We have previously demonstrated in HT29 human colonic epithelial cells that muscarinic M₃ signals are transduced by G protein βγ-subunits and that purinergic P_2U signals are transduced by α-subunits. We have also observed that the carbachol (CCh) (M₃) response displays a sustained plateau, while the UTP (P_2U) response does not, and that infection with a replication-deficient adenovirus expressing the α-subunit of G_q causes the UTP response to become morphologically similar to the CCh response (Cummins et al. 2000).

In the present study, we investigated Ca²⁺ influx following M₃ or P_2U activation in HT29 cells using fura-2 Ca²⁺ imaging and the Mn²⁺ quench technique. Extracellular Mn²⁺ enters the cell via Ca²⁺ channels and quenches fura-2 fluorescence measured at 360 nm. The rate of fura-2 quenching is proportional to the rate of Ca²⁺ influx. Mn²⁺ (100 µM) was added to the perfusion solution after 5 min of a 10 min exposure to either CCh (100 µM) or UTP (100 µM). Results are given as rate of fura-2 quenching min^-1 ± S.E.M. (n).

The rate of Mn²⁺-mediated fura-2 quenching after 5 min of agonist exposure was significantly (P < 0.05, Student’s t test) higher for CCh (-1.97 ± 0.15 min^-1 (10)) than for UTP (-1.42 ± 0.07 min^-1 (10)). Infection with the G_αq virus had no effect on the plateau quenching rate during CCh exposure (-1.89 ± 0.21 min^-1 (10)), but significantly increased the quenching rate during UTP exposure (-2.07 ± 0.21 min^-1 (10)), which reached a level not different from the quenching rate for CCh in uninfected cells (Fig. 1).

To determine if this was a specific G_αq effect, we measured quenching rates during the plateau phase in cells infected with adenoviruses expressing G_α11 or G_αi2. These viruses increased the quenching rates during the UTP response to the level observed with CCh, suggesting that the G protein α-subunits may be acting as non-specific scavengers of βγ-subunits.

To confirm this, we infected cells with an adenovirus expressing the C-terminal of the β-adrenergic receptor kinase (βARK-ct), a specific βγ-subunit scavenger. Infection with the βARK-ct virus increased plateau quenching rates during UTP exposure to that observed with CCh. Co-expression of βARK-ct and G_αq did not produce an additive effect, confirming that these proteins are acting via a common βγ-scavenging mechanism.

Thus we have found that scavenging of G protein βγ-subunits increases Ca²⁺ influx during the plateau phase of the UTP response. We conclude that G protein βγ-subunits inhibit plateau phase Ca²⁺ influx following P_2U activation.

Cummins, M.M., O’Mullane, L.M., Barden, J.A., Cook, D.I. & Poronnik, P. (2000). Cell Calcium 27, 247-255.