S100A1 competes with calmodulin for calmodulin binding proteins, CaV1.2 and SK2 channels
S100 is a structurally similar but functionally diverse family of Ca2+-binding proteins that is associated with conditions such as Alzheimer's disease and heart disease. S100 proteins, such as S100A1, found primarily in brain and heart, change Ca2+ signaling but lack clearly understood mechanisms at the molecular level. The current studies aim to characterize S100A1's potential regulation of CaM-dependent signaling via competition, using representative CaM target proteins: L-type voltage-gated Ca2+ (CaV1.2) and small conductance Ca2+-dependent K+ type 2 (SK2) channels. S100A1 was observed to bind both the full-length and proximal CaV1.2 C-terminus using GST pulldown and co-immunoprecipitation. Similar to CaM, S100A1 could be labeled with dansyl chloride, providing a new tool for fluorescence binding and competition assays. Binding assays showed that S100A1 and CaM bound the CaV1.2 CaM binding domain (L-CaMBD) at a similar affinity (EC50 = 0.25±0.09 and 0.24±0.06 μM, respectively; p=0.71). Both S100A1 and CaM were equally effective in displacing dansyl-labeled CaM from L-CaMBD, although CaM was more effective than S100A1 in competing with dansyl-labeled S100A1 (IC50 = 0.40±0.14 and 1.13±0.21 μM, respectively; p<0.05). To investigate this further, a shorter synthetic L-CaMBD peptide and SK2 channel partner proteins were also tested. S100A1 bound and competed for this shorter L-CaMBD peptide at a similar affinity to CaM. S100A1 bound the SK2 channel CaM binding domain (SK-CaMBD) at a similar affinity to CaM (EC50 = 0.19±0.03 and 0.15±0.05 μM, respectively; p=0.45). However, S100A1 did not compete as effectively as CaM for SK-CaMBD (IC50 = 1.15±0.17 and 0.10±0.001 μM, respectively; p<0.05). Differences in EC50 and IC50 absolute values are possibly due to dansyl labeling altering the affinity of CaM and/or S100A1 for the target proteins. Nonetheless, these data demonstrate that S100A1 binds the CaM binding domain in both CaV1.2 and SK2 and that S100A1 may effectively compete with CaM for interacting with these CaM target proteins. This suggests a role and potential mechanism for S100A1 modulation of CaM signaling processes in CaV1.2 and SK2 channels, namely by competitive binding, and raises the possibility that S100A1 may modulate other CaM target proteins as well.
Metzler-Wilson, Kristen, "S100A1 competes with calmodulin for calmodulin binding proteins, CaV1.2 and SK2 channels" (2010). ETD Collection for Thomas Jefferson University. AAI3397772.