A PQ-Channel Mutation Associated with Epilepsy Alters the Voltage Dependence of Channel Inactivation

Abstract

Epilepsy affects 1 in 140 people, or nearly 50 million people worldwide. It is characterized by seizures that often result from neuronal hyper-excitability. A recent genome-wide study uncovered many mutations associated with epilepsy in voltage-gated calcium channels (VGCC). However, the molecular and cellular consequences of these mutations, and hence, their epileptogenic mechanisms remain unknown. Here, we investigated two mutations that occur in PQ-type voltage-gated calcium channels, which are responsible for neurotransmitter release: R477H (in the I-II loop) and Q1957X (a truncation mutation). The mutations were introduced into a human PQ channel in the pGEMHE vector. The DNA for WT or mutant channels, together with the DNA for other VGCC subunits required for proper channel expression, was subjected to in vitro cRNA synthesis. The resulting cRNA was subsequently injected into Xenopus oocytes, and channel currents were examined using Two-Electrode Voltage Clamp four days later. We thus compared the biophysical properties of WT and mutant channels. We found that the R477H mutation shifts the voltage dependence of inactivation to more depolarized voltages, rendering the mutant channels more difficult to inactivate compared to the WT channel. In addition, the time constant of inactivation was slower for the mutant. At the same time, the voltage dependence of activation was unchanged. These results suggest a potential mechanism for epileptogenesis: reduced channel inactivation would contribute to increased cellular excitability and excitotoxicity - two hallmarks of epilepsy. The results also highlight the role of the I-II loop of VGCC in channel inactivation. As for the Q1957X mutation, our current hypothesis is that this mutant generates no currents. In addition, we hypothesize that the Q1957X mutant will act as a dominant negative regulator of coexpressed WT channels via the unfolded protein response.