The HOOK region of voltage-gated Ca²⁺ channel β subunits senses and transmits PIP₂ signals to the gate

The β subunit of voltage-gated Ca²⁺ (CaV) channels plays an important role in regulating gating of the a1 pore-forming subunit and its regulation by phosphatidylinositol 4,5-bisphosphate (PIP₂). Subcellular localization of the CaV β subunit is critical for this effect; N-terminal-dependent membrane targeting of the β subunit slows inactivation and decreases PIP2 sensitivity. Here, we provide evidence that the HOOK region of the β subunit plays an important role in the regulation of CaV biophysics. Based on amino acid composition, we broadly divide the HOOK region into three domains: S (polyserine), A (polyacidic), and B (polybasic). We show that a β subunit containing only its A domain in the HOOK region increases inactivation kinetics and channel inhibition by PIP2 depletion, whereas a β subunit with only a B domain decreases these responses. When both the A and B domains are deleted, or when the entire HOOK region is deleted, the responses are elevated. Using a peptide-to-liposome binding assay and confocal microscopy, we find that the B domain of the HOOK region directly interacts with anionic phospholipids via polybasic and two hydrophobic Phe residues. The β2c-short subunit, which lacks an A domain and contains fewer basic amino acids and no Phe residues in the B domain, neither associates with phospholipids nor affects channel gating dynamically. Together, our data suggest that the flexible HOOK region of the β subunit acts as an important regulator of Ca_V channel gating via dynamic electrostatic and hydrophobic interaction with the plasma membrane.