Effects of G‐protein‐specific antibodies and Gβγ subunits on the muscarinic receptor‐operated cation current in guinea‐pig ileal smooth muscle cells

Abstract

1 The effects on the whole-cell carbachol-induced muscarinic cationic current (mIcat) of antibodies against the a-subunits of various G proteins, as well as the effect of a Gbg subunit, were studied in single guinea-pig ileal smooth muscle cells voltage-clamped at À50 mV. Ionized intracellular calcium concentration, [Ca 2+ ] i , was clamped at 100 nM using a 1,2-bis(2-aminophenoxyl-ethane-N,N,N 0 ,N 0 -tetraacetic acid)/Ca 2+ mixture. 2 Application of ascending concentrations of carbachol (1 -300 mM) activated mIcat (mean amplitude 0.83 nA at 300 mM carbachol; EC 50 8 mM; Hill slope 1.0). A 20 min or longer intracellular application via the pipette solution of G i3 /G o or G o antibodies resulted in about a 70% depression of the maximum response without change in the EC 50 value. In contrast, antibodies against a-subunits of G i1 , G i1 /G i2 , G i3 , G q /G 11 or G s protein over a similar or longer period did not significantly reduce mIcat. Antibodies to common Gb or infusion of the Gbg subunit itself had no effect on mIcat.3 If cells were exposed briefly to carbachol (50 or 100 mM) at early times (o3 min) after infusion of antibodies to Ga i3 /Ga o or to Ga o had begun, carbachol responses remained unchanged even after 20 -60 min; that is, the depression of mIcat by these antibodies was prevented. 4 These data show that Ga o protein couples the muscarinic receptor to the cationic channel in guinea-pig ileal longitudinal smooth muscle and that Gbg is not involved. They also show that prior activation of the muscarinic receptor presumably causes a long-lasting postactivation change of the G protein, which is not reflected in mIcat, but acts to hinder antibody binding.

Isoform-specific Inhibition of TRPC4 Channel by Phosphatidylinositol 4,5-Bisphosphate

“…This is similar to mI CAT for which internal Ca 2ϩ is also required (36). Furthermore, mI CAT is blocked by PTX treatment (37,38), an effect ascribed to G o protein involvement (39,40). We therefore hypothesized that the failure of carbachol to reliably induce I TRPC4 could be due to the lack of a G o -coupled mAChR in HEK293 cells.…”

Phospholipase C Involvement in Activation of the Muscarinic Receptor-Operated Cationic Current in Guinea Pig Ileal Smooth Muscle Cells

“…In these cell types, the I cat response is abolished by pertussis toxin (PTX) which selectively uncouples G i / G o proteins from the associated receptors (2, 7 -10). With the use of antibodies, the G protein involved in the signal transduction was identified as G o protein (11,12). Based on these results and the general thought that M2 receptors preferencially couple PTX-sensitive G proteins (8), the M2 subtype is supposed to mediate cationic channel opening through G o proteins.…”

“…Similar tests were made for Ga q / Ga 11 -antibody, where cells were dialyzed with the antibody (1:100 v / v) for 18 -20 min in order to allow it to diffuse adequately into the cell (12). With such antibody treatment, carbachol (100 m M) evoked no noticeable I K-Ca , whereas subsequent caffeine did evoke sizable I K-Ca (3.68 ± 0.64 nA, n = 6) (Fig.…”

“…Antibodies against a subunits of G q and G 11 proteins (Ga q / Ga 11 -antibody) were obtained from Perkin-Elmer Life Sciences (NEN) (Boston, MA, USA); each antibody can recognize a specific sequence of the C-terminal of the specific a subunit (12). All other chemicals were obtained from Sigma or Wako.…”

“…DMSO (up to 0.3%), the highest concentration exposed to cells in the present experiments had no effect on the basal current or carbachol-evoked I K-Ca or I cat . GTPgS and Ga q / Ga 11 -antibody were diluted with pipette solution to the respective concentrations 400 m M and 1:100 v / v, included into patch pipettes and intracellularly applied after breakthrough of the patched membrane, as described previously (8,12). Drug concentrations in the text and figures are expressed as final concentrations applied to cells.…”