Long QT syndrome (LQTS) is a major cause of sudden cardiac death, and is characterised by dysfunctional ion channel activity. Genetic screening of clinical populations has identified mutations in the calcium sensor calmodulin (CaM). However, little is currently known about the molecular mechanism of CaM-associated LQTS. A small but ubiquitous protein, CaM is involved in the calcium-dependent regulation of multiple targets such as Cav1.2, where it mediates calcium-dependent inactivation by interaction with CaM binding domains present on the channel, including the N-terminal spatial calcium transforming element (NSCaTE) and the C-terminal IQ domain. Ergo, CaM mutations that affect binding to Cav1.2 may affect channel modulation. We investigated the interaction of clinically relevant CaM mutants D132E, D134H, Q136P and E141G with Cav1.2. Here, we show that these mutations in the fourth EF hand of CaM differentially alter CaM's stability and structure, in addition to its interaction with Cav1.2 CaM binding domains. Circular dichroism experiments reveal alterations to thermal stability and secondary structure content. This is corroborated by HSQC NMR spectra showing differences in conformation which are exacerbated by interaction with NSCaTE or IQ domains. Furthermore, isothermal titration calorimetry demonstrates that in calcium-saturating conditions, Q136P and E141G isoforms have a significantly decreased affinity for NSCaTE (up to 6-fold), whilst D132E, D134H and E141G have an increased affinity for IQ (up to 3-fold). Therefore, mutations have clear but distinct effects on the interaction of CaM with Cav1.2, suggesting differences in the mechanisms underlying the pathophysiology of the disease. This is likely to disrupt channel function, leading to prolonged calcium influx and manifestation of the LQTS phenotype. This work is funded by the Wellcome Trust PhD Studentship (to NG) and the British Heart Foundation Intermediate Basic Science Research Fellowship FS/17/56/32925 (to NH).