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Although several ionic mechanisms are known to control rate and regularity of the slow pacemaker in dopamine (DA) neurons, the core mechanism of pacing is controversial. Here we tested the hypothesis that pacemaking of SNc DA neurons is enabled by an unconventional conductance. We found that 1-(2,4-xylyl)guanidinium (XG), an established blocker of gating pore currents, selectively inhibits pacemaking of DA neurons. The compound inhibited all slow pacemaking DA neurons that were tested, both in the substantia nigra pars compacta, and in the ventral tegmental area. Interestingly, bursting behavior was not affected by XG. Furthermore, the drug did not affect fast pacemaking of GABAergic neurons from substantia nigra pars reticulata neurons or slow pacemaking of noradrenergic neurons. In DA neurons, current-clamp analysis revealed that XG did not appear to affect ion channels involved in the action potential. Its inhibitory effect persisted during blockade of all ion channels previously suggested to contribute to pacemaking. RNA sequencing and voltage-clamp recordings yielded no evidence for a gating pore current to underlie the conductance. However, we could isolate a small subthreshold XG-sensitive current, which was carried by both Na+ and Cl− ions. Although the molecular target of XG remains to be defined, these observations represent a step towards understanding pacemaking in DA neurons.

Although several ionic mechanisms are known to control rate and regularity of the pacemaker in dopamine (DA) neurons from the substantia nigra pars compacta (SNc), a conductance essential for pacing has yet to be defined. Here we provide pharmacological evidence that pacemaking of SNc DA neurons is enabled by an unconventional conductance. We found that 1-(2,4-xylyl)guanidine (XG), an established blocker of gating pore currents in mutant voltage gated sodium channels, selectively stops pacemaking of DA SNc neurons and is without effect on the main pore of their voltage-gated channels. We isolated a voltage-dependent, non-inactivating XG-sensitive current of 20-25 pA which operates in the relevant subthreshold range and is carried by both Na+ and Cl- ions. While the molecular identity of this conductance remains to be determined, we show that this XG-sensitive current is crucial to sustain pacemaking in these neurons.

AIM: Small-conductance Ca(2+)-activated potassium (SK) channels are activated exclusively by increases in intracellular Ca(2+), that binds to calmodulin constitutively associated with the channel. Wild-type SK2 channels are activated by Ca(2+) with an EC50 value of ~0.3 μM. Here, we investigate hydrophobic interactions between the HA helix and the S4–S5 linker as a major determinant of channel apparent Ca(2+) sensitivity. METHODS: site-directed mutagenesis, electrophysiological recordings and molecular dynamic (MD) simulations were utilized. RESULTS: Mutations that decrease hydrophobicity at the HA-S4–S5 interface lead to Ca(2+) hyposensitivity of SK2 channels. Mutation that increase hydrophobicity results in hypersensitivity to Ca(2+). The Ca(2+) hypersensitivity of the V407F mutant relies on the interaction of the cognate phenylalanine with the S4–S5 linker in the SK2 channel. Replacing the S4–S5 linker of the SK2 channel with the S4–S5 linker of the SK4 channel results in loss of the hypersensitivity caused by V407F. This difference between the S4–S5 linkers of SK2 and SK4 channels can be partially attributed to I295 equivalent to a valine in the SK4 channel. A N293A mutation in the S4–S5 linker also increases hydrophobicity at the HA-S4–S5 interface and elevates the channel apparent Ca(2+) sensitivity. The double N293A/V407F mutations generate a highly Ca(2+) sensitive channel, with an EC(50) of 0.02 μM. The MD simulations of this double mutant channel revealed a larger channel cytoplasmic gate. CONCLUSION: The electrophysiological data and MD simulations collectively suggest a crucial role of the interactions between the HA helix and S4–S5 linker in the apparent Ca(2+) sensitivity of SK2 channels.