Phosphorylation of the Kv7.4 B helix reorganizes calmodulin interactions and reduces PIP2 binding
- 1University of Arkansas
Abstract
Voltage-gated Kv7 (KCNQ, M-type) potassium channels regulate cellular excitability through interactions with phosphatidylinositol 4,5-bisphosphate (PIP2;) 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 PIP2;-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 PIP2; binding to both the isolated B helix and CaM-associated Kv7.4 regulatory complexes. Mutation of the distal B-helix KRK motif similarly impaired PIP2; 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 PIP2; 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.
Related
- CD36 phosphorylation alters the thrombospondin binding site and reduces internal cavity accessibility and volume
- Affinity and Conformations of Ca2+-Calmodulin Bound to Peptides Containing the Calmodulin-Binding Motif of HIV-1 Matrix Protein
- Substrate binding reorganizes the energetic landscape of Plasmodium falciparum hexose transporter PfHT1
- Geniposidic Acid Reduces Lipid Accumulation and Is Associated with Altered AMPK Phosphorylation in 3T3-L1 Adipocytes
- Opposing Regulation of Tau Condensation by Phosphorylation Levels and Molecular Chaperone Interactions
BibTeX
@misc{phan2026phosphorylation,
title = {{Phosphorylation of the Kv7.4 B helix reorganizes calmodulin interactions and reduces PIP2 binding}},
author = {P. Phan and C. Okose and D. Sapkota and W. Munyao and D. Girodat and C. Archer},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.18.752758},
url = {https://www.biorxiv.org/content/10.64898/2026.09.18.752758v1},
note = {CC BY},
}
RIS
TY - UNPB
TI - Phosphorylation of the Kv7.4 B helix reorganizes calmodulin interactions and reduces PIP2 binding
AU - P. Phan
AU - C. Okose
AU - D. Sapkota
AU - W. Munyao
AU - D. Girodat
AU - C. Archer
PY - 2026
DA - 2026/09/18
PB - bioRxiv
DO - 10.64898/2026.09.18.752758
UR - https://www.biorxiv.org/content/10.64898/2026.09.18.752758v1
AB - Voltage-gated Kv7 (KCNQ, M-type) potassium channels regulate cellular excitability through interactions with phosphatidylinositol 4,5-bisphosphate (PIP2;) 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 PIP2;-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 PIP2; binding to both the isolated B helix and CaM-associated Kv7.4 regulatory complexes. Mutation of the distal B-helix KRK motif similarly impaired PIP2; 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 PIP2; 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.
ER -