Cysteines in the loop between IS5 and the pore helix of CaV3.1 are essential for channel gating

Abstract

The role of six cysteines of Ca(V)3.1 in channel gating was investigated. C241, C271, C282, C298, C313, and C323, located in the extracellular loop between segment IS5 and the pore helix, were each mutated to alanine; the resultant channels were expressed and studied by patch clamping in HEK293 cells. C298A and C313A conducted calcium currents, while the other mutants were not functional. C298A and C313A as well as double mutation C298/313A significantly reduced the amplitude of the calcium currents, shifted the activation curve in the depolarizing direction and slowed down channel inactivation. Redox agents dithiothreitol (DTT) and 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) shifted the current activation curve of wild-type channels in the hyperpolarizing direction. Activation curve for all mutated channels was shifted in hyperpolarizing direction by DTT while DTNB caused a depolarizing shift. Our study reveals that the cysteines we studied have an essential role in Ca(V)3.1 gating. We hypothesize that cysteines in the large extracellular loop of Ca(V)3.1 form bridges within the loop and/or neighboring channel segments that are essential for channel gating.

Carbon monoxide inhibition of Cav3.2 T‐type Ca 2+ channels reveals tonic modulation by thioredoxin

“…However, it is important to note that redox modulation of these sites (which are conserved among T‐type channel subtypes; ref. 44) also results in large changes in voltage dependence and kinetics (44), yet inhibition by CO was not accompanied by such changes (Fig. 1).…”

“…T‐type channels are known to have extracellular redox‐sensitive sites in addition to the H191 residue of Cav3.2; Karmazinova et al (44), for example, have reported 16 extracellular cysteine residues in Cav3.1, providing much potential for redox modulation. To investigate this further, we examined the effects of cysteine.…”

“…Like other voltage‐gated Ca 2+ channels, T‐type channel α subunits consist of 4 domains (I‐IV), each made up of 6 transmembrane regions (S1‐S6). Lacinova and colleagues (44) have indicated that the extracellular region of the channel that forms the linker in domain I between regions S5 and S6 is highly conserved between the Cav3‐channel subtypes, and contains functionally important cysteine residues with the potential to form disulfide bridges (44). These authors indicated that such sites are redox sensitive, consistent with another report that the extracellularly acting oxidant DTNB can inhibit all three T‐type channel isoforms (48).…”

Congenital Heart Block Maternal Sera Autoantibodies Target an Extracellular Epitope on the α1G T-Type Calcium Channel in Human Fetal Hearts

“…An alignment of the α 1G and α 1H proteins revealed that α 1H contains a similar peptide sequence in the S5–S6 loop in repeat I with sequence identity for NWNQY, as well as nearby amino acids, with the conserved Cys residue being implicated previously in channel gating activation, inactivation, and deactivation [63].…”

Hybrid upconversion nanomaterials for optogenetic neuronal control

“…light). 32 Since ChR2 is a light-gated cation channel, the opening of this channel upon light illumination would facilitate the influx of cations and lead to an inward current flow to drive neuronal depolarization. 2, 17 The membrane-localized expression of ChR2 was first confirmed by the presence of tdTomato fluorescence using confocal microscopy (Fig.…”