Exploring voltage-gated sodium channel conformations and protein-protein interactions using AlphaFold2

Voltage-gated sodium (Na _V ) channels are vital regulators of electrical activity in excitable cells. Given their importance in physiology, Na _V channels are key therapeutic targets for treating numerous conditions, yet developing subtype-selective drugs remains challenging due to the high sequence and structural conservation among Na _V subtypes. Recent advances in cryo-electron microscopy have resolved most human Na _V channels, providing valuable insights into their structure and function. However, limitations persist in fully capturing the complex conformational states that underlie Na _V channel gating and modulation. This study explores the capability of AlphaFold2 to sample multiple Na _V channel conformations and assess AlphaFold Multimer’s accuracy in modeling interactions between the Na _V α-subunit and its protein partners, including auxiliary β-subunits and calmodulin. We enhance conformational sampling to explore Na _V channel conformations using a subsampled multiple sequence alignment approach and varying the number of recycles. Our results demonstrate that AlphaFold2 models multiple Na _V channel conformations, including those observed in experimental structures, states that have not been described experimentally, and potential intermediate states. Correlation and clustering analyses uncover coordinated domain behavior and recurrent state ensembles. Furthermore, AlphaFold Multimer models Na _V complexes with auxiliary β-subunits and calmodulin with high accuracy, and the presence of protein partners significantly alters both the modeled conformational landscape of the Na _V α-subunit and the coupling between its functional states. These findings highlight the potential of deep learning-based methods to expand our understanding of Na _V channel structure, gating, and modulation, while also underscoring the limitations of predicted models that remain hypotheses until validated by experimental data.