Unravelling the intricate cooperativity of subunit gating in P2X2 ion channels

Ionotropic purinergic (P2X) receptors are trimeric channels that are activated by the binding of ATP. They are involved in multiple physiological functions, including synaptic transmission, pain and inflammation. The mechanism of activation is still elusive. Here we kinetically unraveled and quantified subunit activation in P2X2 receptors by an extensive global fit approach with four complex and intimately coupled kinetic schemes to currents obtained from wild type and mutated receptors using ATP and its fluorescent derivative 2-[DY-547P1]-AET-ATP (fATP). We show that the steep concentration-activation relationship in wild type channels is caused by a subunit flip reaction with strong positive cooperativity, overbalancing a pronounced negative cooperativity for the three ATP binding steps, that the net probability fluxes in the model generate a marked hysteresis in the activation-deactivation cycle, and that the predicted fATP binding matches the binding measured by fluorescence. Our results shed light into the intricate activation process of P2X channels.

Cross-Talk between P2X and NMDA Receptors

Purinergic P2X receptors (P2X) are ATP-gated ion channels widely expressed in the CNS. While the direct contribution of P2X to synaptic transmission is uncertain, P2X reportedly affect N-methyl-D-aspartate receptor (NMDAR) activity, which has given rise to competing theories on the role of P2X in the modulation of synapses. However, P2X have also been shown to participate in receptor cross-talk: an interaction where one receptor (e.g., P2X2) directly influences the activity of another (e.g., nicotinic, 5-HT3 or GABA receptors). In this study, we tested for interactions between P2X2 or P2X4 and NMDARs. Using two-electrode voltage-clamp electrophysiology experiments in Xenopus laevis oocytes, we demonstrate that both P2X2 and P2X4 interact with NMDARs in an inhibited manner. When investigating the molecular domains responsible for this phenomenon, we found that the P2X2 c-terminus (CT) could interfere with both P2X2 and P2X4 interactions with NMDARs. We also report that 11 distal CT residues on the P2X4 facilitate the P2X4–NMDAR interaction, and that a peptide consisting of these P2X4 CT residues (11C) can disrupt the interaction between NMDARs and P2X2 or P2X4. Collectively, these results provide new evidence for the modulatory nature of P2X2 and P2X4, suggesting they might play a more nuanced role in the CNS.

Purinergic P2X1 receptor, purinergic P2X7 receptor, and angiotensin II type 1 receptor interactions in the regulation of renal afferent arterioles in angiotensin II-dependent hypertension

In ANG II-dependent hypertension, ANG II activates ANG II type 1 receptors (AT1Rs), elevating blood pressure and increasing renal afferent arteriolar resistance (AAR). The increased arterial pressure augments interstitial ATP concentrations activating purinergic P2X receptors (P2XRs) also increasing AAR. Interestingly, P2X1R and P2X7R inhibition reduces AAR to the normal range, raising the conundrum regarding the apparent disappearance of AT1R influence. To evaluate the interactions between P2XRs and AT1Rs in mediating the increased AAR elicited by chronic ANG II infusions, experiments using the isolated blood perfused juxtamedullary nephron preparation allowed visualization of afferent arteriolar diameters (AAD). Normotensive and ANG II-infused hypertensive rats showed AAD responses to increases in renal perfusion pressure from 100 to 140 mmHg by decreasing AAD by 26 ± 10% and 19 ± 4%. Superfusion with the inhibitor P2X1Ri (NF4490; 1 μM) increased AAD. In normotensive kidneys, superfusion with ANG II (1 nM) decreased AAD by 16 ± 4% and decreased further by 19 ± 5% with an increase in renal perfusion pressure. Treatment with P2X1Ri increased AAD by 30 ± 6% to values higher than those at 100 mmHg plus ANG II. In hypertensive kidneys, the inhibitor AT1Ri (SML1394; 1 μM) increased AAD by 10 ± 7%. In contrast, treatment with P2X1Ri increased AAD by 21 ± 14%; combination with P2X1Ri plus P2X7Ri (A438079; 1 μM) increased AAD further by 25 ± 8%. The results indicate that P2X1R, P2X7R, and AT1R actions converge at receptor or postreceptor signaling pathways, but P2XR exerts a dominant influence abrogating the actions of AT1Rs on AAR in ANG II-dependent hypertension.

