Identification by mass spectrometry and functional characterization of two phosphorylation sites of KCNQ2/KCNQ3 channels

Neuronal potassium channel subunits of the KCNQ (Kv7) family underlie M-current (I-M), and may also underlie the slow potassium current at the node of Ranvier, I-Ks. I-M and I-Ks, are outwardly rectifying currents that regulate excitability of neurons and myelinated axons, respectively. Studies of native I-M and heterologously expressed Kv7 subunits suggest that, in vivo, KCNQ channels exist within heterogeneous, multicomponent protein complexes. KCNQ channel properties are regulated by protein phosphorylation, protein-protein interactions, and protein-lipid interactions within such complexes. To better understand the regulation of neuronal KCNQ channels, we searched directly for posttranslational modificationson KCNQ2/KCNQ3 channels in vivo by using mass spectrometry. Here we describe two sites of phosphorylation. One site, specific for KCNQ3, appears functionally silent in electrophysiological assays but is located in a domain previously shown to be important for subunit tetramerization. Mutagenesis and electrophysiological studies of the second site, located in the S4-S5 intracellular loop of all KCNQ subunits, reveal a mechanism of channel inhibition.