The Journal of Neuroscience, February 7, 2007, ():
Identification of an Intersubunit Cross-Link between Substituted Cysteine Residues Located in the Putative ATP Binding Site of the P2X1 Receptor
J. Neurosci. Marquez-Klaka et al.
27: 1456
Supplemental Data
Files in this Data Supplement:
- supplemental material
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Supplementary figure 1: Specific inhibition of the intersubunit disulfide bond formation between K68C and F291C substituted residues in P2X1 receptor by ATP. A, Upon reducing treatment of the coexpressed single mutant or the double mutant receptor complexes, spontaneous or H2O2-induced reformation of the disulfide is prevented in the presence of 10 mM (Fig. 3 in the paper) or 100 mM ATP. B, The intersubunit-disulfide bond formation between V48C and I329C substituted residues (Jiang et al. 2003) was neither influenced by 10 mM ATP nor by 10 mM ADP.
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Supplementary figure 2: Comparative analysis of the assembly and glycosylation pattern of the His-tagged P2X1 receptor and wt and single mutated His-tagged P2X2-1 chimeras. After expression and metabolic labeling of the indicated P2X subunits in Xenopus oocytes. P2X complexes were purified via Ni2+-NTA agarose. A, BN-PAGE analysis demonstrates that both P2X1 and P2X2-1 chimeric subunits assemble efficiently into trimeric complexes. B, Deglycosylation analysis reveals a similar pattern of Endo H-sensitive and Endo H-resistant protein fractions and virtually identical size shifts for all three subunits, indicating identical glycosylation patterns in which one of the four N-linked glycosylation sites becomes complex glycosylated (Nicke et al. 1998). Note that the non-mutated chimera was expressed for only one day because its high ATP sensitivity and non-desensitizing properties caused cell death after longer expression.
