Subtype-Specific Mechanisms for Functional Interaction between α6β4* Nicotinic Acetylcholine Receptors and P2X Receptors

Abstract

P2X receptors and nicotinic acetylcholine receptors (nAChRs) display functional and physical interactions in many cell types and heterologous expression systems, but interactions between a6b4-containing (a6b4*) nAChRs and P2X2 receptors and/or P2X3 receptors have not been fully characterized. We measured several types of crosstalk in oocytes coexpressing a6b4 nAChRs and P2X2, P2X3, or P2X2/3 receptors. A novel form of crosstalk occurs between a6b4 nAChRs and P2X2 receptors. P2X2 receptors were forced into a prolonged desensitized state upon activation by ATP through a mechanism that does not depend on the intracellular C terminus of the P2X2 receptors. Coexpression of a6b4 nAChRs with P2X3 receptors shifts the ATP dose-response relation to the right, even in the absence of acetylcholine (ACh). Moreover, currents become nonadditive when ACh and ATP are coapplied, as previously reported for other Cys-loop receptors interacting with P2X receptors, and this crosstalk is dependent on the presence of the P2X3 C-terminal domain. P2X2 receptors also functionally interact with a6b4b3 but through a different mechanism from a6b4. The interaction with P2X3 receptors is less pronounced for the a6b4b3 nAChR than the a6b4 nAChR. We also measured a functional interaction between the a6b4 nAChRs and the heteromeric P2X2/3 receptor. Experiments with the nAChR channel blocker mecamylamine on P2X2-a6b4 oocytes point to the loss of P2X2 channel activity during the crosstalk, whereas the ion channel pores of the P2X receptors were fully functional and unaltered by the receptor interaction for P2X2-a6b4b3, P2X2/3-a6b4, and P2X2/3-a6b4b3. These results may be relevant to dorsal root ganglion cells and to other neurons that coexpress these receptor subunits.

The nicotinic α6 subunit gene determines variability in chronic pain sensitivity via cross-inhibition of P2X2/3 receptors

“…The presence of co-expressed α6* nAChRs changes the dose-response relation of the P2X3 receptor (28). This type of interaction has been previously reported only for the interaction between α3β4 nAChR and P2X2 (22).…”

“…As a result, responses to ATP in the concentration range 10–100 μM are reduced by approximately half to two-thirds, when normalized to maximal responses. These data are summarized in table S4 [see also (28)].…”

“…Other nAChR–P2X receptor interactions increase with the densities of the receptors (21). The details of the contacts, the receptor states involved, and the possible roles of ion flux are not fully known; but modifications to desensitization could play a role (28, 45). Purinergic receptors are important pain processing molecules known to be expressed on nociceptive small diameter neurons in the DRG (46), with important roles having been demonstrated for P2X2/3 (23, 24, 47), P2X4 (48), and P2X7 (49).…”

“…For all cross interaction data on the P2X3(K65A) receptor, co-application of the agonists used the “prolonged plus brief pulse” protocol (28). Averaged ATP-evoked peak current during ACh application ( I ATP *) was subtracted from averaged ATP-evoked current in the absence of ACh ( I ATP ) from the same cell to obtain a Δ*.…”

P2X receptors

“…This calcium entry appears to inhibit AMPA receptor trafficking by a mechanism involving calmodulindependent kinase II and/or a calcium-dependent phosphatase [48]. A wider role for P2X receptors in the operation or trafficking of other ion channels has long been suspected [50][51][52] and is now beginning to be worked out in molecular detail [53][54][55].…”