Recent advances in taste transduction and signaling

In the last few years, single-cell profiling of taste cells and ganglion cells has advanced our understanding of transduction, encoding, and transmission of information from taste buds as relayed to the central nervous system. This review focuses on new knowledge from these molecular approaches and attempts to place this in the context of previous questions and findings in the field. The individual taste cells within a taste bud are molecularly specialized for detection of one of the primary taste qualities: salt, sour, sweet, umami, and bitter. Transduction and transmitter release mechanisms differ substantially for taste cells transducing sour (Type III cells) compared with those transducing the qualities of sweet, umami, or bitter (Type II cells), although ultimately all transmission of taste relies on activation of purinergic P2X receptors on the afferent nerves. The ganglion cells providing innervation to the taste buds also appear divisible into functional and molecular subtypes, and each ganglion cell is primarily but not exclusively responsive to one taste quality.

On the permeation of large organic cations through the pore of ATP-gated P2X receptors

Pore dilation is thought to be a hallmark of purinergic P2X receptors. The most commonly held view of this unusual process posits that under prolonged ATP exposure the ion pore expands in a striking manner from an initial small-cation conductive state to a dilated state, which allows the passage of larger synthetic cations, such as N-methyl-d-glucamine (NMDG+). However, this mechanism is controversial, and the identity of the natural large permeating cations remains elusive. Here, we provide evidence that, contrary to the time-dependent pore dilation model, ATP binding opens an NMDG+-permeable channel within milliseconds, with a conductance that remains stable over time. We show that the time course of NMDG+ permeability superimposes that of Na+ and demonstrate that the molecular motions leading to the permeation of NMDG+ are very similar to those that drive Na+ flow. We found, however, that NMDG+ “percolates” 10 times slower than Na+ in the open state, likely due to a conformational and orientational selection of permeating molecules. We further uncover that several P2X receptors, including those able to desensitize, are permeable not only to NMDG+ but also to spermidine, a large natural cation involved in ion channel modulation, revealing a previously unrecognized P2X-mediated signaling. Altogether, our data do not support a time-dependent dilation of the pore on its own but rather reveal that the open pore of P2X receptors is wide enough to allow the permeation of large organic cations, including natural ones. This permeation mechanism has considerable physiological significance.

Antidepressant-like effects of P2 purinergic antagonist PPADS is dependent on serotonergic and noradrenergic integrity

Depression is a common mental disorder affecting around 350 million of individuals globally. The available antidepressant drug monotherapy is far from ideal since it has an efficiency of approximately 60% and takes around 3-4 week to achieve clinical improvement. Attention has been paid to the purinergic signaling regarding neuropathological mechanisms, since it might be involved in psychiatric disorders, such as depression. In fact, blockade of purinergic P2X receptors induces antidepressant-like effects in preclinical models. However, the mechanisms involved in this effect are not yet completely understood. The present work investigated the interplay between a P2X receptor antagonist (PPADS) and clinically used antidepressant drugs on the forced swimming test, an animal model predictive of antidepressant effect. We observed significant synergistic effect of PPADS combined with sub-effective doses of fluoxetine or reboxetine in the FST. Moreover, depletion of serotonergic or noradrenergic systems, with PCPA and DSP-4 treatment, respectively, blocked the antidepressant-like effect of PPADS. No increase in locomotion, a possible source of confusion on FST data, was detected in any of the treated groups. Our results indicate the antidepressant-like effect of PPADS depends on the integrity of serotonergic and noradrenergic transmission.