It is now clear that ligand-gated ion channels (LGICs) are not “stand alone” functional units, but form complexes with other components, including scaffolding proteins, regulatory proteins and enzymes. Besides their important physiological roles, these modulating proteins are also potential targets for drug discovery. P2X receptors are LGICs for which ATP is the endogenous agonist. Seven P2X subunits have been identified and they form trimers to produce at least twelve different receptor subtypes. Here, the authors combined a genome-wide open reading frame (ORF) collection with high-throughput functional screening, to search for P2X receptor modulators.
They first created a HEK-293 cell line that stably expressed human P2X2 plus P2X3 receptors, then one-by-one, co-expressed each of 17,284 non-redundant ORFs, which represents 90% of the known human protein-coding genes. They then compared the rise in intracellular [Ca2+] induced by the P2X3 agonist α,β-meATP, using a composite score of four measurements; 1) baseline, 2) peak and 3) steady-state [Ca2+], along with 4) time-course of the decay of the peak. The highest scoring ORFs were then co-expressed with P2X3 receptors in Xenopus laevis oocytes and their effects on ATP-induced ion currents determined. This process led to TMEM163 being identified as a modulator of P2X3 receptors.
TMEM163 comprises 289 amino acids, with a molecular weight of 31 kDa and predicted secondary structure of six transmembrane-spanning domains with intracellular NH2– and COOH-termini. When expressed on its own, TMEM163 had no ATP-dependent activity and when co-expressed, had no effect on the activity of AMPA, kainate, muscarinic or P2X2 receptors, nor on endogenous P2Y receptors in HEK-293 cells. It did, however, potentiate ATP-evoked currents mediated by P2X1 and P2X4 receptors and conversely, inhibited currents carried by P2X7 receptors. Thus TMEM163 appears to selectively modulate P2X receptor activity, but in a subtype-specific manner.
More detailed analysis of its actions showed that TMEM163 potentiated P2X3 receptors by shifting the ATP concentration-response curve (CRC) to the left, with a five-fold decrease in the EC50 and slowing the time-course of current decay. Surprisingly, it also substantially reduced the surface expression of P2X3 receptors. In contrast, the inhibitory effects on P2X7 receptors were associated with a decrease in both the potency of ATP and surface expression of the receptor.
Next, the expression and effects of native TM163 was investigated in mice. In situ hybridisation and an anti-TM163 antibody showed TMEM163 to be present in primary cultures of mouse cerebellar neurones. Infection with a TMEM163 shRNA reduced protein expression by almost half and the peak amplitude of ATP-induced ion currents by a third. The effect on P2X3 receptor protein levels was not reported, however. To study its role in vivo, TMEM163 was knocked-out using CRISPR/Cas9 and pain-associated behaviour induced by ATP injection into the hind-paw studied. There was no difference in paw lifting between wild-type and knock-out mice, but freezing behaviour was reduced by about 50%. Notably, and in contrast to the data obtained using recombinant TMEM163, the total amount of P2X3 protein in dorsal root ganglion was unchanged. The ATP CRC was, however, shifted about five-fold to the right by TMEM163 knock-out, consistent with the leftwards shift seen using the recombinant proteins.
By showing that that TMEM163 selectively modulates P2X receptor activity and in a subtype-specific manner, this study greatly improves our understanding of how native P2X receptors function. As such, TMEM163 is potentially a new target for drug discovery.
- Salm EJ, Dunn PJ, Shan L, Yamasaki M, Malewicz NM, Miyazaki T, Park J, Sumioka A, Hamer RRL, He WW, Morimoto-Tomita M, LaMotte RH, Tomita S. (2020). TMEM163 regulates ATP-gated P2X receptor and behaviour. Cell Rep, 31: 107704. PMID: 32492420.