Structures of human pannexin 1 reveal ion pathways and mechanism of gating.

Pannexin 1 (PANX1) is an ATP-permeable channel with critical roles in a variety of physiological functions such as blood pressure regulation1, apoptotic cell clearance2 and human oocyte development3. Here we present several structures of human PANX1 in a heptameric assembly at resolutions of up to 2.8 angström, including an apo state, a caspase-7-cleaved state and a carbenoxolone-bound state. We reveal a gating mechanism that involves two ion-conducting pathways. Under normal cellular conditions, the intracellular entry of the wide main pore is physically plugged by the C-terminal tail. Small anions are conducted through narrow tunnels in the intracellular domain. These tunnels connect to the main pore and are gated by a long linker between the N-terminal helix and the first transmembrane helix. During apoptosis, the C-terminal tail is cleaved by caspase, allowing the release of ATP through the main pore. We identified a carbenoxolone-binding site embraced by W74 in the extracellular entrance and a role for carbenoxolone as a channel blocker. We identified a gap-junction-like structure using a glycosylation-deficient mutant, N255A. Our studies provide a solid foundation for understanding the molecular mechanisms underlying the channel gating and inhibition of PANX1 and related large-pore channels.

Role of pannexin-1 in the cellular uptake, release and hydrolysis of anandamide by T84 colon cancer cells.

The large pore ion channel pannexin-1 (Panx1) has been reported to play a role in the cellular uptake and release of anandamide (AEA) in the hippocampus. It is not known whether this is a general mechanism or limited to the hippocampus. We have investigated this pharmacologically using T84 colon cancer cells. The cells expressed Panx1 at the mRNA level, and released ATP in a manner that could be reduced by treatment with the Panx1 inhibitors carbenoxolone and mefloquine and the Panx1 substrate SR101. However, no significant effects of these compounds upon the uptake or hydrolysis of exogenously applied AEA was seen. Uptake by T84 cells of the other main endocannabinoid 2-arachidonoylglycerol and the AEA homologue palmitoylethanolamide was similarly not affected by carbenoxolone or mefloquine. Total release of tritium from [3H]AEA-prelabelled T84 cells over 10 min was increased, rather than inhibited by carbenoxolone and mefloquine. Finally, AEA uptake by PC3 prostate cancer and SH-SY5Y neuroblastoma cells, which express functional Panx1 channels, was not inhibited by carbenoxolone. Thus, in contrast to the hippocampus, Panx1 does not appear to play a role in AEA uptake and release from the cells studied under the conditions used.

Involvement of aberrant calcium signalling in herpetic neuralgia.

Alpha-herpesviruses, herpes simplex viruses (HSV) and varicella zoster virus (VZV), are pathogens of the peripheral nervous system. After primary infection, these viruses establish latency within sensory ganglia, while retaining the ability to reactivate. Reactivation of VZV results in herpes zoster, a condition characterized by skin lesions that leads to post-herpetic neuralgia. Recurrent reactivations of HSV, which cause mucocutaneous lesions, may also result in neuralgia. During reactivation of alpha-herpesviruses, satellite glial cells (SGCs), which surround neurons in sensory ganglia, become infected with the replicating virus. SGCs are known to contribute to neuropathic pain in a variety of animal pain models. Here we investigated how infection of short-term cultures of mouse trigeminal ganglia with HSV-1 affects communication between SGCs and neurons, and how this altered communication may increase neuronal excitability, thus contributing to herpetic neuralgia. Mechanical stimulation of single neurons or SGCs resulted in intercellular calcium waves, which were larger in cultures infected with HSV-1. Two differences were observed between control and HSV-1 infected cultures that could account for this augmentation. Firstly, HSV-1 infection induced cell fusion among SGCs and neurons, which would facilitate the spread of calcium signals over farther distances. Secondly, using calcium imaging and intracellular electrical recordings, we found that neurons in the HSV-1 infected cultures exhibited augmented influx of calcium upon depolarization. These virally induced changes may not only cause more neurons in the sensory ganglia to fire action potentials, but may also increase neurotransmitter release at the presynaptic terminals in the spinal cord. They are therefore likely to be contributing factors to herpetic neuralgia.

