KCNQ subunits encode for the M current (I-KM), a neuron-specific voltage-dependent K+ current with a well established role in the control of neuronal excitability. In this study, by means of a combined biochemical, pharmacological, and electrophysiological approach, the role of presynaptic IKM in the release of previously taken up tritiated norepineprine (NE), GABA, and D-aspartate (D-ASP) from hippocampal nerve terminals (synaptosomes) has been evaluated. Retigabine (RT) (0.01-30 muM), a specific activator of IKM, inhibited [H-3]NE, [H-3]D-ASP, and [H-3]GABA release evoked by 9 mM extracellular K+ ([K+](e)). RT-induced inhibition of [H-3]NE release was prevented by synaptosomal entrapment of polyclonal antibodies directed against KCNQ2 subunits, an effect that was abolished by antibody preabsorption with the KCNQ2 immunizing peptide; antibodies against KCNQ3 subunits were ineffective. Flupirtine (FP), a structural analog of RT, also inhibited 9 mM [K+](e)-induced [H-3]NE release, although its maximal inhibition was lower than that of RT. Electrophysiological studies in KCNQ2-transfected Chinese hamster ovary cells revealed that RT and FP (10 muM) caused a -19 and -9 mV hyperpolarizing shift, respectively, in the voltage dependence of activation of KCNQ2 K+ channels. In the same cells, the cognition enhancer 10,10-bis(4-pyridinylmethyl)-9(10H)-anthracenone (XE-991) (10 muM) blocked KCNQ2 channels and prevented their activation by RT (1-10 muM). Finally, both XE-991 (10-100 muM) and tetraethylammonium ions (100 muM) abolished the inhibitory effect of RT (1 muM) on [H-3]NE release. These findings provide novel evidence for a major regulatory role of KCNQ2 K+ channel subunits in neurotransmitter release from rat hippocampal nerve endings.