In the bronchioles of the asthmatic lung, hypersensitivity of airway smooth muscle cells (ASMCs) to bronchoconstrictor agonists contributes to airway obstruction, though the mechanisms underlying this pathology remain obscure. Our laboratory recently found that Kv7 potassium channels are expressed in ASMCs. These channels maintain an outward flux of potassium ions (K+) to promote a negative resting membrane voltage in smooth muscle cells, thereby opposing their electrical excitability. In the bronchioles, electrical excitation (membrane depolarization) of ASMCs results in activation of voltage sensitive calcium channels, an increase in cytosolic calcium concentration, ASMC contraction, and bronchoconstriction. We previously found that the selective Kv7 channel blocker, XE991, induced robust bronchiolar constriction in human precision cut lung slices (PCLS). Bronchoconstrictor agonists histamine (Hist) and methacholine (MeCh) were found to inhibit Kv7 K+ currents in guinea pig bronchiolar ASMCs, but the mechanisms by which these agonists regulate Kv7 channels in ASMCs are unknown. In preliminary studies, we found that knocking down Kv7.5 in rat ASMCs resulted in elimination of Hist-sensitive Kv7 currents. In the present study, we tested the hypothesis that protein kinase C (PKC), a downstream effector in Hist and MeCh signaling, phosphorylates and consequently inhibits Kv7.5 channels. To detect phosphorylation of Kv7.5 channels, FLAG-tagged human Kv7.5 (FLAG-hKv7.5) channels were overexpressed in primary cultured human ASMCs derived from trachealis muscle (hASMCs). Following treatment with Hist (1 µM, 10 minutes) or the PKC activator phorbol 12-myristate 13-acetate (PMA) (1 nM, 30 min), channel phosphorylation was monitored using immunoprecipitation with anti-FLAG (n=6) or anti-phosphoserine PKC substrate (n=7) antibodies followed by western blot analyses. Both treatments induced an increase in Kv7.5 phosphorylation. Similar results were obtained using fresh human trachealis strips, in which native Kv7.5 channels were increased in anti-phosphoserine immunoprecipitates following MeCh, Hist or PMA treatment. Hist-induced phosphorylation of FLAG-hKv7.5 was blocked by the PKC inhibitor Ro 31-8220 (1 µM, 1 hour, n=3). hASMCs infected with FLAG-hKv7.5 were also subjected to patch-clamp electrophysiology to monitor the Kv7.5 K+ currents. Treatment with either Hist (1 µM, n=4/6) or PMA (1 nM, n=2) inhibited Kv7.5 currents. Hist-induced inhibition was attenuated upon pretreatment with the PKC inhibitor Calphostin C (250nM, 30 min). PMA (100 nM) also induced profound bronchoconstriction in human PCLS (n=2). These findings suggest that PKC induces phosphorylation of Kv7.5 potassium channels in ASM, resulting in channel inhibition and contributing to agonist induced bronchoconstriction.