We examined the concentration dependence of currents through CaV3.1 T-type calcium channels, varying Ca2+ and Ba2+ over a wide concentration range (100 nM to 110 mM) while recording whole-cell currents over a wide voltage range from channels stably expressed in HEK 293 cells. To isolate effects on permeation, instantaneous current–voltage relationships (IIV) were obtained following strong, brief depolarizations to activate channels with minimal inactivation. Reversal potentials were described by PCa/PNa = 87 and PCa/PBa = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent Kd values were similar for Ca2+ and Ba2+, both for block of currents carried by Na+ (3 μM for Ca2+ vs. 4 μM for Ba2+, at −30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for Ca2+ vs. 2.5 mM for Ba2+; nearly voltage independent). Block by 3–10 μM Ca2+ was time dependent, described by bimolecular kinetics with binding at ∼3 × 108 M−1s−1 and voltage-dependent exit. Ca2+o, Ba2+o, and Mg2+o also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 e− per 98 Å2 from Gouy-Chapman theory. Additionally, inward currents inactivated ∼35% faster in Ba2+o (vs. Ca2+o or Na+o). The accelerated inactivation in Ba2+o correlated with the transition from Na+ to Ba2+ permeation, suggesting that Ba2+o speeds inactivation by occupying the pore. We conclude that the selectivity of the “surface charge” among divalent cations differs between calcium channel families, implying that the surface charge is channel specific. Voltage strongly affects the concentration dependence of block, but not of permeation, for Ca2+ or Ba2+.

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 (CaV3.1) 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.

Iron is a biologically essential metal, but excess iron can cause damage to the cardiovascular and nervous systems. We examined the effects of extracellular Fe 2ϩ on permeation and gating of Ca V 3.1 channels stably transfected in HEK293 cells, by using whole-cell recording. Precautions were taken to maintain iron in the Fe 2ϩ state (e.g., use of extracellular ascorbate). With the use of instantaneous I-V currents (measured after strong depolarization) to isolate the effects on permeation, extracellular Fe 2ϩ rapidly blocked currents with 2 mM extracellular Ca 2ϩ in a voltage-dependent manner, as described by a Woodhull model with K D ϭ 2.5 mM at 0 mV and apparent electrical distance ␦ ϭ 0.17. Extracellular Fe 2ϩ also shifted activation to more-depolarized voltages (by ϳ10 mV with 1.

Ni2+ inhibits current through calcium channels, in part by blocking the pore, but Ni2+ may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of Ni2+ on CaV3.1 is affected by permeant ions. We find two components to block by Ni2+, a rapid block with little voltage dependence, and a slow block most visible as accelerated tail currents. Rapid block is weaker for outward vs. inward currents (apparent Kd = 3 vs. 1 mM Ni2+, with 2 mM Ca2+ or Ba2+) and is reduced at high permeant ion concentration (110 vs. 2 mM Ca2+ or Ba2+). Slow block depends both on the concentration and on the identity of the permeant ion (Ca2+ vs. Ba2+ vs. Na+). Slow block is 2–3× faster in Ba2+ than in Ca2+ (2 or 110 mM), and is ∼10× faster with 2 vs. 110 mM Ca2+ or Ba2+. Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations (∼3 μM Ca2+). We conclude that both fast and slow block of CaV3.1 by Ni2+ are most consistent with occlusion of the pore. The exit rate of Ni2+ for slow block is reduced at high Ni2+ concentrations, suggesting that the site responsible for fast block can “lock in” slow block by Ni2+, at a site located deeper within the pore. In contrast to the complex pore block observed for CaV3.1, inhibition of CaV3.2 by Ni2+ was essentially independent of voltage, and was similar in 2 mM Ca2+ vs. Ba2+, consistent with inhibition by a different mechanism, at a site outside the pore.