Diminished Dihydropyridine Block of Timothy Syndrome Cav1.2 Channels Independent from Mutation-Altered Open State Inactivation

Abstract

Timothy syndrome (TS) is a multisystem developmental disorder presenting clinical phenotypes of autism and cardiac arrhythmias. The disease is linked to single amino acid mutations (G402S or G406R) in the Cav1.2 L-type calcium channel which dramatically disrupt voltage-gated inactivation from the open state, as seen in electrophysiological recordings with barium as the charge carrier to remove calcium-dependent inactivation. Initial reports suggested that inactivation-deficient TS channels are less sensitive to DHP antagonists, presumably because these drugs preferentially inhibit inactivated channels. Here we thoroughly investigated inactivation and isradipine inhibition of G406R channels at voltages where the channel inactivates directly from closed states. Interestingly, despite dramatic differences in open state inactivation, closed state inactivation during long 25s conditioning pulses is minimal and not distinguishable between WT and G406R channels. Nevertheless, TS channels are still less sensitive than WT channels to DHP block at these voltages: 10nM isradipine blocks 10% of G406R channels vs. 30% of WT channels at −100mV and 35% of G406R channels vs. 70% of WT channels at −40mV. Investigation of −100mV block by multiple concentrations of isradipine revealed that G406R channels in deep closed states have a greatly reduced affinity (Kd ∼4.5 greater) for isradipine. To test how altered −100mV block affects block at modestly depolarized potentials, a drug concentration was chosen for TS channels (50nM isradipine) that produced the same 30% block at −100mV. Strikingly, when the change in affinity at −100mV was accounted for, block at −40mV prior to channel opening was identical between mutant and WT. These results suggest that the G406R mutation alters rested state block and that this accounts for the reduced drug block at modestly depolarized voltages, independent of the severe disruption in open state inactivation of the channel.