BPS2026 – Regulation of the KCNQ4 channel via the interplay between phosphorylation, calmodulin, and PIP2

The KCNQ4 potassium channel regulates neuronal excitability in the sensory cells of the inner ear and is implicated in hearing loss. Regulation of KCNQ4 involves the A and B helices at the protein's C terminus (Q4AB) and other mediators such as phosphatidylinositol-4,5-bisphosphate (PIP2), calcium-regulated calmodulin (CaM), and protein kinase C (PKC). However, the precise interplay among Q4AB, CaM, PIP2, and PKC remains poorly understood. We investigated these interactions using molecular dynamics (MD) simulation of the Q4AB-CaM complex and biophysical methods such as microscale thermophoresis (MST). MD simulations revealed that phosphorylation of T552 within Q4AB induced only minor structural changes at the Q4AB-CaM interface. However, phosphorylated T552 alone did not alter Q4AB binding affinity to apoCaM compared to unphosphorylated Q4AB-apoCaM ( K d = 2.0 ± 0.5 μM and 2.1 ± 0.4 μM, respectively). In contrast, mutations in the KRK region of Q4AB, a PIP2-binding site, reduced binding to apoCaM ( K d = 9.0 ± 2.0 μM). Simulations of Q4AB-CaM with a PIP2 analog altered binding interactions at the KRK region. Moreover, phosphorylation at T552 on Q4AB-CaM-PIP2 produced additional structural changes compared to the unphosphorylated system. These findings suggest that phosphorylation alone may not affect apoCaM-Q4AB binding. Instead, the polycationic KRK cluster on Q4AB may serve as the competitive binding site for CaM and PIP2, and may play a role in coordinating the regulation of KCNQ4 activities through CaM, PIP2, and PKC phosphorylation.