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    • The modulation of the activity of the Gardos channel

    2021-10-08

    The modulation of the activity of the Gardos channel by the chemokines IL-8 and RANTES was observed in the Duffy-positive but not in the Duffy-negative SSRBCs. This finding is consistent with the previous observation on normal red blood LJI308 (AARBCs) described by Rivera et al. [10]. Altogether, these data suggest a coupling, promoted by the sickling process, between DARC and the Gardos channel. The physiological role of DARC remains unknown. The binding of the chemokines to DARC on RBCs does not trigger a signaling cascade because the receptor lacks the ability to couple with G proteins and to induce cell responses beyond chemokine internalization that are typical for classical chemokine receptors [26]. The mechanism of action of IL-8 and RANTES has not been investigated and cannot be deduced from this study. However, the greatest dehydration risk induced by the chemokines in the Duffy-positive SSRBCs could partly contribute to the clinical heterogeneity of sickle cell disease.
    Acknowledgments
    This work was supported by funds from the Conseil Régional de la Guadeloupe (fellowship to M.C.), institutional funds from Inserm, and grant no. R07103HS from the Agence Nationale de la Recherche (ANR—SCADHESION project).
    Introduction The effect of oxidants on red cell membrane permeability is important for two main reasons. First, they have been used experimentally to study mechanisms of ion permeation [1]. Second, they may accumulate in vivo, for example, in various haemoglobinopathies and oxidant toxicities (e.g. onion poisoning in dogs: Ref. [2]). In this context, they have been associated with the increase in cation permeability observed in red cells from sickle cell patients [3] and may be involved in the pathophysiology of sickle cell disease (SCD, e.g. Ref. [4]). It is therefore relevant to understand their action as fully as possible. 1-Chloro-2,4-dinitrobenzene (CDNB) and phenazine methosulphate (PMS) are two reagents which have been shown to increase passive K+ permeability in human red cells [5], [6], [7], [8], [9]. Effects are mediated via both of the major passive K+ pathways, the K+–Cl− cotransporter (KCC, probably KCC1 isoform: [10], [11]) and the Ca2+-activated K+ channel (Gardos channel, probably IK1/SK4: [12], [13]). Many red cell membrane transporters are also affected by O2 tension [14] and the effect of oxidants may vary with this parameter. The interaction of these compounds with changes in O2 tension, however, has been largely ignored. In this paper, we consider the effects of CNBD and PMS on Gardos channel activity, at different O2 tensions. We correlate activation of the channel with changes in passive and active Ca2+ transport, and changes in Ca2+ sensitivity of the channel.
    Materials and methods
    Results
    Discussion This paper compares the effects of CDNB and PMS on Gardos channel activity in normal human red cells. We show that both stimulate channel activity, both are dependent on the presence of extracellular Ca2+, and neither is affected by inhibitors of protein (de)phosphorylation. Of the two, PMS has a considerably greater effect. In addition, a major difference is that whilst CDNB has a greater stimulatory effect in oxygenated cells, by contrast, PMS is more effective in deoxygenated cells. These actions are correlated with ca. 30% inhibition of the plasma membrane Ca2+ pump (PMCA) and an increased sensitivity of the Gardos channel to Ca2+ (EC50 falling to about 150 nM). These findings are important in understanding how oxidants alter red cell cation permeability and may be relevant to the abnormal permeability phenotype shown by deoxygenated sickle cells. Human red cells have two main passive K+ permeability pathways, KCC and the Gardos channel. The former is regulated by a phosphorylation cascade [23], [24], [25], whilst for the Gardos channel Ca2+ is the important stimulus [12]. We have presented elsewhere findings on the effect of these oxidants on KCC activity [9], [26]. We show here that the activation of the Gardos channel by CDNB and PMS is independent of changes in protein phosphorylation, as prior treatment of cells with either NEM or calyculin A has no effect. Rather, as expected, Ca2+ plays a central role. For both CDNB and PMS, extracellular Ca2+ is required for activity, implying that entry of Ca2+ from outside the cell is critical. In addition, the action of both oxidants correlated with inhibition of PMCA (by about 30%) and an increase in sensitivity of the Gardos channel to Ca2+ (by about threefold). For CDNB, these changes occurred in oxygenated cells, whilst for PMS, they were observed for deoxygenated cells. CDNB stimulation of the Gardos channel has been previously noted for human sickle cells [8]. Previous studies with PMS, however, show some discrepancy, with stimulation of the Gardos channel present [5] or absent [6]. The difference is probably explained by the presence or absence of extracellular Ca2+, which we show here to be necessary.