CRH depolarized the membrane, leading to initial single spiking and sustained bursting; in active cells, CRH further facilitated or inhibited electrical activity and calcium spiking, depending on the initial activity pattern and CRH concentration. The stimulatory but not inhibitory action of CRH on electrical activity was mimicked by cAMP independently of the presence or absence of arachidonic acid. Removal of bath sodium silenced spiking and hyperpolarized the majority of cells; in contrast, the removal of bath calcium did not affect RMP but reduced CRH-induced depolarization, which abolished bursting electrical activity and decreased the spiking frequency but not the amplitude of single spikes. Corticotrophs with inhibited voltage-gated sodium channels fired calcium-dependent action potentials, whereas cells with inhibited L-type calcium channels fired sodium-dependent spikes; blockade of both channels abolished spiking without affecting the RMP. These results indicate that the background voltage-insensitive sodium conductance influences RMP, the CRH-depolarization current is driven by a cationic conductance, and the interplay between voltage-gated sodium and calcium channels plays a critical role in determining the status and pattern of electrical activity and calcium signaling.

Functional characterization of mutants in the transmembrane domains of the rat P2X7 receptor that regulate pore conductivity and agonist sensitivity.

In the sustained presence of agonist, the opening of P2X7R channel is followed by pore dilatation, which causes an increase in its permeability to larger organic cations, accompanied by receptor sensitization. To explore the molecular mechanisms by which the conductivity and sensitivity are increased, we analyzed the electrophysiological properties and YO-PRO-1 uptake of selected alanine mutants in the first and second transmembrane domains of the rat P2X7R. Substitution of residues Y40, F43, G338, and D352 with alanine reduced membrane trafficking, and the D352A was practically non-functional. The Y40A and F43A mutants that were expressed in the membrane lacked pore dilation ability. Moreover, the Y40A and Y40F displayed desensitization, whereas the Y40W partially recovered receptor function. The G338A/S mutations favored the open state of the channel and displayed instantaneous permeability to larger organic cations. The G338P was non-functional. The L341A and G345A displayed normal trafficking, current amplitude, and sensitization, but both mutations resulted in a decreased pore formation and dye uptake. These results showed that the increase in P2X7R conductivity and sensitivity is critically dependent on residues Y40 and F43 in the TM1 domain and that the region located at the intersection of TM2 helices controls the rate of large pore opening. We investigated the mechanism of the proapoptotic receptor P2X7R's large pore opening and its sensitization. We found that aromatic residues in the upper part of the first transmembrane domain (TM1) are critical for both the P2X7R channel pore opening and receptor sensitization, and residues located at or below the intersection of the second transmembrane domains (TM2) control the rate of pore opening. These findings identify new residues involved in pore formation of P2X7R.

Allosteric regulation of the P2X4 receptor channel pore dilation.

Allosteric modulators of ligand-gated receptor channels induce conformational changes of the entire protein that alter potencies and efficacies for orthosteric ligands, expressed as the half maximal effective concentration (EC50) and maximum current amplitude, respectively. Here, we studied the influence of allostery on channel pore dilation, an issue not previously addressed. Experiments were done using the rat P2X4 receptor expressed in human embryonic kidney 293T cells and gated by adenosine 5'-triphosphate (ATP) in the presence and absence of ivermectin (IVM), an established positive allosteric regulator of this channel. In the absence of IVM, this channel activates and deactivates rapidly, does not show transition from open to dilated states, desensitizes completely with a moderate rate, and recovers only fractionally during washout. IVM treatment increases the efficacy of ATP to activate the channel and slows receptor desensitization during sustained ATP application and receptor deactivation after ATP washout. The rescue of the receptor from desensitization temporally coincides with pore dilation, and the dilated channel can be reactivated after washout of ATP. Experiments with vestibular and transmembrane domain receptor mutants further established that IVM has distinct effects on opening and dilation of the channel pore, the first accounting for increased peak current amplitude and the latter correlating with changes in the EC50 and kinetics of receptor deactivation. The corresponding kinetic (Markov state) model indicates that the IVM-dependent transition from open to dilated state is coupled to receptor sensitization, which rescues the receptor from desensitization and subsequent internalization. Allosterically induced sensitization of P2X4R thus provides sustained signaling during prolonged and repetitive ATP stimulation.

Identification of functionally important residues of the rat P2X4 receptor by alanine scanning mutagenesis of the dorsal fin and left flipper domains.

