Effects of ATP, Mg²⁺, and redox agents on the Ca²⁺ dependence of RyR channels from rat brain cortex

Abstract

Despite their relevance for neuronal Ca(2+)-induced Ca(2+) release (CICR), activation by Ca(2+) of ryanodine receptor (RyR) channels of brain endoplasmic reticulum at the [ATP], [Mg(2+)], and redox conditions present in neurons has not been reported. Here, we studied the effects of varying cis-(cytoplasmic) free ATP concentration ([ATP]), [Mg(2+)], and RyR redox state on the Ca(2+) dependence of endoplasmic reticulum RyR channels from rat brain cortex. At pCa 4.9 and 0.5 mM adenylylimidodiphosphate (AMP-PNP), increasing free [Mg(2+)] up to 1 mM inhibited vesicular [(3)H]ryanodine binding; incubation with thimerosal or dithiothreitol decreased or enhanced Mg(2+) inhibition, respectively. Single RyR channels incorporated into lipid bilayers displayed three different Ca(2+) dependencies, defined by low, moderate, or high maximal fractional open time (P(o)), that depend on RyR redox state, as we have previously reported. In all cases, cis-ATP addition (3 mM) decreased threshold [Ca(2+)] for activation, increased maximal P(o), and shifted channel inhibition to higher [Ca(2+)]. Conversely, at pCa 4.5 and 3 mM ATP, increasing cis-[Mg(2+)] up to 1 mM inhibited low activity channels more than moderate activity channels but barely modified high activity channels. Addition of 0.5 mM free [ATP] plus 0.8 mM free [Mg(2+)] induced a right shift in Ca(2+) dependence for all channels so that [Ca(2+)] <30 microM activated only high activity channels. These results strongly suggest that channel redox state determines RyR activation by Ca(2+) at physiological [ATP] and [Mg(2+)]. If RyR behave similarly in living neurons, cellular redox state should affect RyR-mediated CICR.

Ischemia Enhances Activation by Ca2+and Redox Modification of Ryanodine Receptor Channels from Rat Brain Cortex

“…9C). The frequencies of emergence of the three responses to Ca 2ϩ after fusion of ER isolated with DTT (77, 22, and 1% for low, moderate, and high activity channels, respectively) (Bull et al, 2007) differs from the frequencies found here with control ER isolated using physiological GSH/GSSG ratios ( p ϭ 0.02). The very low endogenous levels of S-glutathionylation and/or S-nitrosylation displayed by the RyR isoforms present in ER isolated with DTT may underlie their lower degree Single RyR channels recorded after fusion of ER vesicles obtained from control or ischemic brains and of ER vesicles from control brains preincubated with NADPH or NOR-3.…”

“…After incubation, the brain cortex was quickly dissected and homogenized at 4°C. Total and reduced glutathione were measured, and the GSH/GSSG ratio was calculated (see Materials and Methods (Marengo et al, 1996;Bull et al, 2003Bull et al, , 2007. Because low and moderate activity channels gated with fast kinetics between closed and open states, P o values (denoted as P o *) were calculated as the ratio between the mean current of the entire channel recording and the single-channel current amplitude measured in longlasting (Ͼ30 ms) fully open events (Bull et al, 2003).…”

“…As discussed below, this increase is possibly the result of RyR redox modifications, which are known to modify RyR activity (Donoso et al, 2000;Eu et al, 2000;Xia et al, 2000;Pessah et al, 2002;Aracena et al, 2003). In particular, oxidation or alkylation of critical SH residues with dithiodipyridine or thimerosal, respectively, causes single RyR channel from rat brain cortex to display higher degrees of activation in response to Ca 2ϩ (Marengo et al, 1998;Bull et al, 2003Bull et al, , 2007.…”

“…The redox state of highly reactive cysteine residues regulates RyR channel activation, enabling these channels to act as cellular redox sensors (Eu et al, 2000;Xia et al, 2000;Pessah et al, 2002). In particular, RyR redox state determines the three types of response (low, moderate, or high) to cytoplasmic [Ca 2ϩ ] increases of single RyR channels from rat brain cortex ER (Marengo et al, 1996(Marengo et al, , 1998Bull et al, 2003Bull et al, , 2007. Thus, sulfhydryl (SH) modifying agents added while recording channel activity cause a single low activity channel to adopt sequentially and stepwise, first the moderate and then the high activity response (Marengo et al, 1998).…”

Efectos neurotóxicos del timerosal, a dosis de vacuna, sobre el encéfalo y el desarrollo en hámsteres de 7 días de nacidos

“…De otro lado, recientemente se ha descrito la importancia de los canales de calcio intracitoplasmáticos de neuronas, células cerebelosas y células endoteliales capilares cerebrales (las mismas que conforman la barrera hemato-encefálica) de ratones, en el proceso de diferenciación neuronal y progresión de la neurogénesis ( 73,74 ). Precisamente, la activación de dichos canales está mediada por la oxidación coordinada de radicales SH, sobre los cuales el timerosal ejerce un potente efecto modulador, incrementando las concentraciones de calcio intracitoplasmático; de esta manera, se ha señalado que este último puede alterar la fisiología de las células nerviosas, gracias a sus efectos sobre el estado de óxido-reducción, conduciendo a trastornos funcionales, depleción de glutatión y aumento del estrés oxidativo ( [75][76][77][78][79] ).…”