Two-pore potassium ion channels (K_2Ps) are a sub-group of the potassium ion channel superfamily which are proposed to contribute to the setting and modulation of the neuronal resting membrane potential (Goldstein et al. 2001). We have shown previously that the group III metabotropic glutamate receptor subtype 4 (mGlu4) induces robust potentiation of the K_2P2.1 (TREK-1) current in expression systems and cultured striatal neurons, via a cAMP dependant mechanism (Cain et al. 2005). In the present study, we have investigated the potential phosphorylation sites involved in this potentiation. Perforated patch whole cell recordings were made from Chinese hamster ovary (CHO) cells stably transfected with human mGlu4 cDNA and transiently transfected with human K_2P2.1 or K_2P2.1 mutant channel cDNA. Cells were held at -70mV and voltage ramps (-120mV to 40mV, 500ms) were applied every 30s. All drugs were added via the perfusate. Data are expressed as mean ± S.E.M. and statistical significance determined using Student’s paired t tests, n = number of neurons. In CHO cells co-expressing wild-type K_2P2.1 (K_2P2.1_WT) and mGlu4, the mGlu group III selective agonist L-(+)-2-amino-4-phosphonobutyric acid (L-AP4; 10μM) induced potentiations of 65.1 ± 14.3% (n=11; p<0.05). Mutation of K_2P2.1 serine 333 (reported as a protein kinase A phosphorylation site) to alanine (K_2P2.1_S333A) significantly attenuated L-AP4 induced potentiations to 11.9 ± 3.6% (n=7; p<0.01), whilst mutation of K_2P2.1 serine 300 (reported as a protein kinase A/C phosphorylation site) to alanine (K_2P2.1_S300A) significantly attenuated L-AP4 induced potentiations of the K_2P2.1 current to 34.2 ± 5.2% (n=4; p<0.05). Furthermore, following mutation of both S333 and S300 to alanine (K_2P2.1_S333A/S300A) L-AP4 induced potentiations were completely abolished (2.7 ± 2.9%; n=4; p>0.05). Finally, mutating K_2P2.1 serine 351 (reported as a protein kinase G phosphorylation site) to alanine (K_2P2.1_S351A), resulted in L-AP4 induced potentiations of 75.2 ± 15.6% (n=4;) which were not significantly different from controls (p>0.05). These data indicate that the robust potentiation of K_2P2.1 by activation of mGlu4 is mediated via modulation of the PKA pathway with both S300 and S333 playing a significant role. However, modulation of the PKG pathway does not appear to be involved.Thus, our increased understanding of the second messenger systems underlying this modulation may help elucidate the role of receptor induced activation of K2Ps in controlling neuronal excitability.