Reply to Pitt and Lee: Occupancies of Ca²⁺ in complexes of calmodulin with voltage-gated sodium channels

In PNAS, we describe a crystal structure of Ca²⁺-loaded calmodulin (Ca²⁺/CaM) in complex with the C-terminal region (CT) of the voltage-gated sodium channel Na_V1.5 (1). We contrasted our findings with those of Wang et al. (2) for the following reasons: The title and abstract had suggested a structure of Ca²⁺/CaM in complex with Na_V CTs, and the corresponding Protein Data Bank (PDB) files (4JPZ and 4JQ) contain fully occupied Ca²⁺ ions in EF hands 3 and 4, which is not supported by the conformation and electron density. A similar comparison was previously made by Hovey et al. (3), who concluded that the structures by Wang et al. (2) did not support Ca²⁺ binding to the C-lobes.

In response to Pitt and Lee (4), we acknowledge that Wang et al. (2) had also stated that the C-lobes in their structures were not fully occupied with Ca²⁺ and that we fail to convey this message in our manuscript. However, in light of this analysis, as well as by Hovey et al. (3), and Gardill et al. (1), it is our opinion that assigning full occupancy for Ca²⁺ in EF-hands 3 and 4 in the corresponding PDB files is erroneous. Together with a misleading title, this has unfortunately brought confusion to the field. We do want to make it clear that there is no scientific disagreement between our studies. We show that the differences are due to the presence or absence of a fibroblast growth factor homologous factor (FHF), which can allosterically obstruct the binding site for the Ca²⁺/C-lobe we report. Comparing the structures from the 2 studies thus provides mechanistic insights into how FHFs can affect CaM regulation of Na_Vs.

Pitt and Lee (2) claim that we do not acknowledge a previous finding that FHF can change the conformation of the Na_V CT. This statement is wrong, because on several occasions we reference the finding by Gabelli et al. (5), which was the first one to show this effect. For example, in our introduction, we write, “The authors also noted a different relative orientation of the EF-hand domain to the IQ domain depending on the presence of an FHF (33).” We note the efforts by Wang et al. (2), who were the first to describe the presence of a Ca²⁺/N-lobe binding site immediately downstream of the IQ domain. In our manuscript, our goal was to compare this downstream binding site in Na_V1.4 and Na_V1.5, and we concluded that the site is present for Na_V1.5, but not Na_V1.4, a finding also made independently by Yoder et al. (6). We did reference the downstream binding site for the Ca²⁺/N-lobe reported by Wang et al. (2) in the context of the structure, and analyzed the possibility that this site could be compatible with our reported Ca²⁺/C-lobe binding site [see figure 4D in Gardill et al. (1) and the corresponding results section]. We apologize for our neglect to also reference the isothermal titration calorimetry results by Wang et al. (2), which are in agreement with the results of Yoder et al. (6) and our study (1).