Kv3 channel assembly, trafficking and activity are regulated by zinc through different binding sites

Abstract

Key points• Kv3 channels play a critical role in neuronal fast spiking and their regulation can profoundly influence the functions of various neural circuits.• Zinc reversibly inhibits fast spiking in cultured neurons by inhibiting Kv3 channels. Surprisingly, we found that the zinc inhibition is not through its known site in the N-terminal T1 domain of Kv3.1.• We have identified a new zinc-binding site in the Kv3.1 C-terminus, involved in regulating channel activity and targeting, but not the zinc inhibition.• We have also identified the junction between the first transmembrane segment and the first extracellular loop as the major zinc-sensing site in regulating Kv3 activity. This result allows us to reconstitute zinc-resistant fast spiking.• Kv3 channel assembly, localization and activity are regulated by zinc through intracellular and extracellular binding sites.Abstract Zinc, a divalent heavy metal ion and an essential mineral for life, regulates synaptic transmission and neuronal excitability via ion channels. However, its binding sites and regulatory mechanisms are poorly understood. Here, we report that Kv3 channel assembly, localization and activity are regulated by zinc through different binding sites. Local perfusion of zinc reversibly reduced spiking frequency of cultured neurons most likely by suppressing Kv3 channels. Indeed, zinc inhibited Kv3.1 channel activity and slowed activation kinetics, independent of its site in the N-terminal T1 domain. Biochemical assays surprisingly identified a novel zinc-binding site in the Kv3.1 C-terminus, critical for channel activity and axonal targeting, but not for the zinc inhibition. Finally, mutagenesis revealed an important role of the junction between the first transmembrane (TM) segment and the first extracellular loop in sensing zinc. Its mutant enabled fast spiking with relative resistance to the zinc inhibition. Therefore, our studies provide novel mechanistic insights into the multifaceted regulation of Kv3 channel activity and localization by divalent heavy metal ions.

Kv3 Channels: Enablers of Rapid Firing, Neurotransmitter Release, and Neuronal Endurance

“…In addition to altering axonal targeting, this mutation also slowed the time constant of activation of Kv3.1b channels and altered voltage dependence. In contrast, mutation of the other two histidine residues downstream of the targeting motif did not affect trafficking into the axon, or significantly impact voltage dependence or kinetics (87). Thus the function of these two downstream sites is not yet known.…”

“…Histidine residues at these sites are essential for metal binding, and simultaneous mutation of all three histidines fully abolishes zinc binding (87). In the Kv3.1b channel isoform, mutation of the histidine located within the axonal targeting motif abolished targeting of Kv3.1b to the axon, suggesting that zinc binding at this site controls either the interaction of the targeting motif with ankyrin G or with the NH 2 terminus of the Kv3.1 channel (87). In addition to altering axonal targeting, this mutation also slowed the time constant of activation of Kv3.1b channels and altered voltage dependence.…”

“…Extracellular zinc (1 mM ZnCl 2 ) rapidly suppresses Kv3.1a or Kv3.1b currents in a mammalian cell line, and this effect is reversed within 5 min of washout (87). Application of a lower concentration of zinc ions (100 M) to cerebellar granule cells, which express Kv3.1, suppresses their ability to fire at high rates.…”

TRPM2 ion channels regulate macrophage polarization and gastric inflammation during Helicobacter pylori infection

“…Whole-cell patch-clamp recording was performed on BMDM obtained from WT and Trpm2 −/− mice, using the same internal solution, Hank’s buffer, recording protocols and the setup as previously described 57 . In voltage-clamp recording, the cells were initially held at −80 mV, given 200-ms voltage episodes in 10-mV increment from −100 mV to +100 mV, and then the I-V relationship was obtained.…”

Fibrotic scar model and TGF‐β1 differently modulate action potential firing and voltage‐dependent ion currents in hippocampal neurons in primary culture

“…Increase in A‐type potassium current due to hypothyroidism was accompanied by decreased AP width, lower repolarization potential and increased number of APs in a series (Sanchez‐Alonso et al ., ). In a primary culture of hippocampal neurons, increased expression of K V 3.1 channels increased the firing activity (Martina et al ., ; Gu et al ., ) and inhibition of those channels attenuated AP firing (Gu et al ., ).…”