AbstractThe heteromeric Kir4.1–Kir5.1 channel is a candidate sensing molecule for central CO2chemoreception. Since central CO2chemoreception is subject to neural modulations, we performed studies to test the hypothesis that the Kir4.1–Kir5.1 channel is modulated by the neurotransmitters critical for respiratory control, including serotonin (5‐HT), substance‐P (SP), and thyrotropin releasing hormone (TRH). The heteromeric Kir4.1–Kir5.1 channel was strongly inhibited by SP, TRH, and 5‐HT when expressed inXenopusoocytes, whereas these neurotransmitters had no effect on the homomeric Kir4.1 channel. Such an inhibition was dose‐dependent and relied on specific Gαq‐protein‐coupled receptors and protein kinase C (PKC). No direct interaction of the channel with G‐proteins was found. Channel sensitivity to CO2/pH was not compromised with the inhibition by these neurotransmitters, as the channel remained to be inhibited by acidic pH following an exposure to the neurotransmitters. The firing rate of CO2‐sensitive brainstem neurons cultured in microelectrode arrays was augmented by SP or a 5‐HT2A receptor agonist, which was blocked by PKC inhibitors suggesting that PKC underscores the inhibitory effect of SP and 5‐HT in cultured brainstem neurons as well. Immunostaining showed that both Kir4.1 and Kir5.1 proteins were co‐localized in the cultured brainstem neurons. These results therefore indicate that the heteromeric Kir4.1–Kir5.1 channel is modulated by the neurotransmitters critical for respiratory control, suggesting a novel neuromodulatory mechanism for the chemosensitivity of brainstem neurons to elevated PCO2and acidic pH. J. Cell. Physiol. 214:84–95, 2008. © 2007 Wiley‐Liss, Inc.