Binary architecture of the Na_v 1.2-β2 signaling complex

To investigate the mechanisms by which β-subunits influence Nav channel function, we solved the crystal structure of the β2 extracellular domain at 1.35Å.We combined these data with known bacterial Nav channel structural insights and novel functional studies to determine the interactions of specific residues in β2 with Na_v1.2. We identified a flexible loop formed by⁷²Cys and⁷⁵Cys, a unique feature among the four b-subunit isoforms. Moreover, we found that⁵⁵Cys helps to determine the influence of β2 on Na_v1.2 toxin susceptibility. Further mutagenesis combined with the use of spider toxins reveals that⁵⁵Cys forms a disulfide bond with⁹¹⁰Cys in the Na_v1.2 domain II pore loop, thereby suggesting a 1:1 stoichiometry. Our results also provide clues as to which disulfide bonds are formed between adjacent Na_v1.2^912/918Cys residues. The concepts emerging from this work will help to form a model reflecting the βc-subunit location in a Na_v channel complex.