Little is known about the voltage-dependent potassium currents underlying spike repolarization in midbrain dopaminergic neurons. Studying mouse substantia nigra pars compacta dopaminergic neurons both in brain slice and after acute dissociation, we found that BK calcium-activated potassium channels and Kv2 channels both make major contributions to the depolarization-activated potassium current. Inhibiting Kv2 or BK channels had very different effects on spike shape and evoked firing. Inhibiting Kv2 channels increased spike width and decreased the afterhyperpolarization, as expected for loss of an action potential-activated potassium conductance. BK inhibition also increased spike width but paradoxically increased the afterhyperpolarization. Kv2 channel inhibition steeply increased the slope of the frequency-current ( f-I) relationship, whereas BK channel inhibition had little effect on the f-I slope or decreased it, sometimes resulting in slowed firing. Action potential clamp experiments showed that both BK and Kv2 current flow during spike repolarization but with very different kinetics, with Kv2 current activating later and deactivating more slowly. Further experiments revealed that inhibiting either BK or Kv2 alone leads to recruitment of additional current through the other channel type during the action potential as a consequence of changes in spike shape. Enhancement of slowly deactivating Kv2 current can account for the increased afterhyperpolarization produced by BK inhibition and likely underlies the very different effects on the f-I relationship. The crossregulation of BK and Kv2 activation illustrates that the functional role of a channel cannot be defined in isolation but depends critically on the context of the other conductances in the cell.

SNX-482, a peptide toxin isolated from tarantula venom, has become widely used as an inhibitor of Cav2.3 voltage-gated calcium channels. Unexpectedly, we found that SNX-482 dramatically reduced the A-type potassium current in acutely dissociated dopamine neurons from mouse substantia nigra pars compacta. The inhibition persisted when calcium was replaced by cobalt, showing that it was not secondary to a reduction of calcium influx. Currents from cloned Kv4.3 channels expressed in HEK-293 cells were inhibited by SNX-482 with an IC 50 of Ͻ3 nM, revealing substantially greater potency than for SNX-482 inhibition of Cav2.3 channels (IC 50 20 -60 nM). At sub-saturating concentrations, SNX-482 produced a depolarizing shift in the voltage dependence of activation of Kv4.3 channels and slowed activation kinetics. Similar effects were seen on gating of cloned Kv4.2 channels, but the inhibition was less pronounced and required higher toxin concentrations. These results reveal SNX-482 as the most potent inhibitor of Kv4.3 channels yet identified. Because of the effects on both Kv4.3 and Kv4.2 channels, caution is needed when interpreting the effects of SNX-482 on cells and circuits where these channels are present.

Galanthamine and physostigmine are clinically used cholinomimetics that both inhibit acetylcholinesterase and also interact directly with and potentiate nicotinic acetylcholine receptors (nAChR). As with most nAChRs positive allosteric modulators, the location and number of their binding site(s) within nAChRs are unknown. In this study we use the intrinsic photoreactivities of [3H]physostigmine and [3H]galanthamine upon irradiation at 312 nm to directly identify amino acids contributing to their binding sites in the Torpedo californica nAChR. Protein sequencing of fragments isolated from proteolytic digests of [3H]physostigmine- or [3H]galanthamine-photolabeled nAChR establish that in the presence of agonist (carbamylcholine), both drugs photolabeled amino acids on the complementary (non-α) surface of the transmitter binding sites (γTyr-111/γTyr-117/δTyr172). They also photolabeled δTyr-212 at the δ-β subunit interface and γTyr-105 in the vestibule of the ion channel, with photolabeling of both residues enhanced in the presence of agonist. Furthermore, [3H]physostigmine photolabeling of γTyr-111, γTyr-117, δTyr-212, and γTyr-105 was inhibited in the presence of non-radioactive galanthamine. The locations of the photolabeled amino acids in the nAChR structure and the results of computational docking studies provide evidence that in the presence of agonist, physostigmine and galanthamine bind to at least three distinct sites in the nAChR extracellular domain: at the α-γ interface (I) in the entry to the transmitter binding site and (II) in the vestibule of the ion channel near the level of the transmitter binding site, and at the δ-β interface (III) in a location equivalent to the benzodiazepine binding site in GABAA receptors.