To examine the role of G(o) in modulation of ion channels by neurotransmitter receptors, we characterized modulation of ionic currents in hippocampal CA3 neurons from mice lacking both isoforms of Galpha(o). In CA3 neurons from Galpha(o)(-/-) mice, 2-chloro-adenosine and the GABA(B)-receptor agonist baclofen activated inwardly rectifying K(+) currents and inhibited voltage-dependent Ca(2+) currents just as effectively as in Galpha(o)(+/+) littermates. However, the kinetics of transmitter action were dramatically altered in Galpha(o)(-/-) mice in that recovery on washout of agonist was much slower. For example, recovery from 2-chloro-adenosine inhibition of calcium current was more than fourfold slower in neurons from Galpha(o)(-/-) mice [time constant of 12.0 +/- 0.8 (SE) s] than in neurons from Galpha(o)(+/+) mice (time constant of 2.6 +/- 0.2 s). Recovery from baclofen effects was affected similarly. In neurons from control mice, effects of both baclofen and 2-chloro-adenosine on Ca(2+) currents and K(+) currents were abolished by brief exposure to external N-ethyl-maleimide (NEM). In neurons lacking Galpha(o), some inhibition of Ca(2+) currents by baclofen remained after NEM treatment, whereas baclofen activation of K(+) currents and both effects of 2-chloro-adenosine were abolished. These results show that modulation of Ca(2+) and K(+) currents by G protein-coupled receptors in hippocampal neurons does not have an absolute requirement for Galpha(o). However, modulation is changed in the absence of Galpha(o) in having much slower recovery kinetics. A likely possibility is that the very abundant Galpha(o) is normally used but, when absent, can readily be replaced by G proteins with different properties.