Abstract
The human P2X1 receptor (hP2X1R) is a trimeric ligand-gated ion channel opened by extracellular ATP. The intracellular amino and carboxyl termini play significant roles in determining the time-course and regulation of channel gating-for example, the C terminus regulates recovery from the desensitized state following agonist washout. This suggests that the intracellular regions of the channel have distinct structural features. Studies on the hP2X3R have shown that the intracellular regions associate to form a cytoplasmic cap in the open state of the channel. However, intracellular features could not be resolved in the agonist-free apo and ATP-bound desensitized structures. Here we investigate the organization of the intracellular regions of hP2X1R in the apo and ATP-bound desensitized states following expression in HEK293 cells. We couple cysteine scanning mutagenesis of residues R25-G30 and H355-R360 with the use of bi-functional cysteine reactive cross-linking compounds of different lengths (MTS-2-MTS, BMB, and BM(PEG)2), which we use as molecular calipers. If two cysteine residues come into close proximity, we predict they will be cross-linked and result in ∼66% of the receptor subunits running on a Western blot as dimers. In the control construct (C349A) that removed the free cysteine C349, and some cysteine-containing mutants, cross-linker treatment does not result in dimerization. However, we detect efficient dimerization for R25C, G30C, P358C, K359C, and R360C. This selective pattern indicates that there is structural organization to these regions in the apo and desensitized states in a native membrane environment. The existence of such precap (apo) and postcap (desensitized) organization of the intracellular domains would facilitate efficient gating of the channel.
Highlights
Extracellular ATP acting at cell surface P2X receptors (P2XRs) plays an important role in a variety of physiological and pathophysiological conditions (Kaczmarek-Hájek et al, 2012)
At the C349A human P2X1 receptor (hP2X1R) mutant following treatment with the crosslinkers MTS-2-MTS, BMB, or BM(PEG)2 (300 μM for 30 min), there was no change in the proportion of the receptor on the gel running as a dimer (2.1 ± 0.86, 0.78 ± 0.41, 0.8 ± 0.54, and 0.6 ± 0.19% for apyrase control, MTS-2-MTS, BMB, and BM(PEG)2 respectively; Fig. 1)
These results suggest that the cross-linker interferes with these interactions to inhibit function of the hP2X1R C349A receptor, and so it was not possible to interpret whether the cross-linkers have an additional effect on the G30C C349A mutant
Summary
Extracellular ATP acting at cell surface P2X receptors (P2XRs) plays an important role in a variety of physiological and pathophysiological conditions (Kaczmarek-Hájek et al, 2012). The P2XR subunits assemble to form homo- and heterotrimeric channels often with distinct properties in terms of agonist sensitivity and/or the time-course of the response. P2X1 and P2X3Rs have EC50 values of ∼1 μM ATP, and evoked currents decay rapidly (
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