Redox Modulation of Ion Channels

Redox modulation of ion channel activity seems to be an important regulatory mechanism under physiological conditions for several vasomotor functions, including regulation of cell Em and vascular tone, activation of transcription factors required for the expression of genes, apoptosis and necrosis, and cellular protective mechanisms against ischemic or hypoxic insults.

Several studies have provided evidence that 02-sensing in different tissues is mediated by effector K+ channels with cytosolic redox as a sensor. G-protein-coupled inwardly rectifiying K+ (KIR) channels, present in excitable and endocrine tissues, have been shown to be activated by the reducing agent dithiothreitol (DTT) (84). For DTT-dependent activation, a cysteine-residue located in the N-terminal cytoplasmic domain of the channel seems to be essential. Interestingly, when this cysteine-residue is mutated, DTT-dependent activation is abolished, but receptor-mediated channel activation is not affected, suggesting that intracellular redox potential acts in a receptor-independent manner. It is possible that G-protein signaling may involve redox changes (57). Opening of channels can slow cardiac pacemaking, shut down secretion or inhibit neuronal firing in the central nervous system and thus can protect cells against ischemic or hypoxic insults.

In the lung NEB cells and the H-146 cell line, the 02-sensitive K+ currents are augmented by H202 or by activation of the NADPH oxidase with phorbol esters (51, 69). In the HERG channel (which generates the cardiac KIR currents) activation is accelerated by H202 (10). Consequently oxidation tends to increase the potassium current and reduction to decrease it.

The large conductance channels in different tissues have also been reported to be hypoxia and redox sensitive. In this case, the reducing agent GSH reversibly activates the neuronal KCa channel by increasing the activation rate constant and decreasing the deactivation rate constant (20). In myocytes of the taenia caeci, DTT and GSH significantly increases and the oxidizing agent thimerosal decreases the open probability of KCa channels in excised patches (39). In contrast, the activity of large conductance Kq, channels in CA1 pyramidal neurons is markedly increased by the oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB). DTT has no effect on channel activity but can reverse DTNB-induced enhancement (32). The different behavior of these channels in response to redox changes could be explained by the heterogeneity of the KCa channel in different tissues.

3.1. Redox Control of Pulmonary Vascular Ion Channels

The Em of arterial smooth muscle cells is an important regulator of arterial tone and hence arterial diameter. These cells have steady or slowly changing resting Em around -65 to -50 mV in vitro, close to the predicted equilibrium potential for K+ ions (EK is approximately -85 mV with physiological extracellular K+, ~5 mM) (49). The opening of K+ channels in the cell membrane increases K+ efflux, which causes membrane hyperpolarization. This closes voltage-dependent Ca2+ channels, decreasing Ca2+ entry and leading to pulmonary vasodilatation. Conversely, inhibition of K+ channels activity causes membrane depolarization, leads to cell contraction and vasoconstriction. Therefore, alterations in K+ channel activity play an important role in the development of hypoxic pulmonary vasoconstriction (72). It has been proposed that the reduction in Po2 from normoxic to hypoxic levels, which causes reversible hypoxic pulmonary vasoconstriction, shifts the ratio of redox couples toward a more reduced state in PASMCs, leading to inhibition of K+ current, membrane depolarization, Ca2+ influx via L-type Ca2+ channels, an increase in [Ca2+]cyt and pulmonary vasoconstriction.

Three classes ofK+ channels have been identified in PASMCs: voltage-gated K+ (Kv) channels (25, 58, 95), Ca2+-activated K+ (KCa) channels (3,56) and ATP-sensitive K+ (KATP) channels (49). The K+ channels which control Em in PASMCs, and inhibition of which initiates HPV, conduct an outward current which activates at potentials less negative then -50 mV, is slowly inactivating and blocked by the Kv channel blocker 4-aminopyridine (4-AP) but not by inhibitors of KCa or KATP channels (5, 8, 79). The large conductance KCa channels are activated by intracellular Ca2+ and membrane depolarization. The opening of channels hyperpolarizes the cell membrane and thus prevents excessive vasoconstriction. KATP channels are activated after the depletion of intracellular ATP level for example, by hypoxic vasoconstriction associated with anoxia or after exposure to metabolic poisons. Therefore, the activity of KATP channels could bear an important role for the regulation of basal vascular tone under severe pathological conditions.

