TRPC3 and NALCN channels drive pacemaking in substantia nigra dopaminergic neurons

Midbrain dopamine (DA) neurons are slow pacemakers that maintain extracellular DA levels. During the interspike intervals, subthreshold slow depolarization underlies autonomous pacemaking and determines its rate. However, the ion channels that determine slow depolarization are unknown. Here we show that TRPC3 and NALCN channels together form sustained inward currents responsible for the slow depolarization of nigral DA neurons. Specific TRPC3 channel blockade completely blocked DA neuron pacemaking, but the pacemaking activity in TRPC3 knock-out (KO) mice was perfectly normal, suggesting the presence of compensating ion channels. Blocking NALCN channels abolished pacemaking in both TRPC3 KO and wild-type mice. The NALCN current and mRNA and protein expression are increased in TRPC3 KO mice, indicating that NALCN compensates for TRPC3 currents. In normal conditions, TRPC3 and NALCN contribute equally to slow depolarization. Therefore, we conclude that TRPC3 and NALCN are two major leak channels that drive robust pacemaking in nigral DA neurons.

TRPC3 and NALCN channels drive pacemaking in substantia nigra dopaminergic neurons

Midbrain dopamine (DA) neurons are slow pacemakers that maintain extracellular DA levels. During the interspike intervals, subthreshold slow depolarization underlies autonomous pacemaking and determines its rate. However, the ion channels that determine slow depolarization are unknown. Here we show that TRPC3 and NALCN channels together form sustained inward currents responsible for the slow depolarization of nigral DA neurons. Specific TRPC3 channel blockade completely blocked DA neuron pacemaking, but the pacemaking activity in TRPC3 knock-out (KO) mice was perfectly normal, suggesting the presence of compensating ion channels. Blocking NALCN channels abolished pacemaking in both TRPC3 KO and wild-type mice. The NALCN current and mRNA and protein expression are increased in TRPC3 KO mice, indicating that NALCN compensates for TRPC3 currents. In normal conditions, TRPC3 and NALCN contribute equally to slow depolarization. Therefore, we conclude that TRPC3 and NALCN are two major leak channels that drive robust pacemaking in nigral DA neurons.

Direct Activation of TRPC3 Channels by the Antimalarial Agent Artemisinin

(1) Background: Members of the TRPC3/TRPC6/TRPC7 subfamily of canonical transient receptor potential (TRP) channels share an amino acid similarity of more than 80% and can form heteromeric channel complexes. They are directly gated by diacylglycerols in a protein kinase C-independent manner. To assess TRPC3 channel functions without concomitant protein kinase C activation, direct activators are highly desirable. (2) Methods: By screening 2000 bioactive compounds in a Ca2+ influx assay, we identified artemisinin as a TRPC3 activator. Validation and characterization of the hit was performed by applying fluorometric Ca2+ influx assays and electrophysiological patch-clamp experiments in heterologously or endogenously TRPC3-expressing cells. (3) Results: Artemisinin elicited Ca2+ entry through TRPC3 or heteromeric TRPC3:TRPC6 channels, but did not or only weakly activated TRPC6 and TRPC7. Electrophysiological recordings confirmed the reversible and repeatable TRPC3 activation by artemisinin that was inhibited by established TRPC3 channel blockers. Rectification properties and reversal potentials were similar to those observed after stimulation with a diacylglycerol mimic, indicating that artemisinin induces a similar active state as the physiological activator. In rat pheochromocytoma PC12 cells that endogenously express TRPC3, artemisinin induced a Ca2+ influx and TRPC3-like currents. (4) Conclusions: Our findings identify artemisinin as a new biologically active entity to activate recombinant or native TRPC3-bearing channel complexes in a membrane-confined fashion.