Effect of halothane on the guanosine 5′ triphosphate binding activity of G-protein α_i subunits

Background: Receptor-mediated increases in the force produced by airway smooth muscle are attenuated by anesthetics such as halothane. Guanosine 5′-triphosphate (GTP) binding protein α subunits (Gα_i) are known to participate in the regulation of force in airway smooth muscle. The authors hypothesized that halothane would inhibit the ability of Gα_i subunits to bind a nonhydrolyzable analog of GTP (GTPγS). Methods: The effect of halothane on both GTPase-specific activity and [³⁵S]GTPγS binding were assayed using purified, recombinant Gα_i1. In separate experiments, [³⁵S]GTPγS binding to Gα_i in crude airway smooth muscle membrane preparations was assayed using an immunoprecipitation technique in the presence and absence of halothane. Results: The steady state GTPase-specific activity of the recombinant Gα_i1 was 0.033 ± 0.018 (mean ± SD) mole P_i mole Gα_i1^-1 min^-1 under control conditions and 0.035 ± 0.015 mole P_i mole Gα_i1^-1 min^-1 in the presence of 1.1 ± 0.2 mM halothane, a difference that is not significant. The mole fractions of recombinant Gα_i1 bound to [³⁵S]GTPγS were 0.49 ± 0.02 and 0.60 ± 0.02 at 10 and 20 min, respectively. The addition of halothane (1.26 ± 0.07 mM) did not significantly change these values. Halothane did not affect the binding of [³⁵S]GTPγS to Gα_i subunits in membrane fractions of airway smooth muscle as measured using immunoprecipitation. Validity of the assays was confirmed using suramin, an inhibitor of GTP binding. Conclusion: These results suggest that halothane, which inhibits receptor-activated Gα_i-coupled pathways in intact airway smooth muscle, must functionally target a component of the G protein-coupled receptor complex other than Gα_i.