Recent work has identified missense mutations in calmodulin (CaM) that are associated with severe early-onset long-QT syndrome (LQTS), leading to the proposition that altered CaM function may contribute to the molecular etiology of this subset of LQTS. To date, however, no experimental evidence has established these mutations as directly causative of LQTS substrates, nor have the molecular targets of CaM mutants been identified. Here, therefore, we test whether expression of CaM mutants in adult guinea-pig ventricular myocytes (aGPVM) induces action-potential prolongation, and whether affiliated alterations in the Ca2+ regulation of L-type Ca2+ channels (LTCC) might contribute to such prolongation. In particular, we first overexpressed CaM mutants in aGPVMs, and observed both increased action potential duration (APD) and heightened Ca2+ transients. Next, we demonstrated that all LQTS CaM mutants have the potential to strongly suppress Ca2+/CaM-dependent inactivation (CDI) of LTCCs, whether channels were heterologously expressed in HEK293 cells, or present in native form within myocytes. This attenuation of CDI is predicted to promote action-potential prolongation and boost Ca2+ influx. Finally, we demonstrated how a small fraction of LQTS CaM mutants (as in heterozygous patients) would nonetheless suffice to substantially diminish CDI, and derange electrical and Ca2+ profiles. In all, these results highlight LTCCs as a molecular locus for understanding and treating CaM-related LQTS in this group of patients.

SUMMARY Voltage-gated Na and Ca2+channels comprise distinct ion-channel superfamilies, yet the carboxy tails of these channels exhibit high homology hinting at a long-shared and purposeful module. For different Ca2+ channels, carboxyl-tail inter actions with calmodulin do elaborate robust and similar forms of Ca2+ regulation. However, Na channels have only shown subtler Ca2+modulation that differs among reports, challenging attempts at unified understanding. Here, by rapid Ca2+photoreleaseon to Na channels, we reset this view of Na channel regulation. For cardiac muscle channels (NaV1.5), reported effects from which most mechanistic proposals derive, we observe no Ca2+modulation. Conversely, for skeletal-muscle channels (NaV1.4), we uncover fast Ca2+ regulation eerily similar to that of Ca2+ channels. Channel opathic myotonia mutations halve NaV1.4 Ca2+ regulation, and transplanting the NaV1.4 carboxy tail onto Ca2+ channels recapitulates Ca2+ regulation. Thus we argue for the persistence and physiological relevance of an ancient Ca2+ regulatory module across Na and Ca2+ channels.

Timothy Syndrome (TS) is a multisystem disorder, prominently featuring cardiac action potential prolongation with paroxysms of life-threatening arrhythmias. The underlying defect is a single de novo missense mutation in CaV1.2 channels, either G406R or G402S. Notably, these mutations are often viewed as equivalent, as they produce comparable defects in voltage-dependent inactivation and cause similar manifestations in patients. Yet, their effects on calcium-dependent inactivation (CDI) have remained uncertain. Here, we find a significant defect in CDI in TS channels, and uncover a remarkable divergence in the underlying mechanism for G406R versus G402S variants. Moreover, expression of these TS channels in cultured adult guinea pig myocytes, combined with a quantitative ventricular myocyte model, reveals a threshold behaviour in the induction of arrhythmias due to TS channel expression, suggesting an important therapeutic principle: a small shift in the complement of mutant versus wild-type channels may confer significant clinical improvement.