Antisense co-suppression of Gαq and Gα11 demonstrates that both isoforms mediate M3-receptor-activated Ca2+ signalling in intact epithelial cells

We used replication-deficient adenoviruses overexpressing antisense against G(q) class alpha-subunits to determine the roles of G(q) and G(11) in mediating M-3-receptor-coupled Ca2+ mobilization in intact HT29 human colonic carcinoma epithelial cells. Western blot analysis and confocal microscopy showed that the viruses expressing antisense directed against the alpha-subunits of G(q) or G(11) produced isoform-specific reductions in the levels of these alpha-subunits. Fura-2 was used to measure changes in the Ca2+ response following activation of the M-3 receptors by carbachol. The G(alphaq) antisense virus suppressed the peak Ca2+ response by 70%, whereas the G(alpha11) antisense virus reduced it by 34%. We then used co-infection with both viruses to determine the effect of concomitant suppression of both G(alphaq) and G(alpha11). Overexpression of antisense to both alpha-subunits reduced by approximately 50% the levels of both G(alphaq) and G(alpha11). It also almost completely inhibited the Ca2+ response to carbachol. These data show that both G(q) and G(11) are involved in mediating the action of the M-3 receptor on cytosolic Ca2+ in HT29 cells. Furthermore, they suggest that the coupling of the M-3 receptor to these G proteins is specific, in that G(alphaq) cannot substitute for G(alpha11), and vice versa.