Distinctive Inactivation Defects of Differing Mutant Calcium Channels Underlying Timothy Syndrome

Abstract

Timothy Syndrome (TS) is a multisystem disorder characterized by autism, immune deficiencies, and cardiac arrhythmias. Intriguingly, the underlying defect comes down to a single point mutation (either G402S or G406R) in the IS6 region of CaV1.2 channels. These channels are critical conduits of Ca2+ entry into the heart, smooth muscle and brain. As such, these channels employ two forms of feedback regulation−voltage-dependent inactivation (VDI) and Ca2+/calmodulin-dependent inactivation (CDI). In TS, these regulatory mechanisms are disrupted, resulting in inappropriate Ca2+ feedback. Given that the pattern of multisystem pathology differs for the two types of mutant channels, we here undertook in-depth biophysical analysis of the altered inactivation in each of these constructs. As reported, both mutants exhibited strongly attenuated VDI. Rather surprisingly, however, both constructs also demonstrated a clear reduction of CDI, in contrast to a previous study reporting selective weakening of VDI (PNAS105:11987). Further analysis revealed that the CDI deficits in the two mutants may arise from very different mechanisms. For G406R, voltage-dependent activation is strongly shifted to more negative potentials, while estimated maximal open probability (PO/max) at saturating depolarization was only slightly altered. According to an allosteric mechanism of CDI (Biophys J96:222a), this favoring of channel activation would reduce CDI, because opening would be enhanced even within inactivated channels (i.e., the current decrease seen upon channel inactivation would be lessened). By contrast, the G402S mutation caused a marked depolarizing shift in voltage-dependent activation, with largely unchanged PO/max. This outcome would sharply diminish channel opening at physiological voltages, yielding attenuated CDI via decreased entry into inactivated states. Recognizing these divergent mechanisms of CDI disruption may shed light on the differing disease phenotypes elaborated by the two mutations, and ultimately prove beneficial in tailoring treatments for each TS population.