Arrhythmia-associated calmodulin variants interact with KCNQ1 to confer aberrant membrane trafficking and function.

Missense variants in calmodulin (CaM) predispose patients to arrhythmias associated with high mortality rates ("calmodulinopathy"). As CaM regulates many key cardiac ion channels, an understanding of disease mechanism associated with CaM variant arrhythmias requires elucidating individual CaM variant effects on distinct channels. One key CaM regulatory target is the KCNQ1 (KV7.1) voltage-gated potassium channel that carries the IKs current. Yet, relatively little is known as to how CaM variants interact with KCNQ1 or affect its function. Here, we take a multipronged approach employing a live-cell fluorescence resonance energy transfer binding assay, fluorescence trafficking assay, and functional electrophysiology to characterize >10 arrhythmia-associated CaM variants for effect on KCNQ1 CaM binding, membrane trafficking, and channel function. We identify one variant (G114W) that exhibits severely weakened binding to KCNQ1 but find that most other CaM variants interact with similar binding affinity to KCNQ1 when compared with CaM wild-type over physiological Ca2+ ranges. We further identify several CaM variants that affect KCNQ1 and IKs membrane trafficking and/or baseline current activation kinetics, thereby delineating KCNQ1 dysfunction in calmodulinopathy. Lastly, we identify CaM variants with no effect on KCNQ1 function. This study provides extensive functional data that reveal how CaM variants contribute to creating a proarrhythmic substrate by causing abnormal KCNQ1 membrane trafficking and current conduction. We find that CaM variant regulation of KCNQ1 is not uniform with effects varying from benign to significant loss of function, suggesting how CaM variants predispose patients to arrhythmia via the dysregulation of multiple cardiac ion channels. Classification: Biological, Health, and Medical Sciences, Physiology.

Arrhythmia-associated Calmodulin Variants Interact with KCNQ1 to Confer Aberrant Membrane Trafficking and Function.

Rationale: Missense variants in calmodulin (CaM) predispose patients to arrhythmias associated with high mortality rates. As CaM regulates several key cardiac ion channels, a mechanistic understanding of CaM variant-associated arrhythmias requires elucidating individual CaM variant effect on distinct channels. One key CaM regulatory target is the KCNQ1 (K V 7.1) voltage-gated potassium channel that underlie the I Ks current. Yet, relatively little is known as to how CaM variants interact with KCNQ1 or affect its function. Objective: To observe how arrhythmia-associated CaM variants affect binding to KCNQ1, channel membrane trafficking, and KCNQ1 function. Methods and Results: We combine a live-cell FRET binding assay, fluorescence trafficking assay, and functional electrophysiology to characterize >10 arrhythmia-associated CaM variants effect on KCNQ1. We identify one variant (G114W) that exhibits severely weakened binding to KCNQ1 but find that most other CaM variants interact with similar binding affinity to KCNQ1 when compared to CaM wild-type over physiological Ca 2+ ranges. We further identify several CaM variants that affect KCNQ1 and I Ks membrane trafficking and/or baseline current activation kinetics, thereby contextualizing KCNQ1 dysfunction in calmodulinopathy. Lastly, we delineate CaM variants with no effect on KCNQ1 function. Conclusions: This study provides comprehensive functional data that reveal how CaM variants contribute to creating a pro-arrhythmic substrate by causing abnormal KCNQ1 membrane trafficking and current conduction. We find that CaM variant regulation of KCNQ1 is not uniform with effects varying from benign to significant loss of function. This study provides a new approach to collecting details of CaM binding that are key for understanding how CaM variants predispose patients to arrhythmia via the dysregulation of multiple cardiac ion channels.