Genetic alteration of the metal/redox modulation of Cav3.2 T‐type calcium channel reveals its role in neuronal excitability

Abstract

Key pointsr In this study, we describe a new knock-in (KI) mouse model that allows the study of the H191-dependent regulation of T-type Cav3.2 channels. Sensitivity to zinc, nickel and ascorbate of native Cav3.2 channels is significantly impeded in the dorsal root ganglion (DRG) neurons of this KI mouse. Importantly, we describe that this H191-dependent regulation has discrete but significant effects on the excitability properties of D-hair (down-hair) cells, a sub-population of DRG neurons in which Cav3.2 currents prominently regulate excitability.r Overall, this study reveals that the native H191-dependent regulation of Cav3.2 channels plays a role in the excitability of Cav3.2-expressing neurons. This animal model will be valuable in addressing the potential in vivo roles of the trace metal and redox modulation of Cav3.2 T-type channels in a wide range of physiological and pathological conditions. Abstract Cav3.2 channels are T-type voltage-gated calcium channels that play important roles in controlling neuronal excitability, particularly in dorsal root ganglion (DRG) neurons where they are involved in touch and pain signalling. Cav3.2 channels are modulated by low concentrations of metal ions (nickel, zinc) and redox agents, which involves the histidine 191 (H191) in the channel's extracellular IS3-IS4 loop. It is hypothesized that this metal/redox modulation would contribute to the tuning of the excitability properties of DRG neurons. However, the precise role of this H191-dependent modulation of Cav3.2 channel remains unresolved. Towards this goal, we have generated a knock-in (KI) mouse carrying the mutation H191Q in the Cav3.2 protein.Electrophysiological studies were performed on a subpopulation of DRG neurons, the D-hair cells, which express large Cav3.2 currents. We describe an impaired sensitivity to zinc, nickel and ascorbate of the T-type current in D-hair neurons from KI mice. Analysis of the action potential and low-threshold calcium spike (LTCS) properties revealed that, contrary to that observed in WT D-hair neurons, a low concentration of zinc and nickel is unable to modulate (1) the rheobase threshold current, (2) the afterdepolarization amplitude, (3) the threshold potential necessary to trigger an LTCS or (4) the LTCS amplitude in D-hair neurons from KI mice. Together, our data demonstrate that this H191-dependent metal/redox regulation of Cav3.2 channels can tune neuronal excitability. This study validates the use of this Cav3.2-H191Q mouse model for further investigations of the physiological roles thought to rely on this Cav3.2 modulation.

Activity-dependent regulation of T-type calcium channels by submembrane calcium ions

“…In order to evaluate whether T-type channels would be regulated by a rise in intracellular Ca 2+ ([Ca 2+ ] INT ), we first used the Ca 2+ ionophore ionomycin. In D-hair mechanoreceptor sensory neurons, which specifically express a high density of T-type channels ( Shin et al, 2003 ; Dubreuil et al, 2004 ; Voisin et al, 2016 ), extracellular perfusion of 10 µM ionomycin induced a potent decrease of the T-type current ( Figure 1a ). This current inhibition (~85% in average) occurred in the minute range ( Figure 1b ) and was associated with a large hyperpolarizing shift in the steady-state inactivation curve (~22 mV, p < 0.001, Figure 1c ).…”

“…All animal use procedures were done in accordance with the directives of the French Ministry of Agriculture (A 34-172-41). Dorsal root ganglion (DRG) neurons were prepared as described earlier ( Voisin et al, 2016 ). Briefly, adult male C57BL/6J mice were anaesthetized with pentobarbital injection and transcardially perfused with HBSS (pH 7.4, 4°C).…”

Cell-Type Specific Distribution of T-Type Calcium Currents in Lamina II Neurons of the Rat Spinal Cord

“…Besides ethosuximide, Zonisamide and Z944 also have been recruited in the preclinical trial for pain treatment (Zamponi, 2016 ). Up to now, Ni 2+ is still the most often used T-type channel blockers for animal experiments, possibly due to its high efficiency and reversible action (Chen et al, 2015 ; Voisin et al, 2016 ; Zhang et al, 2017 ). We observed that NiCl 2 (0.2 mM) decreased the amplitude of T-type calcium current to 29.6 ± 4.6% of control in SG neurons, which was lower than that of the observation in lamina II–V SDH neurons (Ryu and Randic, 1990 ), possibly due to the different holding potential (−90 mV in Ryu’s study vs. −110 mV in our study) used.…”

Granulocyte-colony–stimulating factor (G-CSF) signaling in spinal microglia drives visceral sensitization following colitis

“…3B). DRG neurons were exposed to factors secreted from G-CSF-primed microglia cells for a further 16 h, after which both spontaneous and evoked neuronal activity was recorded by patch clamp electrophysiology (26,27). As shown in Fig.…”