P2X₇ R578Q displays defective oligomerization.

<p><b>A,</b> HEK293 cells were treated with the cell-permeable chemical cross-linker DSS to demonstrate that intracellular P2X₇ R578Q does not oligomerize as efficiently as wild-type receptor. The samples were blotted with anti-vinculin antibody as a loading control. The average difference in the amount of P2X₇ wild-type trimers that formed following treatment with DSS was calculated and compared to the amount of P2X₇ R578Q trimers that formed. A Student’s <i>t</i>-test was performed to determine statistical significance. These data are representative of five experiments. <b>B,</b> Proteins from HEK293 lysates were cross-linked with BS³, and the difference in the amount of P2X₇ wild-type trimers that formed upon cross-linking with BS³ was compared to the amount of P2X₇ R578Q trimers that formed. A Student’s <i>t</i>-test was performed to determine statistical significance. These data are representative of five experiments. <b>C,D,</b> HEK293 cells were incubated in the presence of DSS or BS³, respectively, or the appropriate vehicle control. The cells were stained with trypan blue and counted to determine the percent viability. The error bars represent the standard error of the mean. These data are representative of three experiments. <b>E,</b> Human monocytes were treated with BS³ to demonstrate that upon cross-linking, endogenous P2X₇ displays a banding pattern that is consistent with dimer and trimer formation. These data are representative of four experiments. <b>F,</b> Proteins from untreated lysates were resolved on 12.5% SDS-polyacrylamide gels to demonstrate the apparent size difference between P2X₇ wild-type and R578Q. These data are representative of at least fifteen experiments. <b>G,</b> The molecular masses of the P2X₇ wild-type and R578Q monomers and their putative dimers and trimers were extrapolated using the distance the molecular weight standards migrated. The results were averaged and the standard error of the mean was determined. Seven replicates were used to calculate the average mass of the monomers, and three replicates were used to calculate the mass of the dimers and trimers.