Phosphorylation of the Kv7.4 B helix reorganizes calmodulin interactions and reduces PIP ₂ binding

Voltage-gated Kv7 (KCNQ, M-type) potassium channels regulate cellular excitability through interactions with phosphatidylinositol 4,5-bisphosphate (PIP ₂ ) and calmodulin (CaM). The distal B helix of Kv7.2-5 channels contains a conserved protein kinase C (PKC) phosphorylation site, suggesting that phosphorylation may regulate channel activity by altering CaM- and PIP ₂ -dependent mechanisms. Here, we investigated the effects of phosphorylation of Thr552 within the Kv7.4 B helix using electrophysiology, biophysical assays, NMR spectroscopy, and molecular dynamics simulations. Phosphomimetic substitution of Thr552 produced a loss-of-function phenotype characterized by reduced current density and a depolarizing shift in voltage-dependent activation without altering channel surface expression. Biophysical studies demonstrated that phosphorylation produced only modest effects on overall CaM association. However, NMR spectroscopy revealed phosphorylation-dependent changes in apoCaM interactions, and molecular dynamics simulations identified remodeling of the local interaction network surrounding the distal B-helix polybasic “KRK motif” region. In contrast, phosphorylation markedly reduced PIP ₂ binding to both the isolated B helix and CaM-associated Kv7.4 regulatory complexes. Mutation of the distal B-helix KRK motif similarly impaired PIP ₂ binding, identifying this region as an important determinant of lipid recognition. Together, these findings support a model in which phosphorylation of Thr552 suppresses Kv7.4 activity primarily by reducing PIP ₂ interactions while reorganizing, rather than disrupting, CaM binding. These results identify Thr552 as a critical regulatory site linking phosphorylation-dependent signaling to Kv7.4 channel gating.