Impaired CaV1.2 inactivation reduces the efficacy of calcium channel blockers in the treatment of LQT8.

Mutations in the CaV1.2 L-type calcium channel can cause a profound form of long-QT syndrome known as long-QT type 8 (LQT8), which results in cardiac arrhythmias that are often fatal in early childhood. A growing number of such pathogenic mutations in CaV1.2 have been identified, increasing the need for targeted therapies. As many of these mutations reduce channel inactivation; resulting in excess Ca2+ entry during the action potential, calcium channel blockers (CCBs) would seem to represent a promising treatment option. Yet CCBs have been unsuccessful in the treatment of LQT8. Here, we demonstrate that this lack of efficacy likely stems from the impact of the mutations on CaV1.2 channel inactivation. As CCBs are known to preferentially bind to the inactivated state of the channel, mutation-dependent deficits in inactivation result in a decrease in use-dependent block of the mutant channel. Further, application of the CCB verapamil to induced pluripotent stem cell (iPSC) derived cardiomyocytes from an LQT8 patient demonstrates that this loss of use-dependent block translates to a lack of efficacy in correcting the LQT phenotype. As a growing number of channelopathic mutations demonstrate effects on channel inactivation, reliance on state-dependent blockers may leave a growing population of patients without a viable treatment option. This biophysical understanding of the interplay between inactivation deficits and state-dependent block may provide a new avenue to guide the development of improved therapies.

CaV1.2 channelopathic mutations evoke diverse pathophysiological mechanisms.

The first pathogenic mutation in CaV1.2 was identified in 2004 and was shown to cause a severe multisystem disorder known as Timothy syndrome (TS). The mutation was localized to the distal S6 region of the channel, a region known to play a major role in channel activation. TS patients suffer from life-threatening cardiac symptoms as well as significant neurodevelopmental deficits, including autism spectrum disorder (ASD). Since this discovery, the number and variety of mutations identified in CaV1.2 have grown tremendously, and the distal S6 regions remain a frequent locus for many of these mutations. While the majority of patients harboring these mutations exhibit cardiac symptoms that can be well explained by known pathogenic mechanisms, the same cannot be said for the ASD or neurodevelopmental phenotypes seen in some patients, indicating a gap in our understanding of the pathogenesis of CaV1.2 channelopathies. Here, we use whole-cell patch clamp, quantitative Ca2+ imaging, and single channel recordings to expand the known mechanisms underlying the pathogenesis of CaV1.2 channelopathies. Specifically, we find that mutations within the S6 region can exert independent and separable effects on activation, voltage-dependent inactivation (VDI), and Ca2+-dependent inactivation (CDI). Moreover, the mechanisms underlying the CDI effects of these mutations are varied and include altered channel opening and possible disruption of CDI transduction. Overall, these results provide a structure-function framework to conceptualize the role of S6 mutations in pathophysiology and offer insight into the biophysical defects associated with distinct clinical manifestations.

Allosteric regulators selectively prevent Ca2+-feedback of CaV and NaV channels.

Calmodulin (CaM) serves as a pervasive regulatory subunit of CaV1, CaV2, and NaV1 channels, exploiting a functionally conserved carboxy-tail element to afford dynamic Ca2+-feedback of cellular excitability in neurons and cardiomyocytes. Yet this modularity counters functional adaptability, as global changes in ambient CaM indiscriminately alter its targets. Here, we demonstrate that two structurally unrelated proteins, SH3 and cysteine-rich domain (stac) and fibroblast growth factor homologous factors (fhf) selectively diminish Ca2+/CaM-regulation of CaV1 and NaV1 families, respectively. The two proteins operate on allosteric sites within upstream portions of respective channel carboxy-tails, distinct from the CaM-binding interface. Generalizing this mechanism, insertion of a short RxxK binding motif into CaV1.3 carboxy-tail confers synthetic switching of CaM regulation by Mona SH3 domain. Overall, our findings identify a general class of auxiliary proteins that modify Ca2+/CaM signaling to individual targets allowing spatial and temporal orchestration of feedback, and outline strategies for engineering Ca2+/CaM signaling to individual targets.