The existence of redox modulation in KCa channel activity has been shown in studies carried out in PASMCs. Reducing agents, such as DTT, reduced glutathione, and reduced nicotinamide adenine dinucleotide (NADH) decreased, and oxidizing agents, such as DTNB, oxidized glutathione and NAD, increased the KCa channel activity in PASMCs. The increased activity due to oxidizing agents was diminished by applying reducing agents (55). In contrast, in isolated PASMCs from large pulmonary arteries the KCa channel activity was unaffected by NAD and GSSG or NADH and GSH (66). This suggests that the change in the intracellular redox state, which would be expected during acute hypoxia, does not alter the activity of KCa channel in PASMCs from large conduit pulmonary arteries.

A variety of redox-active agents have been studied in PASMCs, showing that exogenous reducing agents mimic the effect of hypoxia on several types of Oj-sensitive K+ channels, Em and [Ca2+]cyt. Yuan (82) provided support for the role of a redox-based 02-sensing system in PASMCs. Reduced glutathione caused an inhibition in the whole-cell K+ current and significant membrane depolarization. In the presence of GSH, hypoxia had no further effect on K+ current or resting Em, suggesting that both hypoxia and GSH block the same K+ channels. In addition, oxidized gluthatione (GSSG), co-enzyme Q,0 and duroquinone, on the other hand, increase whole cell K+ current and hyperpolarize the resting Em (61, 72). Park et al. showed, that DTT partially blocks Kv current in PASMCs and accelerates the inactivation kinetics, but does not affect steady-state activation and inactivation (54). On the contrary, the oxidizing agent 2,2'-dithio-bis(5-nitropyridine) (DTNBP) increases Kv current and accelerates activation kinetics (54). The effect of DTT and DTNB on whole-cell K+ current and resting Em of adult rat PASMCs is shown in Figure 2. Under normoxic conditions superfusion of PA SMCs with experimental solution containing DTT reduced K+ current and depolarized the resting Em. DTNB showed a time-dependent membrane hyperpolarization of these cells under hypoxic conditions, which is consistent with the opening of K+ channels.

Figure 2. Effect of the reducing agent dithiothreito! (DTT) and oxidizing agent dithionitrobenzoic-acid (DTNB) on whole-cell outward K+ current (/J and resting £mof adult rat PASMCs. Currents were evoked from a holding potential of-70 mV to +50 mV in 20-mV steps. A: Representative traces demonstrate IK under normoxic conditions (left) and after treatment with 3 mM DTT (right). B: DTT depolarizes the resting Em in normoxia (n=5). C: The figure shows the augmenting effect of DTNB on /K during hypoxia. D: DTNB causes PASMC hyperpolarization under hypoxic conditions (n=4). Data are mean±SE. * P<0.05 vs. control.

Figure 2. Effect of the reducing agent dithiothreito! (DTT) and oxidizing agent dithionitrobenzoic-acid (DTNB) on whole-cell outward K+ current (/J and resting £mof adult rat PASMCs. Currents were evoked from a holding potential of-70 mV to +50 mV in 20-mV steps. A: Representative traces demonstrate IK under normoxic conditions (left) and after treatment with 3 mM DTT (right). B: DTT depolarizes the resting Em in normoxia (n=5). C: The figure shows the augmenting effect of DTNB on /K during hypoxia. D: DTNB causes PASMC hyperpolarization under hypoxic conditions (n=4). Data are mean±SE. * P<0.05 vs. control.

The studies presented here support the hypothesis that K+ channels and in particular Kv channels are a target for redox active agents. These results suggest that pulmonary vascular tone can be modulated by electron-transfer agents at the level of K+ channels. In addition, there appear to be similarities between the effects of hypoxia and reducing agents in PASMCs. Shifting the cellular redox status to a more reduced state, in both cases, causes pulmonary vasoconstriction by inhibition of K+ channels and subsequent Em depolarization.

Was this article helpful?

0 0
Reducing Blood Pressure Naturally

Reducing Blood Pressure Naturally

Do You Suffer From High Blood Pressure? Do You Feel Like This Silent Killer Might Be Stalking You? Have you been diagnosed or pre-hypertension and hypertension? Then JOIN THE CROWD Nearly 1 in 3 adults in the United States suffer from High Blood Pressure and only 1 in 3 adults are actually aware that they have it.

Get My Free Ebook


Post a comment