Long QT syndrome (LQTS) is an inherited and often life-threating cardiac disorder characterized by abnormal prolongation of the QT interval on an electrocardiogram (ECG). Affecting approximately 1 in 2000 individuals, LQTS-diagnosed patients are at a greater risk of developing fatal arrhythmic symptoms, typically resulting in cardiac arrest and sudden cardiac death (SCD). Multiple genetic variations in the highly conserved calcium (Ca²⁺) sensing protein Calmodulin (CaM) have recently been identified in human patients exhibiting LQTS phenotypes, highlighting a key role of CaM in the molecular causation of LQTS. CaM is a ubiquitous intracellular protein responsible for regulating the activity of several ion channels involved in cardiac excitation-contraction coupling and cardiac muscle contraction. However, the mechanistic aetiology of CaM-associated LQTS remains unclear and is still under investigation. One of the major cardiac ion channels CaM modulates is the L-type-voltage-gated Ca²⁺ channel, Cav1.2 which contributes to cellular depolarisation and ventricular myocyte contraction. CaM binding domains have been identified at the N-terminus (NSCaTE) and C-terminus (IQ domain) of Cav1.2, allowing for the modulation/regulation of the ion channel. Small synthetic peptides containing CaM recognition sequences were used for the characterisation of CaM-target interactions. The binding parameters of CaM with each Cav1.2 peptides in Ca2+-free (5 mM EGTA) and Ca²⁺-bound (5 mM CaCl₂) conditions were determined using isothermal titration calorimetry (ITC). Secondary protein structure and thermostability details of CaM variants were determined by circular dichroism (CD), whilst proteolytic stability was determined using SDS-PAGE and densitometry analysis. We showed by using ITC that CaM binds to Cav1.2-NSCaTE and Cav1.2-IQ in a Ca²⁺-dependent manner. CaM has a nM range binding affinity for the Cav1.2-IQ domain and a μM range for Cav1.2-NSCaTE (K_d = 2.0 ± 0.19 μM). The binding parameters of the LQTS-associated variants are significantly altered, with a reduction in affinity of up to 3-fold for Cav1.2-NSCaTE, when compared to wild-type. Using CD, we revealed that the missense mutations caused by single amino acid substitutions caused significant changes in the secondary structure content, thermostability and susceptibility to protease digestion for both CaM variants. We present novel data showing that mutations within the highly conserved calcium co-ordinating domains of CaM perturb protein structure and interaction with Cav1.2, resulting in aberrated Cav1.2/CaM complex formation. Here we are beginning to develop a mechanistic insight into the structure-function relationship of CaM-associated LQTS variants and its role in Ca²⁺ signalling dysfunction and arrhythmogenesis.