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[Ni.sup.2+] inhibits current through calcium channels, in part by blocking the pore, but [Ni.sup.2+] may also allosterically affect channel activity via sites outside the permeation pathway. As a test for pore blockade, we examined whether the effect of [ faster with 2 vs. 110 mM [Ca.sup.2+] or [Ba.sup.2+]. Slow block is orders of magnitude slower than the diffusion limit, except in the nominal absence of divalent cations (~3 [micro]M [Ca.sup.2+]). We conclude that both fast and slo 1 at a site outside the pore.

We examined the concentration dependence of currents through [Ca.sub.v]3.1 T-type calcium channels, varying [Ca.sup.2+] and [Ba.sup.2+] 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 [P.sub.Ca]/[P.sub.Na] = 87 and [P.sub.Ca]/[P.sub.Ba] = 2, based on Goldman-Hodgkin-Katz theory. However, analysis of chord conductances found that apparent [[K].sub.d] values were similar for [Ca.sup.2+] and [Ba.sup.2+], both for block of currents carried by [Na.sup.+] (3 [micro]M for [Ca.sup.2+] vs. 4 [micro]M for [Ba.sup.2+], at -30 mV; weaker at more positive or negative voltages) and for permeation (3.3 mM for [Ca.sup.2+] vs. 2.5 mM for [Ba.sup.2+]; nearly voltage independent). Block by 3-10 [micro]M [Ca.sup.2+] was time dependent, described by bimolecular kinetics with binding at ~3 x [10.sup.8] [M.sup.-1] [s.sup.-1] and voltage-dependent exit. [Ca.sup.2+.sub.o], [Ba.sup.2+.sub.o], and [Mg.sup.2+.sub.o] also affected channel gating, primarily by shifting channel activation, consistent with screening a surface charge of 1 [e.sup.-] per 98 [[Angstrom].sup.2] from Gouy-Chapman theory. Additionally, inward currents inactivated ~35% faster in [Ba.sup.2+.sub.o] (vs. [Ca.sup.2+.sub.o] or [Na.sup.2+.sub.o]). The accelerated inactivation in [Ba.sup.2+.sub.o] correlated with the transition from [Na.sup.+] to [Ba.sup.2+] permeation, suggesting that [Ba.sup.2+.sub.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 [Ca.sup.2+] or [Ba.sup.2+].

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 [Y.sup.3+] as a tool to locate the activation gate of the [alpha]1G ([Ca.sub.v]3.1) T-type calcium channel, [Y.sup.3+] 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 [Y.sup.3+] at a concentration-dependent rate, only eightfold slower than open-channel block. When extracellular [Ca.sup.2+] was replaced with [Ba.sup.2+], the rate of open block by [Y.sup.3+] was unaffected, but closed block was threefold faster than in [Ca.sup.2+], 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. KEY WORDS: channel block * selectivity filter * patch clamp * voltage clamp * permeation

J Neurol Neurosurg Psychiatry 2003;74: 1555-1561 Objective: Multifocal motor neuropathy with persistent conduction blocks is classically described as a chronic neuropathy with progressive onset, and acute forms have not previously [...]