Molecular Mechanisms of Subtype-Specific Inhibition of Neuronal T-Type Calcium Channels by Ascorbate

Abstract

T-type Ca2ϩ channels (T-channels) are involved in the control of neuronal excitability and their gating can be modulated by a variety of redox agents. Ascorbate is an endogenous redox agent that can function as both an anti-and pro-oxidant. Here, we show that ascorbate selectively inhibits native Ca v 3.2 T-channels in peripheral and central neurons, as well as recombinant Ca v 3.2 channels heterologously expressed in human embryonic kidney 293 cells, by initiating the metal-catalyzed oxidation of a specific, metal-binding histidine residue in domain 1 of the channel. Our biophysical experiments indicate that ascorbate reduces the availability of Ca v 3.2 channels over a wide range of membrane potentials, and inhibits Ca v 3.2-dependent low-threshold-Ca 2ϩ spikes as well as burst-firing in reticular thalamic neurons at physiologically relevant concentrations. This study represents the first mechanistic demonstration of ion channel modulation by ascorbate, and suggests that ascorbate may function as an endogenous modulator of neuronal excitability.

GABAB receptors inhibit low-voltage activated and high-voltage activated Ca2+ channels in sensory neurons via distinct mechanisms

“…Indeed, Kv7 channels are highly sensitive to oxidation by ROS due to the specific cysteine pocket in the intracellular S2-S3 linker of the channel [27,28]. Interestingly, T-type Ca 2+ channels are also sensitive to redox modulation, albeit in contrast to Kv7 channels, oxidizing compounds and ROS do not activate but inhibit T-type channel activity [21,29,30]. Since ROS generation by NK1 receptor triggering was sensitive to PTX [26] we hypothesized that unconventional signaling cascade causing inhibition of LVA current in DRG neurons by baclofen may also be mediated by a ROSdependent mechanism.…”

“…Acting as intracellular second messengers, such ROS augmented the activity of inhibitory Kv7 channels providing a mechanism for substance P mediated peripheral endogenous analgesia [26]. Because i) T-type channels are inhibited by oxidizing agents and ROS [21,29,30];…”

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

“…We recently identified His 191 in the IS3-IS4 extracellular linker as a critical determinant of nickel, copper, zinc, and redox sensitivity of Ca v 3.2 (15)(16)(17). To localize other partners of His 191 required for interacting with high affinity zinc, we systematically transferred various portions in domain I of Ca v 3.2 into Ca v 3.1/ Q172H , which was only slightly more sensitive to zinc than wild-type Ca v 3.1.…”

“…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).…”

“…Therefore, we investigated additional residue(s) involved in zinc block of Ca v 3.2, focusing on zinc inhibition rather than copper to avoid complications of redox reactions (16). We found that the residues that precede His 191 , Asp 189 , and Gly 190 , were also critical residues for determining the high zinc sensitivity of Ca v 3.2.…”