Multilayer spheroids to quantify drug uptake and diffusion in 3D.

There is a need for new quantitative in vitro models of drug uptake and diffusion to help assess drug toxicity/efficacy as well as new more predictive models for drug discovery. We report a three-dimensional (3D) multilayer spheroid model and a new algorithm to quantitatively study uptake and inward diffusion of fluorescent calcein via gap junction intercellular communication (GJIC). When incubated with calcein-AM, a substrate of the efflux transporter P-glycoprotein (Pgp), spheroids from a variety of cell types accumulated calcein over time. Accumulation decreased in spheroids overexpressing Pgp (HEK-MDR) and was increased in the presence of Pgp inhibitors (verapamil, loperamide, cyclosporin A). Inward diffusion of calcein was negligible in spheroids that lacked GJIC (OVCAR-3, SK-OV-3) and was reduced in the presence of an inhibitor of GJIC (carbenoxolone). In addition to inhibiting Pgp, verapamil and loperamide, but not cyclosporin A, inhibited inward diffusion of calcein, suggesting that they also inhibit GJIC. The dose response curves of verapamil's inhibition of Pgp and GJIC were similar (IC50: 8 µM). The method is amenable to many different cell types and may serve as a quantitative 3D model that more accurately replicates in vivo barriers to drug uptake and diffusion.

Cell density-dependent changes in intracellular Ca2+ mobilization via the P2Y2 receptor in rat bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) are an interesting subject of research because they have characteristics of mesenchymal stem cells. We investigated intracellular Ca(2+) signaling in rat BMSCs. Agonists for purinergic receptors increased intracellular Ca(2+) levels ([Ca(2+)](i)). The order of potency followed ATP = UTP > ADP = UDP. ATP-induced rise in [Ca(2+)](i) was suppressed by U73122 and suramin, but not by pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS), suggesting the functional expression of G protein-coupled P2Y(2) receptors. RT-PCR and immunohistochemical studies also showed the expression of P2Y(2) receptors. [Ca(2+)](i) response to UTP changed with cell density. The UTP-induced rise in [Ca(2+)](i) was greatest at high density. V(max) (maximum Ca(2+) response) and EC(50) (agonist concentration that evokes 50% of V(max)) suggest that the amount and property of P2Y(2) receptors were changed by cell density. Note that UTP induced Ca(2+) oscillation at only medium cell density. Pharmacological studies indicated that UTP-induced Ca(2+) oscillation required Ca(2+) influx by store-operated Ca(2+) entry. Carbenoxolone, a gap junction blocker, enhanced Ca(2+) oscillation. Immunohistochemical and quantitative real-time PCR studies revealed that proliferating cell nuclear antigen (PCNA)-positive cells declined but the mRNA expression level of the P2Y(2) receptor increased as cell density increased. Co-application of fetal calf serum with UTP induced Ca(2+) oscillation at high cell density. These results suggest that the different patterns observed for [Ca(2+)](i) mobilization with respect to cell density may be associated with cell cycle progression.

Pan1b (17betaHSD11)-enzymatic activity and distribution in the lung.

We describe a new member of the 17beta-hydroxysteroid dehydrogenase group of enzymes. Human Pan1b displays greatest activity with 5alpha-androstan-3alpha,17beta-diol (3alpha-Diol) as substrate, suggesting that it may be important in androgen metabolism. Enzymic activity was non-saturable with 3alpha-Diol but saturable with retinoids, although retinoids were not metabolized. Immunohistochemical studies on 10% formalin fixed and paraffin embedded sections of human tissues showed that Pan1b was present in acini and ciliated epithelia of the lung. In the fetus immuno reactivity was present in ciliated epithelia throughout gestation and staining appeared to be stronger in the second half of pregnancy. Pan1b was also expressed in the nonpigmented epithelium of the ciliary body, and in adrenocortical tumor cells. Although 3alpha-Diol is generally considered a degradation product of androgen metabolism it could have its own biological function. Pan1b may be an important modulator of the endocrine, or intracrine activity of this steroid.