Redox regulation of the yeast voltage-gated Ca2+ channel homolog Cch1p by glutathionylation of specific cysteine residues

Abstract

Cch1p, the yeast homolog of the pore-forming subunit α 1 of the mammalian voltage-gated Ca 2+ channel (VGCC), is located on the plasma membrane and mediates the redox-dependent influx of Ca 2+ . Cch1p is known to undergo both rapid activation (after oxidative stress and or a change to high pH) and slow activation (after ER stress and mating pheromone activation), but the mechanism of activation is not known. We demonstrate here that both the fast activation (exposure to pH 8-8.5 or treatment with H 2 O 2 ) and the slow activation (treatment with tunicamycin or α-factor) are mediated through a common redoxdependent mechanism. Furthermore, through mutational analysis of all 18 exposed cysteine residues in the Cch1p protein, we show that the four mutants C587A, C606A, C636A and C642A, which are clustered together in a common cytoplasmic loop region, were functionally defective for both fast and slow activations, and also showed reduced glutathionylation. These four cysteine residues are also conserved across phyla, suggesting a conserved mechanism of activation. Investigations into the enzymes involved in the activation reveal that the yeast glutathione S-transferase Gtt1p is involved in the glutathionylation of Cch1p, while the thioredoxin Trx2p plays a role in the Cch1p deglutathionylation.

Highly conserved extracellular residues mediate interactions between pore-forming and regulatory subunits of the yeast Ca2+ channel related to the animal VGCC/NALCN family

“…One of the nine Cys residues in repeat II, Cys-798, is not conserved, even in yeasts and fungi, and was not subsequently examined. Note that the transmembrane topology model is completely identical to those of animal VGCCs and NALCNs and partly differs from the previously proposed model of Cch1, in which the four Cys residues in repeat I is located in the cytoplasm ( 27 ).…”

In Vivo Imaging with Genetically Encoded Redox Biosensors

“…Pichia pastoris E GSH roGFP1 [37,197] roGFP1-iE [37,197,198] roGFP1-iL [198] general redox state Redoxfluor [44] Saccharomyces cerevisiae E GSH rxYFP [31,43,46,[199][200][201] roGFP1 [25][26][27][28][29]202,203] roGFP2 [12,19,33,[204][205][206] roGFP [207] eroGFP [35,36,[38][39][40][41][42]208] Grx1-roGFP2 [15,24,32,47,48,[209][210][211][212][213][214] general redox state rxRFP [30] Redoxfluor [44,215] H 2 O 2 HyPer…”

“…Several research groups conducted studies devoted to various transport proteins in microorganisms which turned out to be regulated by redox-sensitive mechanisms [43,209,211,258,263]. By recording E GSH using roGFP in catalases and peroxidases deprived S. Typhimurium after H 2 O 2 addition, van der Heijden et al discovered that at a potential of approximately −290 mV a switching point was observed after which the H 2 O 2 influx rapidly dropped to ~8% and ~3% of initial values in log-and stationary-phase bacteria, respectively [258].…”

“…The authors reported that glutathione S-transferase Gtt1p was responsible for glutathionylation of both Yvc1p and Cch1p. The opposite process, degluthathionylation, was mediated by thioredoxin Trx2 for vacuolar calcium channel and by Trx2 and glutaredoxin Grx1p for plasma membrane calcium channel (Figure 7B) [209,211].…”

“…Oxidation of the cellular glutathione pool leads to activation of Cch1p and Yvc1p calcium channels by glutathionylation. Deglutathionylaton is required for inactivation of these channels[209,211]. (C) So-AqpA of S. oligofermentans is involved in transport of H2O2 and expression of this gene is up-regulated in the presence of H2O2.…”