Y³⁺ Block Demonstrates an Intracellular Activation Gate for the α1G T-type Ca²⁺ Channel

Classical electrophysiology and contemporary crystallography suggest that the activation gate of voltage-dependent channels is on the intracellular side, but a more extracellular “pore gate” has also been proposed. We have used the voltage dependence of block by extracellular Y³⁺ as a tool to locate the activation gate of the α1G (Ca_V3.1) T-type calcium channel. Y³⁺ block exhibited no clear voltage dependence from −40 to +40 mV (50% block at 25 nM), but block was relieved rapidly by stronger depolarization. Reblock of the open channel, reflected in accelerated tail currents, was fast and concentration dependent. Closed channels were also blocked by Y³⁺ at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular Ca²⁺ was replaced with Ba²⁺, the rate of open block by Y³⁺ was unaffected, but closed block was threefold faster than in Ca²⁺, suggesting the slower closed-block rate reflects ion–ion interactions in the pore rather than an extracellularly located gate. Since an extracellular blocker can rapidly enter the closed pore, the primary activation gate must be on the intracellular side of the selectivity filter.