Homodimeric anoctamin-1, but not homodimeric anoctamin-6, is activated by calcium increases mediated by the P2Y1 and P2X7 receptors

Abstract

The P2X7 receptor (P2X7R) is a ligand-gated ion channel that conducts Na(+), K(+), and Ca(2+) when activated by extracellular ATP. In various cell types, such as secretory epithelia, the P2X7R is co-expressed with Ca(2+)-dependent Cl(-) channels of the TMEM16/anoctamin family. Here, we studied whether the P2X7R and TMEM16A/anoctamin-1 (Ano1) or TMEM16F/anoctamin-6 (Ano6) interact functionally and physically, using oocytes of Xenopus laevis and Ambystoma mexicanum (Axolotl) for heterologous expression. As a control, we co-expressed anoctamin-1 with the P2Y1 receptor (P2Y1R), which induces the release of Ca(2+) from intracellular stores via activating phospholipase C through coupling to Gαq. We found that co-expression of anoctamin-1 with the P2Y1R resulted in a small transient increase in Cl(-) conductance in response to ATP. Co-expression of anoctamin-1 with the P2X7R resulted in a large sustained increase in Cl(-) conductance via Ca(2+) influx through the ATP-opened P2X7R in Xenopus and in Axolotl oocytes, which lack endogenous Ca(2+)-dependent Cl(-) channels. P2Y1R- or P2X7R-mediated stimulation of Ano1 was primarily functional, as demonstrated by the absence of a physically stable interaction between Ano1 and the P2X7R. In the pancreatic cell line AsPC-1, we found the same functional Ca(2+)-dependent interaction of P2X7R and Ano1. The P2X7R-mediated sustained activation of Ano1 may be physiologically relevant to the time course of stimulus-secretion coupling in secretory epithelia. No such increase in Cl(-) conductance could be elicited by activating the P2X7 receptor in either Xenopus oocytes or Axolotl oocytes co-expressing Ano6. The lack of function of Ano6 can, at least in part, be explained by its poor cell-surface expression, resulting from a relatively inefficient exit of the homodimeric Ano6 from the endoplasmic reticulum.

Interaction of Purinergic P2X4 and P2X7 Receptor Subunits

“…hP2X4-dependent currents displayed the typical inactivating behavior, leading to a peak current at about 1 s after ATP application. In contrast, currents in hP2X7-expressing oocytes showed the typical slowly activating non-saturating behavior during the 12 s of ATP application (Klapperstück et al, 2001;Stolz et al, 2015). In hP2X4/hP2X7-coexpressing oocytes, a peak ATP-induced FIGURE 7 | Effect of ivermectin on ATP-induced currents of hP2X4/hP2X7-coexpressing oocytes.…”

“…We performed the electrophysiological experiments at room temperature (∼22 • C) as previously described (Stolz et al, 2015). We accomplished rapid, reproducible solution exchange by using a small tube-like chamber (0.1 ml) combined with fast superfusion (75 µl/s).…”

“…We synthesized capped cRNA using a modified method (Klapperstück et al, 2000) involving co-transcriptional incorporation of the anti-reverse cap analog (m 2 7,3 −O GpppG; NU-855; Jena Bioscience, Germany) to ensure the correct orientation at the ATG start codon of the cRNA (GrudzienNogalska et al, 2007;Stolz et al, 2015). We surgically isolated oocytes from tricaine-anesthetized Xenopus laevis (Xenopus Express, Vernassal, France) using sterile surgical techniques and defolliculated them with collagenase NB 4G (Serva, Heidelberg, Germany).…”

“…This was subcloned into a Gateway-compatible pNKS2 vector (Stolz et al, 2015) using the Gateway cloning system (Invitrogen, Karlsruhe, Germany). We previously verified by blue native PAGE that ectopic GluCl cryst efficiently assembles into a homopentamer in Xenopus laevis oocytes (Dopychai et al, 2015).…”

Localization of the gate and selectivity filter of the full-length P2X7 receptor

“…SI Appendix, SI Materials and Methods provides the reagents (40)(41)(42) and the methods, including the oocyte preparation (33); labeling and immunoprecipitation of hP2X7R (41,43,44); recordings of macroscopic and singlechannel currents by TEVC (33) and patch clamping, respectively (17); patch noise analysis (31,32); and homology-modeling (45) …”