Crystallization of the zebrafish P2X4 receptor in both open and closed states revealed conformational differences in the ectodomain structures, including the dorsal fin and left flipper domains. Here, we focused on the role of these domains in receptor activation, responsiveness to orthosteric ATP analogue agonists, and desensitization. Alanine scanning mutagenesis of the R203-L214 (dorsal fin) and the D280-N293 (left flipper) sequences of the rat P2X4 receptor showed that ATP potency/efficacy was reduced in 15 out of 26 alanine mutants. The R203A, N204A, and N293A mutants were essentially non-functional, but receptor function was restored by ivermectin, an allosteric modulator. The I205A, T210A, L214A, P290A, G291A, and Y292A mutants exhibited significant changes in the responsiveness to orthosteric analog agonists 2-(methylthio)adenosine 5'-triphosphate, adenosine 5'-(γ-thio)triphosphate, 2'(3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate, and α,ß-methyleneadenosine 5'-triphosphate. In contrast, the responsiveness of L206A, N208A, D280A, T281A, R282A, and H286A mutants to analog agonists was comparable to that of the wild type receptor. Among these mutants, D280A, T281A, R282A, H286A, G291A, and Y292A also exhibited increased time-constant of the desensitizing current response. These experiments, together with homology modeling, indicate that residues located in the upper part of the dorsal fin and left flipper domains, relative to distance from the channel pore, contribute to the organization of the ATP binding pocket and to the initiation of signal transmission towards residues in the lower part of both domains. The R203 and N204 residues, deeply buried in the protein, may integrate the output signal from these two domains towards the gate. In addition, the left flipper residues predominantly account for the control of transition of channels from an open to a desensitized state.

Structural and functional properties of the rat P2X4 purinoreceptor extracellular vestibule during gating.

P2X receptors are ATP-gated cation channels consisting of three subunits that are mutually intertwined and form an upper, central, and extracellular vestibule with three lateral portals and the channel pore. Here we used cysteine and alanine scanning mutagenesis of the rat P2X4R receptor V47-V61 and K326-N338 sequences to study structural and functional properties of extracellular vestibule during gating. Cysteine mutants were used to test the accessibility of these residue side chains to cadmium during closed-open-desensitized transitions, whereas alanine mutants served as controls. This study revealed the accessibility of residues E51, T57, S59, V61, K326, and M336 to cadmium in channels undergoing a transition from a closed-to-open state and the accessibility of residues V47, G53, D331, I332, I333, T335, I337, and N338 in channels undergoing a transition from an open-to-desensitized state; residues E56 and K329 were accessible during both transitions. The effect of cadmium on channel gating was stimulatory in all reactive V47-V61 mutants and inhibitory in the majority of reactive K326-N338 mutants. The rat P2X4 receptor homology model suggests that residues affected by cadmium in the closed-to-open transition were located within the lumen of the extracellular vestibule and toward the central vestibule; however, the residues affected by cadmium in the open-to-desensitized state were located at the bottom of the vestibule near the pore. Analysis of the model assumed that there is ion access to extracellular and central vestibules through lateral ports when the channel is closed, with residues above the first transmembrane domain being predominantly responsible for ion uptake. Upon receptor activation, there is passage of ions toward the residues located on the upper region of the second transmembrane domain, followed by permeation through the gate region.

P2X receptor channels in endocrine glands.

The endocrine system is the system of ductless glands and single cells that synthetize hormones and release them directly into the bloodstream. Regulation of endocrine system is very complex and ATP and its degradable products ADP and adenosine contribute to its regulation acting as extracellular messengers for purinergic receptors. These include P2X receptors, a family of ligand-gated ion channels which expression and roles in endocrine tissues are reviewed here. There are seven mammalian purinergic receptor subunits, denoted P2X1 through P2X7, and the majority of these subunits are also expressed in secretory and non-secretory cells of endocrine system. Functional channels have been identified in the neuroendocrine hypothalamus, the posterior and anterior pituitary, the thyroid gland, the adrenals, the endocrine pancreas, the gonads and the placenta. Native channels are capable of promoting calcium influx through its pore in both excitable and non-excitable cells, as well as of increasing electrical activity in excitable cells by membrane depolarization. This leads to generation of calcium transients and stimulation of hormone release. The pattern of expression and action of P2XRs in endocrine system suggests that locally produced ATP amplifies and synchronizes the secretory responses of individual cells.

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.