Reducing Agents Sensitize C-Type Nociceptors by Relieving High-Affinity Zinc Inhibition of T-Type Calcium Channels

Abstract

Recent studies have demonstrated an important role for T-type Ca2ϩ channels (T-channels) in controlling the excitability of peripheral pain-sensing neurons (nociceptors). However, the molecular mechanisms underlying the functions of T-channels in nociceptors are poorly understood. Here, we demonstrate that reducing agents as well as endogenous metal chelators sensitize C-type dorsal root ganglion nociceptors by chelating Zn 2ϩ ions off specific extracellular histidine residues on Ca v 3.2 T-channels, thus relieving tonic channel inhibition, enhancing Ca v 3.2 currents, and lowering the threshold for nociceptor excitability in vitro and in vivo. Collectively, these findings describe a novel mechanism of nociceptor sensitization and firmly establish reducing agents, as well as Zn 2ϩ , Zn 2ϩ -chelating amino acids, and Zn 2ϩ -chelating proteins as endogenous modulators of Ca v 3.2 and nociceptor excitability.

Structural Determinants of the High Affinity Extracellular Zinc Binding Site on Cav3.2 T-type Calcium Channels

“…The average IC 50 values for the Ca v 3.1 and Ca v 3.2 currents were 82.2 Ϯ 6.9 and 3.0 Ϯ 0.2 M, respectively (n ϭ 13, 42; open symbols in Fig. 1, E and F), being consistent with the previously reported findings performed in HEK-293 cells (14,17). The half-potentials for their activation and steady-state inactivation were not significantly changed by zinc ( (Fig.…”

“…We first confirmed the Xenopus oocyte expression system could be used to measure the higher potency of zinc to inhibit Ca v 3.2 over Ca v 3.1 channels, which was originally observed in mammalian cells (14,17). Application of serial zinc solutions demonstrated that Ca v 3.2 currents were sensitively inhibited by low micromolar concentrations of zinc, whereas Ca v 3.1 currents required much higher concentrations (Fig.…”

“…Notably, Ca v 3.2 channels are among the most sensitive targets of ion channel targets to zinc block (12). Our recent experiments using chimeric channels between Ca v 3.1 and Ca v 3.2 channels identified His 191 in the extracellular loop connecting S3 and S4 of domain I as a major structural determinant for inhibition of the Ca v 3.2 by nickel, zinc, copper, and redox agents (15)(16)(17).…”

Optimization of ADME Properties for Sulfonamides Leading to the Discovery of a T-Type Calcium Channel Blocker, ABT-639

“…16 Modulation of the Ca v 3.2 (α1H) channel controls the sensitization of nociceptors, the peripheral painsensing neurons. 17 These results further support Ca v 3.2 T-type channels as a mechanism for modulating nociceptive sensitivity.High throughput screening (HTS) generated a number of sulfonamide hits including 1 (IC 50 = 3 μM) and 2 (IC 50 = 5 μM) (Figure 1) against Ca v 3.2 T-type channel in a FLIPR based Ca 2+ flux assay. 18,19 However, HTS hits 1 and 2 are metabolically unstable in rats and both have very poor oral bioavailability (F = 0.5% and 1.9%, respectively) ( Table 1).…”

“…16 Modulation of the Ca v 3.2 (α1H) channel controls the sensitization of nociceptors, the peripheral painsensing neurons. 17 These results further support Ca v 3.2 T-type channels as a mechanism for modulating nociceptive sensitivity.…”